Bio

Academic Appointments


Research & Scholarship

Current Research and Scholarly Interests


Dr. Falkow is no longer taking students or postdoctoral fellows in his laboratory. He is actively engaged in undergraduate teaching and course development in the medical school

Teaching

2013-14 Courses


Publications

Journal Articles


  • The Lessons of Asilomar and the H5N1 "Affair" MBIO Falkow, S. 2012; 3 (5)

    Abstract

    In mid-1974, soon after the first recombinant DNA molecules were replicated in Escherichia coli, scientists called for, and observed, a voluntary moratorium on certain experiments. One goal of the moratorium was to hold a conference (Asilomar) to evaluate the risks, if any, of this new technology. The Asilomar conference concluded that recombinant DNA research should proceed but under strict guidelines. The furor surrounding the recent genetic manipulation of the transmissibility of avian influenza virus H5N1 led to a short-term moratorium that has been extended indefinitely. The question is how long should the moratorium remain in place, or should it be permanent? Voltaire observed, "History never repeats itself; man always does." I believe the parallels of Asilomar can be applied to the problem facing biomedical science today. We should move forward to establish standardized guidelines, using common sense and scientific creativity. The onus of responsibility falls on the individual scientist and involves the education of a new generation of scientists into the social and ethical implications of genetic engineering in a new age of genomics and synthetic biology. In addition, scientists who work with infectious agents must deal not only with biosafety but also, alas, with bioterrorism. The H5N1 "affair" is not a question of freedom of inquiry or the dissemination of scientific research; it is a question of the social responsibility of science and scientists to ensure that the public understands why this work is beneficial and worthwhile.

    View details for DOI 10.1128/mBio.00354-12

    View details for Web of Science ID 000310585000037

    View details for PubMedID 23047749

  • Transcriptional response in the peripheral blood of patients infected with Salmonella enterica serovar Typhi PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Thompson, L. J., Dunstan, S. J., Dolecek, C., Perkins, T., House, D., Dougan, G., Nguyen Thi Hue, T. H., Tran Thi Phi La, T. P., Du, D. C., Le Thi Phuong, T. P., Nguyen Thi Dung, T. D., Tran Tinh Hien, T. H., Farrar, J. J., Monack, D., Lynn, D. J., Popper, S. J., Falkow, S. 2009; 106 (52): 22433-22438

    Abstract

    We used microarrays and transcriptional profiling of peripheral blood to investigate the host response of 29 individuals who contracted typhoid fever in the Mekong Delta region of Vietnam. Samples were taken over a nine month period encompassing acute disease, convalescence, and recovery. We found that typhoid fever induced a distinct and highly reproducible signature in the peripheral blood that changed during treatment and convalescence, returning in the majority of cases to the "normal" profile as measured in healthy uninfected controls. Unexpectedly, there was a strong, distinct signature of convalescence present at day 9 after infection that remained virtually unchanged one month after acute infection and in some cases persisted as long as nine months despite a complete clinical recovery in all patients. Patients who retain the convalescent signature may be genetically or temporarily incapable of developing an effective immune response and may be more susceptible to reinfection, relapse, or the establishment of a carrier state.

    View details for DOI 10.1073/pnas.0912386106

    View details for Web of Science ID 000273178700071

    View details for PubMedID 20018727

  • Streptococcus pneumoniae nasopharyngeal colonization induces type I interferons and interferon-induced gene expression BMC GENOMICS Joyce, E. A., Popper, S. J., Falkow, S. 2009; 10

    Abstract

    We employed DNA microarray technology to investigate the host response to Streptococcus pneumoniae in a mouse model of asymptomatic carriage. Over a period of six weeks, we profiled transcript abundance and complexity in the Nasal Associated Lymphoid Tissue (NALT) to identify genes whose expression differed between pneumococcal-colonized and uncolonized states.Colonization with S. pneumoniae altered the expression of hundreds of genes over the course of the study, demonstrating that carriage is a dynamic process characterized by increased expression of a set of early inflammatory responses, including induction of a Type I Interferon response, and the production of several antimicrobial factors. Subsequent to this initial inflammatory response, we observed increases in transcripts associated with T cell development and activation, as well as wounding, basement membrane remodeling, and cell proliferation. Our analysis suggests that microbial colonization induced expression of genes encoding components critical for controlling JAK/STAT signaling, including stat1, stat2, socs3, and mapk1, as well as induction of several Type I Interferon-inducible genes and other antimicrobial factors at the earliest stages of colonization.Examining multiple time points over six weeks of colonization demonstrated that asymptomatic carriage stimulates a dynamic host response characterized by temporal waves with distinct biological programs. Our data suggest that the usual response to the presence of the pneumocccus is an initial controlled inflammatory response followed by activation of host physiological processes such as response to wounding, basement membrane remodeling, and increasing cellular numbers that ultimately allow the host to maintain an intact epithelium and eventually mount a preventive adaptive immune response.

    View details for DOI 10.1186/1471-2164-10-404

    View details for Web of Science ID 000270394200002

    View details for PubMedID 19712482

  • I never met a microbe I didn't like NATURE MEDICINE Falkow, S. 2008; 14 (10): 1053-1057

    View details for Web of Science ID 000259892300035

    View details for PubMedID 18841148

  • The Fortunate Professor ANNUAL REVIEW OF MICROBIOLOGY Falkow, S. 2008; 62: 1-18

    Abstract

    My professional life can be summarized by a quote from the Talmud. Much have I learned from my teachers, More from my colleagues, But most from my students. It is the fortunate professor who learns from the student.

    View details for DOI 10.1146/annurev.micro.62.081307.162931

    View details for Web of Science ID 000259968000002

    View details for PubMedID 18345978

  • Is persistent bacterial infection good for your health? CELL Falkow, S. 2006; 124 (4): 699-702

    Abstract

    Certain bacterial pathogens have evolved to survive in their human hosts for long periods without causing harm. Is it possible that these persistent bacterial infections provide a protective benefit to the host?

    View details for DOI 10.1016/j.cell.2006.02.004

    View details for Web of Science ID 000237240900014

    View details for PubMedID 16497581

  • The role of antigenic drive and tumor-infiltrating accessory cells in the pathogenesis of Helicobacter-induced mucosa-associated lymphoid tissue lymphoma AMERICAN JOURNAL OF PATHOLOGY Mueller, A., O'Rourke, J., Chu, P., Chu, A., Dixon, M. F., Bouley, D. M., Lee, A., FALKOW, S. 2005; 167 (3): 797-812

    Abstract

    Gastric B-cell lymphoma of mucosa-associated lymphoid tissue type is closely linked to chronic Helicobacter pylori infection. Most clinical and histopathological features of the tumor can be reproduced by prolonged Helicobacter infection of BALB/c mice. In this study, we have addressed the role of antigenic stimulation in the pathogenesis of the lymphoma by experimental infection with Helicobacter felis, followed by antibiotic eradication therapy and subsequent re-infection. Antimicrobial therapy was successful in 75% of mice and led to complete histological but not "molecular" tumor remission. Although lympho-epithelial lesions disappeared and most gastric lymphoid aggregates resolved, transcriptional profiling revealed the long-term mucosal persistence of residual B cells. Experimental re-introduction of Helicobacter led to very rapid recurrence of the lymphomas, which differed from the original lesions by higher proliferative indices and more aggressive behavior. Immunophenotyping of tumor cells revealed massive infiltration of lesions by CD4(+) T cells, which express CD 28, CD 69, and interleukin-4 but not interferon-gamma, suggesting that tumor B-cell proliferation was driven by Th 2-polarized, immunocompetent, and activated T cells. Tumors were also densely colonized by follicular dendritic cells, whose numbers were closely associated with and predictive of treatment outcome.

    View details for Web of Science ID 000231514500015

    View details for PubMedID 16127158

  • Microarray-based detection of Salmonella enterica serovar typhimurium transposon mutants that cannot survive in macrophages and mice INFECTION AND IMMUNITY Chan, K., Kim, C. C., FALKOW, S. 2005; 73 (9): 5438-5449

    Abstract

    DNA microarrays provide an opportunity to combine the principles of signature-tagged mutagenesis (STM) with microarray technology to identify potentially important bacterial virulence genes. The scope of DNA microarrays allows for less laborious screening on a much larger scale than possible by STM alone. We have adapted a microarray-based transposon tracking strategy for use with a Salmonella enterica serovar Typhimurium cDNA microarray in order to identify genes important for survival and replication in RAW 264.7 mouse macrophage-like cells or in the spleens of BALB/cJ mice. A 50,000-CFU transposon library of S. enterica serovar Typhimurium strain SL1344 was serially passaged in cultured macrophages or intraperitoneally inoculated into BALB/cJ mice. The bacterial genomic DNA was isolated and processed for analysis on the microarray. The novel application of this approach to identify mutants unable to survive in cultured cells resulted in the identification of components of Salmonella pathogenicity island 2 (SPI2), which is known to be critical for intracellular survival and replication. In addition, array results indicated that a number of SPI1-associated genes, currently not associated with intracellular survival, are negatively selected. However, of the SPI1-associated mutants individually tested for intracellular survival, only a sirA mutant exhibited reduced numbers relative to those of wild-type bacteria. Of the mutants unable to survive in mice, significant proportions are either components of the SPI2 pathogenicity island or involved in lipopolysaccharide synthesis. This observation is in agreement with results obtained in the original S. enterica serovar Typhimurium STM screen, illustrating the utility of this approach for the high-throughput identification of virulence factors important for survival in the host.

    View details for DOI 10.1128/IAI.73.9.5438-5449.2005

    View details for Web of Science ID 000231460000017

    View details for PubMedID 16113260

  • Helicobacter pylori and gastric cancer: What can be learned by studying the response of gastric epithelial cells to the infection? CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION Mueller, A., FALKOW, S., Amieva, M. R. 2005; 14 (8): 1859-1864

    Abstract

    The development of gastric adenocarcinoma is closely linked to chronic infection with the bacterial pathogen Helicobacter pylori. One Helicobacter-specific virulence factor in particular, the CagA protein, has emerged as a main effector molecule in the interaction of H. pylori with gastric epithelial cells and has been implicated in gastric carcinogenesis. This review highlights the latest insights that have been gained into the pathogenesis of the disease by transcriptional profiling approaches studying gene expression in normal gastric tissue and gastric cancer tissue from human biopsy material as well as animal models of Helicobacter infection. The potential role of CagA as a bacterial oncoprotein is also discussed.

    View details for DOI 10.1158/1055-9965.EPI-04-0820

    View details for Web of Science ID 000231195600004

    View details for PubMedID 16103426

  • The Campylobacter jejuni stringent response controls specific stress survival and virulence-associated phenotypes MOLECULAR MICROBIOLOGY Gaynor, E. C., Wells, D. H., MacKichan, J. K., FALKOW, S. 2005; 56 (1): 8-27

    Abstract

    Campylobacter jejuni is a highly prevalent food-borne pathogen that causes diarrhoeal disease in humans. A natural zoonotic, it must overcome significant stresses both in vivo and during transmission despite the absence of several traditional stress response genes. Although relatively little is understood about its mechanisms of pathogenesis, its ability to interact with and invade human intestinal epithelial cells closely correlates with virulence. A C. jejuni microarray-based screen revealed that several known virulence genes and several uncharacterized genes, including spoT, were rapidly upregulated during infection of human epithelial cells. spoT and its homologue relA have been shown in other bacteria to regulate the stringent response, an important stress response that to date had not been demonstrated for C. jejuni or any other epsilon-proteobacteria. We have found that C. jejuni mounts a stringent response that is regulated by spoT. Detailed analyses of a C. jejuni delta spoT mutant revealed that the stringent response is required for several specific stress, transmission and antibiotic resistance-related phenotypes. These include stationary phase survival, growth and survival under low CO2/high O2 conditions, and rifampicin resistance. A secondary suppressor strain that specifically rescues the low CO2 growth defect of the delta spoT mutant was also isolated. The stringent response additionally proved to be required for the virulence-related phenotypes of adherence, invasion, and intracellular survival in two human epithelial cell culture models of infection; spoT is the first C. jejuni gene shown to participate in longer term survival in epithelial cells. Microarray analyses comparing wild-type to the delta spoT mutant also revealed a strong correlation between gene expression profiles and phenotype differences observed. Together, these data demonstrate a critical role for the C. jejuni stringent response in multiple aspects of C. jejuni biology and pathogenesis and, further, may lend novel insight into unexplored features of the stringent response in other prokaryotic organisms.

    View details for DOI 10.1111/j.1365-2958.2005.04525.x

    View details for Web of Science ID 000227499000002

    View details for PubMedID 15773975

  • The adaptor molecules LAT and SLP-76 are specifically targeted by Yersinia to inhibit T cell activation JOURNAL OF EXPERIMENTAL MEDICINE Gerke, C., FALKOW, S., Chien, Y. H. 2005; 201 (3): 361-371

    Abstract

    T cell responses are critical to the survival of Yersinia-infected animals. Yersinia have the ability to directly suppress T lymphocyte activation through the virulence factor YopH, a tyrosine phosphatase. Using single cell video microscopy and FACS analysis, here we show that even an average of one Yersinia per T cell is sufficient to inhibit or alter T cell responses. This efficient inhibition is traced to specific targeting by YopH of the adaptor proteins, linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kD (SLP-76), which are crucial for T cell antigen receptor (TCR) signaling. A catalytically inactive YopH translocated via the type III secretory pathway from the bacteria into T cells primarily binds to LAT and SLP-76. Furthermore, among the proteins of the TCR signaling pathway, the tyrosine phosphorylation levels of LAT and SLP-76 are the most affected in T cells exposed to low numbers of Yersinia pseudotuberculosis. This is the first example showing that a pathogen targets these adaptor proteins in the TCR signaling pathway, suggesting a novel mechanism by which pathogens may efficiently alter T cell-mediated immune responses.

    View details for DOI 10.1084/jem.20041120

    View details for Web of Science ID 000227092900007

    View details for PubMedID 15699071

  • Mig-14 is an inner membrane-associated protein that promotes Salmonella typhimurium resistance to CRAMP, survival within activated macrophages and persistent infection MOLECULAR MICROBIOLOGY Brodsky, I. E., Ghori, N., FALKOW, S., Monack, D. 2005; 55 (3): 954-972

    Abstract

    Salmonella enterica serovar Typhimurium (S. typhimurium) infects a wide variety of mammalian hosts and in rodents causes a typhoid-like systemic disease involving replication of bacteria inside macrophages within reticuloendothelial tissues. Previous studies demonstrated that the mig-14 and virK genes of Salmonella enterica are important in bacterial resistance to anti-microbial peptides and are necessary for continued replication of S. typhimurium in the liver and spleen of susceptible mice after orogastric inoculation. In this work we report that inflammatory signalling via interferon-gamma (IFN-gamma) is crucial to controlling replication of mig-14 mutant bacteria within the liver and spleen of mice after oral infection. Using a Salmonella persistence model recently developed in our laboratory, we further demonstrate that mig-14 contributes to long-term persistence of Salmonella in the spleen and mesenteric lymph nodes of chronically infected mice. Both mig-14 and virK contribute to the survival of Salmonella in macrophages treated with IFN-gamma and are necessary for resistance to cathelin-related anti-microbial peptide (CRAMP), an anti-microbial peptide expressed at high levels in activated mouse macrophages. We also show that both Mig-14 and VirK inhibit the binding of CRAMP to Salmonella, and demonstrate that Mig-14 is an inner membrane-associated protein. We further demonstrate by transmission electron microscopy that the primary locus of CRAMP activity appears to be intracytoplasmic, rather than at the outer membrane, suggesting that Mig-14 may prevent the penetration of the inner membrane by CRAMP. Together, these data indicate an important role for mig-14 in anti-microbial peptide resistance in vivo, and show that this resistance is important to the survival of Salmonella in systemic sites during both acute and persistent infection.

    View details for DOI 10.1111/j.1365-2958.2004.04444.x

    View details for Web of Science ID 000226457800024

    View details for PubMedID 15661016

  • The Campylobacter jejuni dccRS two-component system is required for optimal in vivo colonization but is dispensable for in vitro growth MOLECULAR MICROBIOLOGY MacKichan, J. K., Gaynor, E. C., Chang, C., Cawthraw, S., NEWELL, D. G., Miller, J. F., FALKOW, S. 2004; 54 (5): 1269-1286

    Abstract

    A Campylobacter jejuni two-component signal transduction system (TCSTS), designated dccR-dccS (diminished capacity to colonize; Cj1223c-Cj1222c), has been found to be important for in vivo colonization but dispensable for in vitro growth. A DeltadccR response regulator mutant generated using the virulent strain 81-176 background exhibited significantly reduced colonization of immunocompetent limited flora (I-LF) mice, severe combined immunodeficient limited flora (SCID-LF) mice, and 1-day-old chicks. A DeltadccS sensor kinase mutant was likewise defective for colonization in the I-LF mouse model. DeltadccR-infected SCID-LF mice also exhibited dramatically reduced inflammation relative to wild type-infected SCID-LF mice. Despite this diminished colonization capacity, the DeltadccRS mutants were indistinguishable from wild type for growth under numerous in vitro conditions as well as for various phenotypes. Microarray analysis identified several genes encoding putative periplasmic and membrane proteins as being regulated by this two-component system; binding of purified His-tagged DccR to the promoter region of two of these genes supports a direct protein-DNA interaction. A conserved repeat sequence was identified in the promoter regions of these genes and in three other promoter regions in the genome, including that of an operon encoding a putative type I secretion system. Two of the regulated target genes were found to be essential for optimal colonization. Both the two-component system and the putative regulated genes have uncharacterized homologues in other Campylobacter and Helicobacter spp., suggesting that they may perform an important function in colonization among a variety of related pathogenic species.

    View details for DOI 10.1111/j.1365-2958.2004.04371.x

    View details for Web of Science ID 000225264900011

    View details for PubMedID 15554967

  • Profiling of microdissected gastric epithelial cells reveals a cell type-specific response to Helicobacter pylori infection GASTROENTEROLOGY Mueller, A., Merrell, D. S., Grimm, J., FALKOW, S. 2004; 127 (5): 1446-1462

    Abstract

    Helicobacter pylori colonizes the epithelial lining of the human stomach and is associated with disorders ranging from chronic gastritis to peptic ulcers and gastric cancer. We have explored the transcriptional response of the epithelium globally by applying a whole-genome approach to a murine model of infection.The 3 major epithelial lineages of the stomach-the parietal, mucus-producing, and chief cells-were harvested from cryosections of infected and uninfected murine stomachs by laser microdissection and subjected to gene expression profiling. The localization and quantity of selected transcripts were verified by in situ hybridization and quantitative real-time reverse-transcription polymerase chain reaction.Each cell type is characterized by a transcriptional signature profile. The parietal cell profile is highly enriched for factors involved in mitochondrial energy generation, whereas the chief cell predominantly expresses digestive enzymes and glycosylation-associated proteins. In contrast, the mucus cell signature is distinguished by an abundance of cell-surface receptors, signaling molecules, and factors involved in antigen presentation. All of these indicate a role in sampling, sensing, and responding to environmental stimuli. In line with this biological function, we measured a strong transcriptional response to Helicobacter pylori infection only in this cell type. The genes that are differentially expressed upon infection are implicated in a proinflammatory and mucosal defense response as well as modulation of angiogenesis, iron availability, and tumor suppression.Laser microdissection-assisted transcriptional profiling is a useful tool to explore the biology of specific cell populations and is sensitive enough to measure the transcriptional response to bacterial infection in vivo.

    View details for DOI 10.1053/j.gastro.2004.08.054

    View details for Web of Science ID 000225049800020

    View details for PubMedID 15521014

  • Phosphorylation-independent effects of CagA during interaction between Helicobacter pylori and T84 polarized monolayers JOURNAL OF INFECTIOUS DISEASES El-Etr, S. H., Mueller, A., Tompkins, L. S., FALKOW, S., Merrell, D. S. 2004; 190 (8): 1516-1523

    Abstract

    To extend our knowledge of host-cell targets of Helicobacter pylori, we characterized the interaction between H. pylori and human T84 epithelial cell polarized monolayers. Transcriptional analysis by use of human microarrays and a panel of isogenic H. pylori mutants revealed distinct responses to infection. Of the 670 genes whose expression changed, most (92%) required the cag pathogenicity island (PAI). Although altered expression of many genes was dependent on CagA (80% of the PAI-dependent genes), expression of >30% of these host genes occurred independent of the phosphorylation state of the CagA protein. Similarly, we found that injected CagA localized to the apical surface of cells and showed preferential accumulation at the apical junctions in a phosphorylation-independent manner. These data suggest the presence of distinct functional domains within the CagA protein that play essential roles in protein targeting and alteration of host-cell signaling pathways.

    View details for Web of Science ID 000223968700020

    View details for PubMedID 15378446

  • Persistent bacterial infections: The interface of the pathogen and the host immune system NATURE REVIEWS MICROBIOLOGY Monack, D. M., Mueller, A., FALKOW, S. 2004; 2 (9): 747-765

    Abstract

    Persistent bacterial infections involving Mycobacterium tuberculosis, Salmonella enterica serovar Typhi (S. typhi) and Helicobacter pylori pose significant public-health problems. Multidrug-resistant strains of M. tuberculosis and S. typhi are on the increase, and M. tuberculosis and S. typhi infections are often associated with HIV infection. This review discusses the strategies used by these bacteria during persistent infections that allow them to colonize specific sites in the host and evade immune surveillance. The nature of the host immune response to this type of infection and the balance between clearance of the pathogen and avoidance of damage to host tissues are also discussed.

    View details for DOI 10.1038/nrmicro955

    View details for Web of Science ID 000223627200019

    View details for PubMedID 15372085

  • Chronic Helicobacter pylori infection with Syndey Strain 1 and a newly identified mouse-adapted strain (Sydney Strain 2000) in C57BL/6 and BALB/c mice INFECTION AND IMMUNITY Thompson, L. J., Danon, S. J., Wilson, J. E., O'Rourke, J. L., Salama, N. R., FALKOW, S., Mitchell, H., Lee, A. 2004; 72 (8): 4668-4679

    Abstract

    The mouse model of Helicobacter pylori-induced disease using Sydney strain 1 (SS1) has been used extensively in Helicobacter research. Herein we describe the isolation and characterization of a new mouse-colonizing strain for use in comparative studies. One strain capable of persistent mouse colonization was isolated from a total of 110 clinical isolates and is named here SS2000 (Sydney strain 2000). Genome typing revealed a number of differences between SS1 and SS2000 as well as between them and the respective original clinical isolates. In particular, SS2000 lacked the entire cag pathogenicity island, while SS1 contained all 27 genes of the island. C57BL/6 and BALB/c mice were infected with SS1 or SS2000 or were treated with broth medium (controls). After 6 months host-specific effects were evident, including lower colonization levels in the BALB/c animals. Few pathological differences were observed between SS1- and SS2000-infected animals. However, by 15 months postinfection, SS1-infected C57BL/6 mice had developed more severe gastritis than the SS2000-infected animals. In contrast SS2000-infected BALB/c mice showed increased accumulation of mucosa-associated lymphoid tissue compared to those infected with SS1. This improved comparative model of H. pylori-induced disease allowed dissection of both host and strain effects and thus will prove useful in further studies.

    View details for DOI 10.1128/IAI.72.8.4668-4679.2004

    View details for Web of Science ID 000222932600042

    View details for PubMedID 15271928

  • Delineation of upstream signaling events in the Salmonella pathogenicity island 2 transcriptional activation pathway JOURNAL OF BACTERIOLOGY Kim, C. C., FALKOW, S. 2004; 186 (14): 4694-4704

    Abstract

    Survival and replication in the intracellular environment are critical components of the ability of Salmonella enterica serovar Typhimurium to establish systemic infection in the murine host. Intracellular survival is mediated by a number of genetic loci, including Salmonella pathogenicity island 2 (SPI2). SPI2 is a 40-kb locus encoding a type III secretion system that secretes effector molecules, which permits bacterial survival and replication in the intracellular environment of host cells. A two-component regulatory system, ssrAB, is also encoded in SPI2 and controls expression of the secretion system and effectors. While the environmental signals to which SPI2 responds in vivo are not known, activation of expression is dependent on OmpR and can be stimulated in vitro by chelation of cations or by a shift from rich to acidic minimal medium. In this work, we demonstrated that SPI2 activation is associated with OmpR in the phosphorylated form (OmpR-P). Mutations in envZ and ackA-pta, which disrupted two distinct sources of OmpR phosphorylation, indicated that SPI2 activation by chelators or a shift from rich to acidic minimal medium is largely dependent on functional EnvZ. In contrast, the PhoPQ pathway is not required for SPI2 activation in the presence of OmpR-P. As in the case of in vitro stimulation, SPI2 expression in macrophages correlates with the presence of OmpR-P. Additionally, EnvZ, but not acetyl phosphate, is required for maximal expression of SPI2 in the intracellular environment, suggesting that the in vitro SPI2 activation pathway is the same as that used in vivo.

    View details for DOI 10.1128/JB.186.14.1694-4704.2004

    View details for Web of Science ID 000222728100030

    View details for PubMedID 15231802

  • LuxS is required for persistent pneumococcal carriage and expression of virulence and biosynthesis genes INFECTION AND IMMUNITY Joyce, E. A., Kawale, A., Censini, S., Kim, C. C., Covacci, A., FALKOW, S. 2004; 72 (5): 2964-2975

    Abstract

    Streptococcus pneumoniae causes several diseases, including otitis media, pneumonia, and meningitis. Although little is known about the regulation of or how individual pneumococcal factors contribute to these disease states, there is evidence suggesting that some factors are regulated by a cell-density-dependent mechanism (quorum sensing). Quorum sensing allows bacteria to couple transcription with changes in cell density; bacteria achieve this by sensing and responding to small diffusible signaling molecules. We investigated how the LuxS signaling system impacts the biology of S. pneumoniae. An analysis of the transcriptional profiles of a serotype 2 strain and an isogenic luxS deletion strain utilizing an S. pneumoniae-specific microarray indicated that LuxS regulates gene expression involved in discrete cellular processes, including pneumolysin expression. Contrary to the paradigm for quorum sensing, we observed pronounced effects on transcription in early log phase, where gene expression was repressed in the mutant. Assessing the mutant for its ability to infect and cause disease in animals revealed a profound defect in ability to persist in the nasopharyngeal tissues. Our analysis of an S. pneumoniae transcriptome revealed a function for LuxS in gene regulation that is not dependent upon high cell density and is likely involved in the maintenance of pneumococcal load in susceptible hosts.

    View details for DOI 10.1128/IAI.72.5.2964.2795.2004

    View details for Web of Science ID 000221120100059

    View details for PubMedID 15102809

  • Evolutionary genetics: CCR5 mutation and plague protection. Nature Mecsas, J., Franklin, G., Kuziel, W. A., Brubaker, R. R., Falkow, S., Mosier, D. E. 2004; 427 (6975): 606-?

    Abstract

    A recent and prevalent mutation in the chemokine receptor CCR5 in humans of northern European ancestry has been proposed to provide protection against bubonic plague. Here we infect both normal and CCR5-deficient mice with the bacterium Yersinia pestis, the cause of the plague epidemics that wiped out one-third of Europeans in the Middle Ages, and find no difference in either bacterial growth or survival time between the two groups. Unless the pathogenesis of Yersinia infection differs markedly between mice and humans, our results indicate that CCR5 deficiency in people is unlikely to protect against plague.

    View details for PubMedID 14961112

  • Breaking into the epithelial apical-junctional complex - news from pathogen hackers CURRENT OPINION IN CELL BIOLOGY Vogelmann, R., Amieva, M. R., FALKOW, S., Nelson, W. J. 2004; 16 (1): 86-93

    Abstract

    The epithelial apical-junctional complex is a key regulator of cellular functions. In addition, it is an important target for microbial pathogens that manipulate the cell to survive, proliferate and sometimes persist within a host. Out of a myriad of potential molecular targets, some bacterial and viral pathogens have selected a subset of protein targets at the apical-junctional complex of epithelial cells. Studying how microbes use these targets also teaches us about the inherent physiological properties of host molecules in the context of normal junctional structure and function. Thus, we have learned that three recently uncovered components of the apical-junctional complex of the Ig superfamily--junctional adhesion molecule, Nectin and the coxsackievirus and adenovirus receptor--are important regulators of junction structure and function and represent critical targets of microbial virulence gene products.

    View details for DOI 10.1016/j.ceb.2003.12.002

    View details for Web of Science ID 000188769900013

    View details for PubMedID 15037310

  • Comparison of Campylobacter jejuni isolates implicated in Guillain-Barre syndrome and strains that cause enteritis by a DNA microarray INFECTION AND IMMUNITY Leonard, E. E., Tompkins, L. S., FALKOW, S., Nachamkin, I. 2004; 72 (2): 1199-1203

    Abstract

    We asked whether Campylobacter jejuni isolated from patients with Guillain-Barré syndrome (GBS) differ from isolates isolated from patients with uncomplicated gastrointestinal infection using DNA microarray analysis. We found that specific GBS genes or regions were not identified, and microarray analysis confirmed significant genomic heterogeneity among the isolates.

    View details for DOI 10.1128/IAI.72.2.1199-1203.2004

    View details for Web of Science ID 000188766400076

    View details for PubMedID 14742576

  • Salmonella typhimurium persists within macrophages in the mesenteric lymph nodes of chronically infected Nramp1(+/+) mice and can be reactivated by IFN gamma neutralization JOURNAL OF EXPERIMENTAL MEDICINE Monack, D. M., Bouley, D. M., FALKOW, S. 2004; 199 (2): 231-241

    Abstract

    Host-adapted strains of Salmonella are capable of establishing a persistent infection in their host often in the absence of clinical disease. The mouse model of Salmonella infection has primarily been used as a model for the acute systemic disease. Therefore, the sites of long-term S. typhimurium persistence in the mouse are not known nor are the mechanisms of persistent infection clearly understood. Here, we show that S. typhimurium can persist for as long as 1 yr in the mesenteric lymph nodes (MLNs) of 129sv Nramp1(+)(/)(+) (Slc11a1(+)(/)(+)) mice despite the presence of high levels of anti-S. typhimurium antibody. Tissues from 129sv mice colonized for 60 d contain numerous inflammatory foci and lesions with features resembling S. typhi granulomas. Tissues from mice infected for 365 d have very few organized inflammatory lesions, but the bacteria continue to persist within macrophages in the MLN and the animals generally remain disease-free. Finally, chronically infected mice treated with an interferon-gamma neutralizing antibody exhibited symptoms of acute systemic infection, with evidence of high levels of bacterial replication in most tissues and high levels of fecal shedding. Thus, interferon-gamma, which may affect the level of macrophage activation, plays an essential role in the control of the persistent S. typhimurium infection in mice.

    View details for DOI 10.1084/jem.20031319

    View details for Web of Science ID 000188369700009

    View details for PubMedID 14734525

  • Molecular Koch's postulates applied to bacterial pathogenicity - a personal recollection 15 years later NATURE REVIEWS MICROBIOLOGY Falkow, S. 2004; 2 (1): 67-72

    Abstract

    Koch's postulates were derived from Robert Koch's work on infectious diseases, such as anthrax and tuberculosis, which still engage us to this day. These guidelines were an attempt to establish a standard for identifying the specific causation of an infectious disease and to convince sceptics that microorganisms could cause disease. They were also established to encourage an increasing number of novice microbiologists to use more rigorous criteria before claiming a causal relationship between a microorganism and a disease.

    View details for DOI 10.1038/nrmicro799

    View details for Web of Science ID 000221401000019

    View details for PubMedID 15035010

  • The genome-sequenced variant of Campylobacter jejuni NCTC 11168 and the original clonal clinical isolate differ markedly in colonization, gene expression, and virulence-associated phenotypes JOURNAL OF BACTERIOLOGY Gaynor, E. C., Cawthraw, S., Manning, G., MacKichan, J. K., FALKOW, S., NEWELL, D. G. 2004; 186 (2): 503-517

    Abstract

    The genome sequence of the enteric bacterial pathogen Campylobacter jejuni NCTC 11168 (11168-GS) was published in 2000, providing a valuable resource for the identification of C. jejuni-specific colonization and virulence factors. Surprisingly, the 11168-GS clone was subsequently found to colonize 1-day-old chicks following oral challenge very poorly compared to other strains. In contrast, we have found that the original clinical isolate from which 11168-GS was derived, 11168-O, is an excellent colonizer of chicks. Other marked phenotypic differences were also identified: 11168-O invaded and translocated through tissue culture cells far more efficiently and rapidly than 11168-GS, was significantly more motile, and displayed a different morphology. Serotyping, multiple high-resolution molecular genotyping procedures, and subtractive hybridization did not yield observable genetic differences between the variants, suggesting that they are clonal. However, microarray transcriptional profiling of these strains under microaerobic and severely oxygen-limited conditions revealed dramatic expression differences for several gene families. Many of the differences were in respiration and metabolism genes and operons, suggesting that adaptation to different oxygen tensions may influence colonization potential. This correlates biologically with our observation that anaerobically priming 11168-GS or aerobically passaging 11168-O caused an increase or decrease, respectively, in colonization compared to the parent strain. Expression differences were also observed for several flagellar genes and other less well-characterized genes that may participate in motility. Targeted sequencing of the sigma factors revealed specific DNA differences undetected by the other genomic methods [corrected].

    View details for Web of Science ID 000188066800028

    View details for PubMedID 14702320

  • Growth phase-dependent response of Helicobacter pylori to iron starvation INFECTION AND IMMUNITY Merrell, D. S., Thompson, L. J., Kim, C. C., Mitchell, H., Tompkins, L. S., Lee, A., FALKOW, S. 2003; 71 (11): 6510-6525

    Abstract

    Iron is an essential nutrient that is often found in extremely limited available quantities within eukaryotic hosts. Because of this, many pathogenic bacteria have developed regulated networks of genes important for iron uptake and storage. In addition, it has been shown that many bacteria use available iron concentrations as a signal to regulate virulence gene expression. We have utilized DNA microarray technology to identify genes of the human pathogen Helicobacter pylori that are differentially regulated on a growth-inhibiting shift to iron starvation conditions. In addition, the growth phase-dependent expression of these genes was investigated by examining both exponential and stationary growth phase cultures. We identified known iron-regulated genes, as well as a number of genes whose regulation by iron concentration was not previously appreciated. Included in the list of regulated factors were the known virulence genes cagA, vacA, and napA. We examined the effect of iron starvation on the motility of H. pylori and found that exponential- and stationary-phase cultures responded differently to the stress. We further found that while growing cells are rapidly killed by iron starvation, stationary-phase cells show a remarkable ability to survive iron depletion. Finally, bioinformatic analysis of the predicted promoter regions of the differentially regulated genes led to identification of several putative Fur boxes, suggesting a direct role for Fur in iron-dependent regulation of these genes.

    View details for DOI 10.1128/IAI.71.11.6510-6525.2003

    View details for Web of Science ID 000186194300049

    View details for PubMedID 14573673

  • Protective immunity against Helicobacter is characterized by a unique transcriptional signature PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mueller, A., O'Rourke, J., Chu, P., Kim, C. C., Sutton, P., Lee, A., FALKOW, S. 2003; 100 (21): 12289-12294

    Abstract

    Immunization with a whole-cell sonicate vaccine of Helicobacter felis in conjunction with cholera toxin as a mucosal adjuvant induces long-term protective immunity in a majority of laboratory mice. We have combined gene expression profiling and immunohistochemical analysis on a set of immunized animals to better understand the mechanism of protection. The stomachs of protected animals exhibited a strikingly different transcriptional profile compared with those of nonprotected or control mice, indicating that vaccination targets the appropriate site and leaves a molecular signature. Among the genes whose up-regulation is significantly correlated with protection are a number of adipocyte-specific factors. These include the fat-cell-specific cytokines adipsin, resistin, and adiponectin and the adipocyte surface marker CD36. Interestingly, potentially protective T and B lymphocytes can be found embedded in the adipose tissue surrounding protected stomachs but never in control or unprotected stomachs. Adipsin-specific immunohistochemical staining of protected stomach sections further revealed molecular cross-talk between adjacent lymphoid and adipose cell populations. We propose a mechanism of protection that involves the effector responses of either or both lymphocyte subclasses as well as the previously unappreciated paracrine functions of adipose tissue surrounding the resident lymphocytes.

    View details for DOI 10.1073/pnas.1635231100

    View details for Web of Science ID 000186024300065

    View details for PubMedID 14528007

  • Modulation of virulence by two acidified nitrite-responsive loci of Salmonella enterica serovar Typhimurium INFECTION AND IMMUNITY Kim, C. C., Monack, D., FALKOW, S. 2003; 71 (6): 3196-3205

    Abstract

    Two acidified nitrite-inducible genes of Salmonella enterica serovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designated nipAB and nipC, which correspond to hcp-hcr (hybrid cluster protein) of Escherichia coli and norA of Alcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. The nipAB promoter was regulated by oxygen in an Fnr-dependent manner. The nipC promoter was also regulated by oxygen but in an Fnr-independent manner. The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS. Although the nipAB and nipC mutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD(50)s) than did the wild type in C57BL/6J mouse infections. The lower LD(50)s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria. We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.

    View details for DOI 10.1128/IAI.71.6.3196-3205.2003

    View details for Web of Science ID 000183116300027

    View details for PubMedID 12761099

  • pH-regulated gene expression of the gastric pathogen Helicobacter pylori INFECTION AND IMMUNITY Merrell, D. S., Goodrich, M. L., Otto, G., Tompkins, L. S., FALKOW, S. 2003; 71 (6): 3529-3539

    Abstract

    Colonization by the gastric pathogen Helicobacter pylori has been shown to be intricately linked to the development of gastritis, ulcers, and gastric malignancy. Little is known about mechanisms employed by the bacterium that help it adapt to the hostile environment of the human stomach. In an effort to extend our knowledge of these mechanisms, we utilized spotted-DNA microarrays to characterize the response of H. pylori to low pH. Expression of approximately 7% of the bacterial genome was reproducibly altered by shift to low pH. Analysis of the differentially expressed genes led to the discovery that acid exposure leads to profound changes in motility of H. pylori, as a larger percentage of acid-exposed bacterial cells displayed motility and moved at significantly higher speeds. In contrast to previous publications, we found that expression of the bacterial virulence gene cagA was strongly repressed by acid exposure. Furthermore, this transcriptional repression was reflected at the level of protein accumulation in the H. pylori cell.

    View details for DOI 10.1128/IAI.71.6.3529-3539.2003

    View details for Web of Science ID 000183116300066

    View details for PubMedID 12761138

  • Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA SCIENCE Amieva, M. R., Vogelmann, R., Covacci, A., Tompkins, L. S., NELSON, W. J., FALKOW, S. 2003; 300 (5624): 1430-1434

    Abstract

    Helicobacter pylori translocates the protein CagA into gastric epithelial cells and has been linked to peptic ulcer disease and gastric carcinoma. We show that injected CagA associates with the epithelial tight-junction scaffolding protein ZO-1 and the transmembrane protein junctional adhesion molecule, causing an ectopic assembly of tight-junction components at sites of bacterial attachment, and altering the composition and function of the apical-junctional complex. Long-term CagA delivery to polarized epithelia caused a disruption of the epithelial barrier function and dysplastic alterations in epithelial cell morphology. CagA appears to target H. pylori to host cell intercellular junctions and to disrupt junction-mediated functions.

    View details for Web of Science ID 000183181800045

    View details for PubMedID 12775840

  • Gene expression profiling of Helicobacter pylori reveals a growth-phase-dependent switch in virulence gene expression INFECTION AND IMMUNITY Thompson, L. J., Merrell, D. S., Neilan, B. A., Mitchell, H., Lee, A., FALKOW, S. 2003; 71 (5): 2643-2655

    Abstract

    The global pattern of growth-phase-dependent gene expression of Helicobacter pylori during in vitro culture was analyzed by using a high-density DNA microarray. To detect consistent coordinated gene expression in this bacterium, temporal changes in transcription were assessed in two independent time courses. Cluster analysis of the expression profiles highlighted a major switch in gene expression during the late log-to-stationary phase transition that we have termed the Log-Stat switch. Statistical analysis of the genes that were significantly induced or repressed during the Log-Stat switch revealed that many of these genes were related to virulence. Among these, expression of the genes for the neutrophil activating protein (napA) and the major flagellin subunit (flaA) were significantly induced. Additionally, the expression of a number of genes involved in iron homeostasis changed dramatically at this switch; the gene for the iron-storage protein, pfr, was induced, while the genes for two putative iron uptake proteins, fecA and frpB, were significantly repressed. These data suggest that the late log phase may correspond to the most virulent phase of growth in H. pylori and may be intimately related to its pathogenesis. The use of microarrays to analyze the kinetics of the transcriptional response of a bacterial pathogen to a changing environment has enabled the discovery of previously unappreciated relationships between genes by elucidation of coordinated gene expression profiles.

    View details for DOI 10.1128/IAI.71.5.2643-2655.2003

    View details for Web of Science ID 000182501500042

    View details for PubMedID 12704139

  • Significance analysis of lexical bias in microarray data BMC BIOINFORMATICS Kim, C. C., FALKOW, S. 2003; 4

    Abstract

    Genes that are determined to be significantly differentially regulated in microarray analyses often appear to have functional commonalities, such as being components of the same biochemical pathway. This results in certain words being under- or overrepresented in the list of genes. Distinguishing between biologically meaningful trends and artifacts of annotation and analysis procedures is of the utmost importance, as only true biological trends are of interest for further experimentation. A number of sophisticated methods for identification of significant lexical trends are currently available, but these methods are generally too cumbersome for practical use by most microarray users.We have developed a tool, LACK, for calculating the statistical significance of apparent lexical bias in microarray datasets. The frequency of a user-specified list of search terms in a list of genes which are differentially regulated is assessed for statistical significance by comparison to randomly generated datasets. The simplicity of the input files and user interface targets the average microarray user who wishes to have a statistical measure of apparent lexical trends in analyzed datasets without the need for bioinformatics skills. The software is available as Perl source or a Windows executable.We have used LACK in our laboratory to generate biological hypotheses based on our microarray data. We demonstrate the program's utility using an example in which we confirm significant upregulation of SPI-2 pathogenicity island of Salmonella enterica serovar Typhimurium by the cation chelator dipyridyl.

    View details for Web of Science ID 000182371100001

    View details for PubMedID 12697067

  • virK, somA and rcsC are important for systemic Salmonella enterica serovar Typhimurium infection and cationic peptide resistance MOLECULAR MICROBIOLOGY Detweiler, C. S., Monack, D. M., Brodsky, I. E., Mathew, H., FALKOW, S. 2003; 48 (2): 385-400

    Abstract

    Salmonella must express and deploy a type III secretion system located in Salmonella pathogenicity island 2 (SPI-2) in order to survive in host phagocytic vacuoles and to cause systemic infection in mouse models of typhoid fever. A genome-wide approach to screening for Salmonella genes that are transcriptionally co-regulated in vitro with SPI-2 genes was used to identify bacterial loci that might function in a mouse model of systemic disease. Strains with mutations in three SPI-2 co-expressed genes were constructed and tested for their ability to cause disease in mice. We found that virK, a homologue of a Shigella virulence determinant, and rcsC, a sensor kinase, are important at late stages of infection. A second Salmonella gene that has VirK homology, somA, is also important for systemic infection in mice. We have shown that expression of both virK and somA requires the transcription factor PhoP, whereas rcsC does not. Additionally, rcsC expression does not require the transcription factor OmpR, but expression of one of the known targets of RcsC, the yojN rcsB putative operon, does require OmpR. virK, somA and rcsC are expressed in tissue culture macrophages and confer Salmonella resistance to the cationic peptide polymyxin B. We conclude that virK, somA and rcsC are important for late stages of Salmonella enteric fever, and that they probably contribute to the remodelling of the bacterial outer membrane in response to the host environment.

    View details for Web of Science ID 000182042800009

    View details for PubMedID 12675799

  • Use of an open-reading frame-specific Campylobacter jejuni DNA microarray as a new genotyping tool for studying epidemiologically related isolates JOURNAL OF INFECTIOUS DISEASES Leonard, E. E., Takata, T., Blaser, M. J., FALKOW, S., Tompkins, L. S., Gaynor, E. C. 2003; 187 (4): 691-694

    Abstract

    Findings from use of an open-reading frame-specific Campylobacter jejuni DNA microarray to investigate genetic diversity among clinical isolates associated with 5 independent clusters of infection were compared with data from random amplified polymeric DNA (RAPD) and Penner serotyping analyses. The DNA microarray provides a highly specific epidemiological typing tool for analysis of C. jejuni isolates and reveals both divergent and highly conserved gene classes among isolates.

    View details for Web of Science ID 000180884500022

    View details for PubMedID 12599089

  • Distinct gene expression profiles characterize the histopathological stages of disease in Helicobacter-induced mucosa-associated lymphoid tissue lymphoma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mueller, A., O'Rourke, J., Grimm, J., Guillemin, K., Dixon, M. F., Lee, A., FALKOW, S. 2003; 100 (3): 1292-1297

    Abstract

    Long-term colonization of humans with Helicobacter pylori can cause the development of gastric B cell mucosa-associated lymphoid tissue lymphoma, yet little is known about the sequence of molecular steps that accompany disease progression. We used microarray analysis and laser microdissection to identify gene expression profiles characteristic and predictive of the various histopathological stages in a mouse model of the disease. The initial step in lymphoma development is marked by infiltration of reactive lymphocytes into the stomach and the launching of a mucosal immune response. Our analysis uncovered molecular markers of both of these processes, including genes coding for the immunoglobulins and the small proline-rich protein Sprr 2A. The subsequent step is characterized histologically by the antigen-driven proliferation and aggregation of B cells and the gradual appearance of lymphoepithelial lesions. In tissues of this stage, we observed increased expression of genes previously associated with malignancy, including the laminin receptor-1 and the multidrug-resistance channel MDR-1. Finally, we found that the transition to destructive lymphoepithelial lesions and malignant lymphoma is marked by an increase in transcription of a single gene encoding calgranulin AMrp-8.

    View details for DOI 10.1073/pnas.242741699

    View details for Web of Science ID 000180838100094

    View details for PubMedID 12552104

  • Genomic comparison of Salmonella enterica serovars and Salmonella bongori by use of an S-enterica serovar Typhimurium DNA microarray JOURNAL OF BACTERIOLOGY Chan, K., Baker, S., Kim, C. C., Detweiler, C. S., Dougan, G., FALKOW, S. 2003; 185 (2): 553-563

    Abstract

    The genus Salmonella consists of over 2,200 serovars that differ in their host range and ability to cause disease despite their close genetic relatedness. The genetic factors that influence each serovar's level of host adaptation, how they evolved or were acquired, their influence on the evolution of each serovar, and the phylogenic relationships between the serovars are of great interest as they provide insight into the mechanisms behind these differences in host range and disease progression. We have used an Salmonella enterica serovar Typhimurium spotted DNA microarray to perform genomic hybridizations of various serovars and strains of both S. enterica (subspecies I and IIIa) and Salmonella bongori to gain insight into the genetic organization of the serovars. Our results are generally consistent with previously published DNA association and multilocus enzyme electrophoresis data. Our findings also reveal novel information. We observe a more distant relationship of serovar Arizona (subspecies IIIa) from the subspecies I serovars than previously measured. We also observe variability in the Arizona SPI-2 pathogenicity island, indicating that it has evolved in a manner distinct from the other serovars. In addition, we identify shared genetic features of S. enterica serovars Typhi, Paratyphi A, and Sendai that parallel their unique ability to cause enteric fever in humans. Therefore, whereas the taxonomic organization of Salmonella into serogroups provides a good first approximation of genetic relatedness, we show that it does not account for genomic changes that contribute to a serovar's degree of host adaptation.

    View details for DOI 10.1128/JB.185.2.553-563.2003

    View details for Web of Science ID 000180272600021

    View details for PubMedID 12511502

  • Cag pathogenicity island-specific responses of gastric epithelial cells to Helicobacter pylori infection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Guillemin, K., Salama, N. R., Tompkins, L. S., FALKOW, S. 2002; 99 (23): 15136-15141

    Abstract

    Helicobacter pylori infects over half the world's population and causes a wide range of diseases, including gastritis, peptic ulcer, and two forms of gastric cancer. H. pylori infection elicits a variety of phenotypic responses in cultured gastric epithelial cells, including the expression of proinflammatory genes and changes in the actin cytoskeleton. Both of these responses are mediated by the type IV secretion system (TFSS) encoded by the cag pathogenicity island (cag PAI). We used human cDNA microarrays to examine the temporal transcriptional profiles of gastric AGS cells infected with H. pylori strain G27 and a panel of isogenic mutants to dissect the contributions of various genes in the cag PAI. Infection with G27 induced expression of genes involved in the innate immune response, cell shape regulation, and signal transduction. A mutant lacking the cagA gene, which encodes an effector molecule secreted by the TFSS and required for the host cell cytoskeletal response, induced the expression of fewer cytoskeletal genes. A mutant lacking cagE, which encodes a structural component of the TFSS, failed to up-regulate a superset of host genes, including the cagA-dependent genes, and many of the immune response genes. A mutant lacking the entire cag PAI failed to induce both the cagE-dependent genes and several transiently expressed cagE independent genes. Host cell transcriptional profiling of infection with isogenic strains offered a detailed molecular picture of H. pylori infection and provided insight into potential targets of individual virulence determinants such as tyrosine kinase and Rho GTPase signaling molecules.

    View details for DOI 10.1073/pnas.182558799

    View details for Web of Science ID 000179224800094

    View details for PubMedID 12411577

  • Helicobacter pylori enter and survive within multivesicular vacuoles of epithelial cells CELLULAR MICROBIOLOGY Amieva, M. R., Salama, N. R., Tompkins, L. S., FALKOW, S. 2002; 4 (10): 677-690

    Abstract

    Although intracellular Helicobacter pylori have been described in biopsy specimens and in cultured epithelial cells, the fate of these bacteria is unknown. Using differential interference contrast (DIC) video and immunofluorescence microscopy, we document that a proportion of cell-associated H. pylori enter large cytoplasmic vacuoles, where they remain viable and motile and can survive lethal concentrations of extracellular gentamicin. Entry into vacuoles occurs in multiple epithelial cell lines including AGS gastric adenocarcinoma, Caco-2 colon adenocarcinoma and MDCK kidney cell line, and depends on the actin cytoskeleton. Time-lapse microscopy over several hours was used to follow the movement of live H. pylori within vacuoles of a single cell. Pulsed, extracellular gentamicin treatments show that the half-life of intravacuolar bacteria is on the order of 24 h. Viable H. pylori repopulate the extracellular environment in parallel with the disappearance of intravacuolar bacteria, suggesting release from the intravacuolar niche. Using electron microscopy and live fluorescent staining with endosomal dyes, we observe that H. pylori-containing vacuoles are similar in morphology to late endosomal multivesicular bodies. VacA is not required for these events, as isogenic vacA- mutants still enter and survive within the intravacuolar niche. The exploitation of an intravacuolar niche is a new aspect of the biological life cycle of H. pylori that could explain the difficulties in eradicating this infection.

    View details for Web of Science ID 000178480600005

    View details for PubMedID 12366404

  • Identification of Streptococcus bovis biotype I strains among S-bovis clinical isolates by PCR JOURNAL OF CLINICAL MICROBIOLOGY Songy, W. B., Ruoff, K. L., Facklam, R. R., Ferraro, M. J., Falkow, S. 2002; 40 (8): 2913-2918

    Abstract

    Streptococcus bovis causes 24% of all streptococcal infective endocarditis cases. There are many reports linking both S. bovis bacteremia and endocarditis with various forms of gastrointestinal disease (primarily colonic cancers). S. bovis is divided into two biotypes: I and II. The biotype I strain is much more frequently isolated from patients with endocarditis, gastrointestinal disease, or both. We describe here the isolation of biotype I-specific DNA sequences and the development of a PCR test which can identify S. bovis biotype I strains among S. bovis clinical isolates.

    View details for DOI 10.1128/JCM.40.8.2913-2918.2002

    View details for Web of Science ID 000177255900034

    View details for PubMedID 12149351

  • Redefining bacterial populations: A post-genomic reformation NATURE REVIEWS GENETICS Joyce, E. A., Chan, K., Salama, N. R., FALKOW, S. 2002; 3 (6): 462-473

    Abstract

    Sexual reproduction and recombination are essential for the survival of most eukaryotic populations. Until recently, the impact of these processes on the structure of bacterial populations has been largely overlooked. The advent of large-scale whole-genome sequencing and the concomitant development of molecular tools, such as microarray technology, facilitate the sensitive detection of recombination events in bacteria. These techniques are revealing that bacterial populations are comprised of isolates that show a surprisingly wide spectrum of genetic diversity at the DNA level. Our new awareness of this genetic diversity is increasing our understanding of population structures and of how these affect host pathogen relationships.

    View details for DOI 10.1038/nrg820

    View details for Web of Science ID 000175942600014

    View details for PubMedID 12042773

  • mig-14 is a Salmonella gene that plays a role in bacterial resistance to antimicrobial peptides JOURNAL OF BACTERIOLOGY Brodsky, I. E., Ernst, R. K., Miller, S. I., FALKOW, S. 2002; 184 (12): 3203-3213

    Abstract

    It was previously demonstrated that the mig-14 gene of Salmonella enterica serovar Typhimurium is necessary for bacterial proliferation in the liver and spleen of mice following intragastric inoculation and that mig-14 expression, which is induced within macrophages, is under the control of the global regulator PhoP. Here we demonstrate that the mig-14 promoter is induced by growth in minimal medium containing low magnesium or acidic pH, consistent with regulation by PhoP. In addition, mig-14 is strongly induced by polymyxin B, protamine, and the mammalian antimicrobial peptide protegrin-1. While phoP is necessary for the induction of mig-14 in response to protamine and protegrin, mig-14 is still induced by polymyxin B in a phoP background. We also demonstrate that mig-14 is necessary for resistance of S. enterica serovar Typhimurium to both polymyxin B and protegrin-1. Gram-negative resistance to a variety of antimicrobial peptides has been correlated with modifications of lipopolysaccharide structure. However, we show that mig-14 is not required for one of these modifications, the addition of 4-aminoarabinose to lipid A. Additionally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of wild-type and mig-14 lipopolysaccharide also shows no detectable differences between the two strains. Therefore, mig-14 contributes to Salmonella resistance to antimicrobial peptides by a mechanism that is not yet fully understood.

    View details for DOI 10.1128/JB.184.12.3203-3213.2002

    View details for Web of Science ID 000175918800007

    View details for PubMedID 12029036

  • Caenorhabditis elegans - Plague bacteria biofilm blocks food intake NATURE Darby, C., Hsu, J. W., Ghori, N., FALKOW, S. 2002; 417 (6886): 243-244

    Abstract

    Bubonic plague is transmitted to mammals, including humans, by the bites of fleas whose digestive tracts are blocked by a mass of the bacterium Yersinia pestis. In these fleas, the plague-causing bacteria are surrounded by an extracellular matrix of unknown composition, and the blockage depends on a group of bacterial genes known as the hmsHFRS operon. Here we show that Y. pestis creates an hmsHFRS-dependent extracellular biofilm to inhibit feeding by the nematode Caenorhabditis elegans. Our results suggest that feeding obstruction in fleas is a biofilm-mediated process and that biofilms may be a bacterial defence against predation by invertebrates.

    View details for DOI 10.1038/417243a

    View details for Web of Science ID 000175592100035

    View details for PubMedID 12015591

  • Complex pattern of Mycobacterium marinum gene expression during long-term granulomatous infection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, K., Knaak, T., Satkamp, L., Humbert, O., FALKOW, S., Ramakrishnan, L. 2002; 99 (6): 3920-3925

    Abstract

    During latent infection of humans with Mycobacterium tuberculosis, bacteria persist in the asymptomatic host within granulomas, organized collections of differentiated macrophages, and other immune cells. The mechanisms for persistence remain poorly understood, as is the metabolic and replicative state of the microbes within granulomas. We analyzed the gene expression profile of Mycobacterium marinum, the cause of fish and amphibian tuberculosis, during its persistence in granulomas. We identified genes expressed specifically when M. marinum persists within granulomas. These granuloma-activated genes were not activated in vitro in response to various conditions postulated to be operant in tuberculous granulomas, suggesting that their granuloma-specific activation was caused by complex conditions that could not be mimicked in vitro. In addition to the granuloma-activated genes, the bacteria resident in granulomas expressed a wide range of metabolic and synthetic genes that are expressed during logarithmic growth in laboratory medium. Our results suggest a dynamic host-pathogen interaction in the granuloma, where metabolically active bacteria are kept in check by the host immune system and where the products of granuloma-specific bacterial genes may thwart the host's attempt to completely eradicate the bacteria.

    View details for DOI 10.1073/pnas.002024599

    View details for Web of Science ID 000174511000100

    View details for PubMedID 11891270

  • Dissecting Host-Pathogen Molecular Interactions with Microarrays In: Methods in Microbiology, Vol. 31, Academic Press, Ltd. S. Falkow., Detweiler, C.S. 2002
  • Improved analytical methods for microarray-based genome-composition analysis GENOME BIOLOGY Kim, C. C., Joyce, E. A., Chan, K., Falkow, S. 2002; 3 (11)

    Abstract

    Whereas genome sequencing has given us high-resolution pictures of many different species of bacteria, microarrays provide a means of obtaining information on genome composition for many strains of a given species. Genome-composition analysis using microarrays, or 'genomotyping', can be used to categorize genes into 'present' and 'divergent' categories based on the level of hybridization signal. This typically involves selecting a signal value that is used as a cutoff to discriminate present (high signal) and divergent (low signal) genes. Current methodology uses empirical determination of cutoffs for classification into these categories, but this methodology is subject to several problems that can result in the misclassification of many genes.We describe a method that depends on the shape of the signal-ratio distribution and does not require empirical determination of a cutoff. Moreover, the cutoff is determined on an array-to-array basis, accounting for variation in strain composition and hybridization quality. The algorithm also provides an estimate of the probability that any given gene is present, which provides a measure of confidence in the categorical assignments.Many genes previously classified as present using static methods are in fact divergent on the basis of microarray signal; this is corrected by our algorithm. We have reassigned hundreds of genes from previous genomotyping studies of Helicobacter pylori and Campylobacter jejuni strains, and expect that the algorithm should be widely applicable to genomotyping data.

    View details for Web of Science ID 000207582200017

    View details for PubMedID 12429064

  • Dynamic nature of host-pathogen interactions in Mycobacterium marinum granulomas INFECTION AND IMMUNITY Bouley, D. M., Ghori, N., Mercer, K. L., FALKOW, S., Ramakrishnan, L. 2001; 69 (12): 7820-7831

    Abstract

    Mycobacterium marinum causes long-term subclinical granulomatous infection in immunocompetent leopard frogs (Rana pipiens). These granulomas, organized collections of activated macrophages, share many morphological features with persistent human tuberculous infection. We examined organs of frogs with chronic M. marinum infection using transmission electron microscopy in conjunction with immunohistochemistry and acid phosphatase cytochemistry to better define the bacterium-host interplay during persistent infection. Bacteria were always found within macrophage phagosomes. These phagosomes were often fused to lysosomes, in sharp contrast to those formed during in vitro infection of J774 macrophage-like cells by M. marinum. The infected macrophages in frog granulomas showed various levels of activation, as evidenced by morphological changes, including epithelioid transformation, recent phagocytic events, phagolysosomal fusion, and disintegration of bacteria. Our results demonstrate that even long-term granulomas are dynamic environments with regard to the level of host cell activation and bacterial turnover and suggest a continuum between constantly replicating bacteria and phagocytic killing that maintains relatively constant bacterial numbers despite an established immune response. Infection with a mutant bacterial strain with a reduced capacity for intracellular replication shifted the balance, leading to a greatly reduced bacterial burden and inflammatory foci that differed from typical granulomas.

    View details for Web of Science ID 000172297600077

    View details for PubMedID 11705964

  • Salmonella pathogenicity island 2-dependent macrophage death is mediated in part by the host cysteine protease caspase-1 CELLULAR MICROBIOLOGY Monack, D. M., Detweiler, C. S., FALKOW, S. 2001; 3 (12): 825-837

    Abstract

    Salmonella typhimurium invades host macrophages and can either induce a rapid cell death or establish an intracellular niche within the phagocytic vacuole. Rapid cell death requires the Salmonella pathogenicity island (SPI)1 and the host protein caspase-1, a member of the pro-apoptotic caspase family of proteases. Salmonella that do not cause this rapid cell death and instead reside in the phagocytic vacuole can trigger macrophage death at a later time point. We show here that the human pathogen Salmonella typhi also triggers both rapid, caspase-1-dependent and delayed cell death in human monocytes. The delayed cell death has previously been shown with S. typhimurium to be dependent on SPI2-encoded genes and ompR. Using caspase-1(-/-) bone marrow-derived macrophages and isogenic S. typhimurium mutant strains, we show that a large portion of the delayed, SPI2-dependent death is mediated by caspase-1. The two known substrates of activated caspase-1 are the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and IL-18, which are cleaved to produce bioactive cytokines. We show here that IL-1beta is released during both SPI1- and SPI2-dependent macrophage killing. Using IL-1beta(-/-) bone marrow-derived macrophages and a neutralizing anti-IL-18 antibody, we show that neither IL-1beta nor IL-18 is required for rapid or delayed macrophage death. Thus, both rapid, SPI1-mediated killing and delayed, SPI2-mediated killing require caspase-1 and result in the secretion of IL-1beta, which promotes inflammation and may facilitate the spread of Salmonella beyond the gastrointestinal tract in systemic disease.

    View details for Web of Science ID 000172601200005

    View details for PubMedID 11736994

  • Salmonella-induced macrophage death: the role of caspase-1 in death and inflammation MICROBES AND INFECTION Monack, D. M., Navarre, W. W., FALKOW, S. 2001; 3 (14-15): 1201-1212

    Abstract

    Salmonella typhimurium invades host macrophages and can induce either an almost immediate cell death or establish an intracellular niche within the phagocytic vacuole. Rapid cell death depends on the Salmonella pathogenicity island SPI1 and the host protein caspase-1, a member of the pro-apoptotic caspase family of proteases. Caspase-1-dependent cell death leads to the activation of the potent pro-inflammatory cytokines interleukin (IL)-1beta and IL-18 to produce bioactive cytokines. Animal studies indicate that the activation of these cytokines is necessary for efficient colonization of the mouse gastrointestinal tract. Salmonella that reside in the phagocytic vacuole do not cause this early cell death and can trigger a macrophage death at a much later time point. This late-phase cell death is dependent on SPI2-encoded genes and ompR.

    View details for Web of Science ID 000173168500004

    View details for PubMedID 11755408

  • Mimicry of a G protein mutation by pertussis toxin expression in transgenic Caenorhabditis elegans INFECTION AND IMMUNITY Darby, C., FALKOW, S. 2001; 69 (10): 6271-6275

    Abstract

    Pathogens produce virulence factors that interact directly with host molecules, but in many cases the host targets are unknown. The genetic and molecular identification of these orphan targets is often not feasible with mammalian experimental models. However, a substantial number of known targets are molecules and pathways that are conserved among eukaryotes, and therefore the use of nonmammalian model hosts to identify orphan targets may prove useful. To demonstrate the feasibility of this approach, we transformed the nematode Caenorhabditis elegans with a gene encoding the catalytic subunit of pertussis toxin (PTX), which in mammals inactivates G(o/i)alpha proteins. Expression of PTX in C. elegans produced phenotypes almost identical to those of a null mutation in the nematode gene encoding G(o/i)alpha. Furthermore, PTX suppressed the phenotype of a constitutively active form of nematode G(o/i)alpha protein. These results indicate that PTX is functional in nematodes and acts specifically on the C. elegans homologue of the mammalian target.

    View details for Web of Science ID 000171121100040

    View details for PubMedID 11553570

  • Host microarray analysis reveals a role for the Salmonella response regulator phoP in human macrophage cell death PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Detweiler, C. S., Cunanan, D. B., FALKOW, S. 2001; 98 (10): 5850-5855

    Abstract

    Bacterial pathogens manipulate host cells to promote pathogen survival and dissemination. We used a 22,571 human cDNA microarray to identify host pathways that are affected by the Salmonella enterica subspecies typhimurium phoP gene, a transcription factor required for virulence, by comparing the expression profiles of human monocytic tissue culture cells infected with either the wild-type bacteria or a phoPTn10 mutant strain. Both wild-type and phoPTn10 bacteria induced a common set of genes, many of which are proinflammatory. Differentially expressed genes included those that affect host cell death, suggesting that the phoP regulatory system controls bacterial genes that alter macrophage survival. Subsequent experiments showed that the phoPTn10 mutant strain is defective for killing both cultured and primary human macrophages but is able to replicate intracellularly. These experiments indicate that phoP plays a role in Salmonella-induced human macrophage cell death.

    View details for Web of Science ID 000168623300086

    View details for PubMedID 11320214

  • Identification of attenuated Yersinia pseudotuberculosis strains and characterization of an orogastric infection in BALB/c mice on day 5 postinfection by signature-tagged mutagenesis INFECTION AND IMMUNITY Mecsas, J., Bilis, I., FALKOW, S. 2001; 69 (5): 2779-2787

    Abstract

    Yersinia pseudotuberculosis localizes to the distal ileum, cecum, and proximal colon of the gastrointestinal tract after oral infection. Using signature-tagged mutagenesis, we isolated 13 Y. pseudotuberculosis mutants that failed to survive in the cecum of mice after orogastric inoculation. Twelve of these mutants were also attenuated for replication in the spleen after intraperitoneal infection, whereas one strain, mutated the gene encoding invasin, replicated as well as wild-type bacteria in the spleen. Several mutations were in operons encoding components of the type III secretion system, including components involved in translocating Yop proteins into host cells. This indicates that one or more Yops may be necessary for survival in the gastrointestinal tract. Three mutants were defective in O-antigen biosynthesis; these mutants were also unable to invade epithelial cells as efficiently as wild-type Y. pseudotuberculosis. Several other mutations were in genes that had not previously been associated with growth in a host, including cls, ksgA, and sufl. In addition, using Y. pseudotuberculosis strains marked with signature tags, we counted the number of different bacterial clones that were present in the cecum, mesenteric lymph nodes, and spleen 5 days postinfection. We find barriers in the host animal that limit the number of bacteria that succeed in reaching and/or replicating in the mesenteric lymph nodes and spleen after breaching the gut mucosa.

    View details for Web of Science ID 000168158400002

    View details for PubMedID 11292689

  • The meaning and impact of the human genome sequence for microbiology TRENDS IN MICROBIOLOGY Relman, D. A., FALKOW, S. 2001; 9 (5): 206-208

    Abstract

    The characterization of life is immeasurably enhanced by determination of complete genome sequences. For organisms that engage in intimate interactions with others, the genome sequence from one participant, and associated tools, provide unique insight into its partner. We discuss how the human genome sequence will further our understanding of microbial pathogens and commensals, and vice versa. We also propose criteria for implicating a host gene in microbial pathogenesis, and urge consideration of a'second human genome project'.

    View details for Web of Science ID 000168719300008

    View details for PubMedID 11336835

  • Bile-induced 'pili' in Campylobacter jejuni are bacteria-independent artifacts of the culture medium MOLECULAR MICROBIOLOGY Gaynor, E. C., Ghori, N., FALKOW, S. 2001; 39 (6): 1546-1549

    Abstract

    In 1996, it was reported that the enteric pathogen Campylobacter jejuni produces pilus-like appendages in response to bile salts such as deoxycholate (DOC), and that the formation of these appendages requires the putative peptidase PspA. Pili were known to be important virulence determinants in other pathogenic bacteria but had never before been observed for C. jejuni. We report here that these appendages are not pili, but are instead a bacteria-independent morphological artifact of the growth medium. Furthermore, the pspA gene is not required for their formation. Broth cultures containing a threshold concentration of DOC inoculated with no bacteria produced identical abundant, fibrous, pilus-like structures as those cultures that had been inoculated with C. jejuni. These fibres were also found in growth media from DOC-containing pspA:CmR mutant cultures. These results are consistent with the absence of candidate pilin monomers in protein gel analyses as well as the dearth of pilin-like genes and pilus formation gene clusters in the C. jejuni genome.

    View details for Web of Science ID 000167644800012

    View details for PubMedID 11260471

  • New approaches for validation of lethal phenotypes and genetic reversion in Helicobacter pylori HELICOBACTER McDaniel, T. K., DeWalt, K. C., Salama, N. R., FALKOW, S. 2001; 6 (1): 15-23

    Abstract

    Because of limited genetic tools for use in Helicobacter pylori, tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism. In a mutational study of putative response regulator genes, we aimed to develop such tools for H. pylori.We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis. For genes that yielded no viable mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would permit the gene's disruption. For genes that yielded mutants with selectable phenotypes, a strategy was developed for reversion whereby an intact copy of the gene is introduced to the organism by transformation with PCR products. Following this procedure, revertants were selected by phenotypic tests then tested for genetic reversion.After failure to attain transformants upon attempted mutation of genes HP0166 and HP1365, we inserted a second copy of each gene within the H. pylori chromosome. In each case the second copy relieved the block of transformation. Mutation of genes HP0703 and HP1021 gave non-motile and small-colony phenotypes, respectively. Following transformation with PCR products containing intact copies of the genes, both phenotype and genotype had reverted following phenotypic selections.The methods used in this study provide new approaches for confirming suspected genotype/phenotype associations and should be widely applicable in the study of H. pylori.

    View details for Web of Science ID 000168272600002

    View details for PubMedID 11328361

  • Identification of Legionella pneumophila genes important for infection of amoebas by signature-tagged mutagenesis INFECTION AND IMMUNITY Polesky, A. H., Ross, J. T., FALKOW, S., Tompkins, L. S. 2001; 69 (2): 977-987

    Abstract

    Legionella pneumophila is a facultative intracellular gram-negative rod that causes pneumonia in humans. Free-living amoebas are thought to serve as a reservoir for Legionella infections. Signature-tagged mutagenesis was employed to identify Legionella pneumophila genes necessary for survival in the amoeba Acanthamoeba castellanii. Six mutant strains were defective in assays of invasion and intracellular growth. Four mutants also exhibited invasion and replication defects in Hartmannella vermiformis, an amoeba linked to hospital outbreaks of Legionella pneumonia. The six mutants also were tested in macrophages derived from peripheral blood mononuclear cells. Two mutants had intracellular replication defects, and two different strains entered cells less efficiently. Two transposon insertions were in known L. pneumophila genes, lspK and aroB. The other four were in novel genes. One gene has similarity to a cytochrome c-type biogenesis protein of Pseudomonas fluorescens. Another has similarity to a transcriptional activator regulating flagellar biosynthesis in Vibrio cholera. The third is similar to traA of Rhizobium sp. strain NGR234, which is involved in conjugal transfer of DNA. The fourth has no homology. By using survival in amoeba as a selection, we have isolated mutant strains with a range of phenotypes; and we have potentially identified new L. pneumophila virulence genes.

    View details for Web of Science ID 000166528700045

    View details for PubMedID 11159993

  • Vacuolating cytotoxin of Helicobacter pylori plays a role during colonization in a mouse model of infection INFECTION AND IMMUNITY Salama, N. R., Otto, G., Tompkins, L., FALKOW, S. 2001; 69 (2): 730-736

    Abstract

    Helicobacter pylori, the causative agent of gastritis and ulcer disease in humans, secretes a toxin called VacA (vacuolating cytotoxin) into culture supernatants. VacA was initially characterized and purified on the basis of its ability to induce the formation of intracellular vacuoles in tissue culture cells. H. pylori strains possessing different alleles of vacA differ in their ability to express active toxin. Those strains expressing higher toxin levels are correlated with more severe gastric disease. However, the specific role(s) played by VacA during the course of infection and disease is not clear. We have used a mouse model of H. pylori infection to begin to address this role. A null mutation of vacA compromises H. pylori in its ability to initially establish infection. If an infection by a vacA mutant is established, the bacterial load and degree of inflammation are similar to those associated with an isogenic wild-type strain. Thus, in this infection model, vacA plays a role in the initial colonization of the host, suggesting that strains of H. pylori expressing active alleles of vacA may be better adapted for host-to-host transmission.

    View details for Web of Science ID 000166528700013

    View details for PubMedID 11159961

  • A whole-genome microarray reveals genetic diversity among Helicobacter pylori strains PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Salama, N., Guillemin, K., McDaniel, T. K., Sherlock, G., Tompkins, L., FALKOW, S. 2000; 97 (26): 14668-14673

    Abstract

    Helicobacter pylori colonizes the stomach of half of the world's population, causing a wide spectrum of disease ranging from asymptomatic gastritis to ulcers to gastric cancer. Although the basis for these diverse clinical outcomes is not understood, more severe disease is associated with strains harboring a pathogenicity island. To characterize the genetic diversity of more and less virulent strains, we examined the genomic content of 15 H. pylori clinical isolates by using a whole genome H. pylori DNA microarray. We found that a full 22% of H. pylori genes are dispensable in one or more strains, thus defining a minimal functional core of 1281 H. pylori genes. While the core genes encode most metabolic and cellular processes, the strain-specific genes include genes unique to H. pylori, restriction modification genes, transposases, and genes encoding cell surface proteins, which may aid the bacteria under specific circumstances during their long-term infection of genetically diverse hosts. We observed distinct patterns of the strain-specific gene distribution along the chromosome, which may result from different mechanisms of gene acquisition and loss. Among the strain-specific genes, we have found a class of candidate virulence genes identified by their coinheritance with the pathogenicity island.

    View details for Web of Science ID 000165993700121

    View details for PubMedID 11121067

  • mig-14 is a horizontally acquired, host-induced gene required for Salmonella enterica lethal infection in the murine model of typhoid fever INFECTION AND IMMUNITY Valdivia, R. H., Cirillo, D. M., Lee, A. K., Bouley, D. M., FALKOW, S. 2000; 68 (12): 7126-7131

    Abstract

    We have characterized a host-induced virulence gene, mig-14, that is required for fatal infection in the mouse model of enteric fever. mig-14 is present in all Salmonella enterica subspecies I serovars and maps to a region of the chromosome that appears to have been acquired by horizontal transmission. A mig-14 mutant replicated in host tissues early after infection but was later cleared from the spleens and livers of infected animals. Bacterial clearance by the host occurred concomitantly with an increase in gamma interferon levels and recruitment of macrophages, but few neutrophils, to the infection foci. We hypothesize that the mig-14 gene product may repress immune system functions by interfering with normal cytokine expression in response to bacterial infections.

    View details for Web of Science ID 000167020000081

    View details for PubMedID 11083839

  • Salmonella exploits caspase-1 to colonize Peyer's patches in a murine typhoid model JOURNAL OF EXPERIMENTAL MEDICINE Monack, D. M., Hersh, D., Ghori, N., Bouley, D., Zychlinsky, A., FALKOW, S. 2000; 192 (2): 249-258

    Abstract

    Salmonella typhimurium invades host macrophages and induces apoptosis and the release of mature proinflammatory cytokines. SipB, a protein translocated by Salmonella into the cytoplasm of macrophages, is required for activation of Caspase-1 (Casp-1, an interleukin [IL]-1beta-converting enzyme), which is a member of a family of cysteine proteases that induce apoptosis in mammalian cells. Casp-1 is unique among caspases because it also directly cleaves the proinflammatory cytokines IL-1beta and IL-18 to produce bioactive cytokines. We show here that mice lacking Casp-1 (casp-1(-/)- mice) had an oral S. typhimurium 50% lethal dose (LD(50)) that was 1,000-fold higher than that of wild-type mice. Salmonella breached the M cell barrier of casp-1(-/)- mice efficiently; however, there was a decrease in the number of apoptotic cells, intracellular bacteria, and the recruitment of polymorphonuclear lymphocytes in the Peyer's patches (PP) as compared with wild-type mice. Furthermore, Salmonella did not disseminate systemically in the majority of casp-1(-/)- mice, as demonstrated by significantly less colonization in the PP, mesenteric lymph nodes, and spleens of casp-1(-/)- mice after an oral dose of S. typhimurium that was 100-fold higher than the LD(50). The increased resistance in casp-1(-/)- animals appears specific for Salmonella infection since these mice were susceptible to colonization by another enteric pathogen, Yersinia pseudotuberculosis, which normally invades the PP. These results show that Casp-1, which is both proapoptotic and proinflammatory, is essential for S. typhimurium to efficiently colonize the cecum and PP and subsequently cause systemic typhoid-like disease in mice.

    View details for Web of Science ID 000088261100011

    View details for PubMedID 10899911

  • Living in stools is not as dumb as you think JOURNAL OF BACTERIOLOGY FALKOW, S. 2000; 182 (12): 3319-3322

    View details for Web of Science ID 000087307700001

    View details for PubMedID 10852859

  • Granuloma-specific expression of Mycobacterium virulence proteins from the glycine-rich PE-PGRS family SCIENCE Ramakrishnan, L., Federspiel, N. A., FALKOW, S. 2000; 288 (5470): 1436-1439

    Abstract

    Pathogenic mycobacteria, including the agent of tuberculosis, Mycobacterium tuberculosis, must replicate in macrophages for long-term persistence within their niche during chronic infection: organized collections of macrophages and lymphocytes called granulomas. We identified several genes preferentially expressed when Mycobacterium marinum, the cause of fish and amphibian tuberculosis, resides in host granulomas and/or macrophages. Two were homologs of M. tuberculosis PE/PE-PGRS genes, a family encoding numerous repetitive glycine-rich proteins of unknown function. Mutation of two PE-PGRS genes produced M. marinum strains incapable of replication in macrophages and with decreased persistence in granulomas. Our results establish a direct role in virulence for some PE-PGRS proteins.

    View details for Web of Science ID 000087270900054

    View details for PubMedID 10827956

  • The good old days are now TRENDS IN MICROBIOLOGY FALKOW, S. 2000; 8 (5): 198-199

    View details for Web of Science ID 000087036100004

    View details for PubMedID 10785631

  • Apoptosis as a common bacterial virulence strategy INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY Monack, D., FALKOW, S. 2000; 290 (1): 7-13

    Abstract

    The comparison of common strategies used by bacterial pathogens to overcome host defenses provides us with the opportunity to analyze the biology of pathogenicity, as well as point out the unique interactions between a particular pathogen and its host. Here we compare and contrast apoptosis induced by three enteric pathogens, Salmonella, Shigella, and Yersinia. We point out that all three enteric pathogens induce apoptosis in macrophages in vitro, but the proposed mechanisms are quite different. Yersinia induces apoptosis by inhibiting the translocation of the transcriptional activator, NF-kappaB, into the nucleus, which results in the suppression of TNFalpha production; whereas Salmonella- and Shigella-induced apoptosis depend on the activation of caspase-1 (casp-1). The result of casp-1 activation is to induce apoptosis as well as to process the proinflammatory cytokines, pro-IL-1beta and pro-IL18 into their mature bioactive forms. Thus, in contrast to Yersinia, Salmonella and Shigella-induced apoptosis results in a proinflammatory cascade.

    View details for Web of Science ID 000086862900002

    View details for PubMedID 11043977

  • OmpR regulates the two-component system SsrA-SsrB in Salmonella pathogenicity island 2 JOURNAL OF BACTERIOLOGY Lee, A. K., Detweiler, C. S., FALKOW, S. 2000; 182 (3): 771-781

    Abstract

    Salmonella pathogenicity island 2 (SPI-2) encodes a putative, two-component regulatory system, SsrA-SsrB, which regulates a type III secretion system needed for replication inside macrophages and systemic infection in mice. The sensor and regulator homologs, ssrAB (spiR), and genes within the secretion system, including the structural gene ssaH, are transcribed after Salmonella enters host cells. We have studied the transcriptional regulation of ssrAB and the secretion system by using gfp fusions to the ssrA and ssaH promoters. We found that early transcription of ssrA, after entry into macrophages, is most efficient in the presence of OmpR. An ompR mutant strain does not exhibit replication within cultured macrophages. Furthermore, footprint analysis shows that purified OmpR protein binds directly to the ssrA promoter region. We also show that minimal medium, pH 4.5, induces SPI-2 gene expression in wild-type but not ompR mutant strains. We conclude that the type III secretion system of SPI-2 is regulated by OmpR, which activates expression of ssrA soon after Salmonella enters the macrophage.

    View details for Web of Science ID 000085003000027

    View details for PubMedID 10633113

  • Altered states: Involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Segal, E. D., Cha, J., Lo, J., FALKOW, S., Tompkins, L. S. 1999; 96 (25): 14559-14564

    Abstract

    Helicobacter pylori, present in half of the world's population, is a very successful pathogen. It can survive for decades in the human stomach with few obvious consequences to the host. However, it is also the cause of gastric diseases ranging from gastritis to ulcers to gastric cancer and has been classified a type 1 carcinogen by the World Health Organization. We have previously shown that phosphorylation of a 145-kDa protein and activation of signal transduction pathways are associated with the attachment of H. pylori to gastric cells. Here we identify the 145-kDa protein as the H. pylori CagA protein. We also show that CagA is necessary to induce a growth-factor-like phenotype (hummingbird) in host gastric cells similar to that induced by hepatocyte growth factor (HGF). Additionally, we identify a second cellular phenotype induced after attachment by H. pylori, which we call SFA (stress fiber associated). SFA is CagA independent and is produced by type I and type II H. pylori.

    View details for Web of Science ID 000084149700075

    View details for PubMedID 10588744

  • Suppression of T and B lymphocyte activation by a Yersinia pseudotuberculosis virulence factor, YopH JOURNAL OF EXPERIMENTAL MEDICINE Yao, T., Mecsas, J., Healy, J. I., FALKOW, S., Chien, Y. H. 1999; 190 (9): 1343-1350

    Abstract

    The acquired immune responses are crucial to the survival of Yersinia-infected animals. Mice lacking T cells are sensitive to Yersinia infection, and a humoral response to Yersinia can be protective. Diverse mechanisms for Yersinia to impair and evade the host innate immune defense have been suggested, but the effects of Yersinia on lymphocytes are not known. Here, we demonstrate that after a transient exposure to Y. pseudotuberculosis, T and B cells are impaired in their ability to be activated through their antigen receptors. T cells are inhibited in their ability to produce cytokines, and B cells are unable to upregulate surface expression of the costimulatory molecule, B7.2, in response to antigenic stimulation. The block of lymphocyte activation results from the inhibition of early phosphorylation events of the antigen receptor signaling complex. Through the use of Y. pseudotuberculosis mutants, we show that the inhibitory effect in both T cells and B cells is dependent on the production of Yersinia outermembrane protein (Yop) H, a tyrosine phosphatase. Our results suggest a mechanism by which the pathogenic bacteria may modulate a wide range of T and B cell-mediated immune responses.

    View details for Web of Science ID 000083552800014

    View details for PubMedID 10544205

  • An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells SCIENCE Cormack, B. P., Ghori, N., FALKOW, S. 1999; 285 (5427): 578-582

    Abstract

    Candida glabrata is an important fungal pathogen of humans that is responsible for about 15 percent of mucosal and systemic candidiasis. Candida glabrata adhered avidly to human epithelial cells in culture. By means of a genetic approach and a strategy allowing parallel screening of mutants, it was possible to clone a lectin from a Candida species. Deletion of this adhesin reduced adherence of C. glabrata to human epithelial cells by 95 percent. The adhesin, encoded by the EPA1 gene, is likely a glucan-cross-linked cell-wall protein and binds to host-cell carbohydrate, specifically recognizing asialo-lactosyl-containing carbohydrates.

    View details for Web of Science ID 000081609500036

    View details for PubMedID 10417386

  • Genomic clues for defining bacterial pathogenicity MICROBES AND INFECTION Salama, N. R., FALKOW, S. 1999; 1 (8): 615-619

    Abstract

    Genomic sequences are becoming available from both pathogenic and nonpathogenic bacteria. Here we analyze an increasing body of information available on the molecular mechanisms Salmonella typhimurium uses to cause disease, in order to divine clues for identifying sequences that play a role in pathogenesis in other bacterial pathogens.

    View details for Web of Science ID 000082407100005

    View details for PubMedID 10611738

  • Cellular Microbiology is launched CELLULAR MICROBIOLOGY FALKOW, S. 1999; 1 (1): 3-6

    View details for Web of Science ID 000088694900002

    View details for PubMedID 11207536

  • Sequence anomalies in the Cag7 gene of the Helicobacter pylori pathogenicity island PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liu, G. Y., McDaniel, T. K., FALKOW, S., Karlin, S. 1999; 96 (12): 7011-7016

    Abstract

    The severity of Helicobacter pylori-related disease is correlated with a pathogenicity island (the Cag region of about 26 genes) whose presence is associated with the up-regulation of an IL-8 cytokine inflammatory response in gastric epithelial cells. Statistical analysis of the Cag gene sequences calculated from the complete genome of strain 26695 revealed several unusual features. The Cag7 sequence (1,927 aa) has two repeat regions. Repeat region I runs 317 aa in a form of AAA proximal to the protein N terminal; repeat region II extends 907 aa in the middle of the protein sequence consisting of 74 contiguous segments composed from selections among six consensus sequences and includes 58 regularly distributed cysteine residues with consecutive cysteines mostly 12, 18, or 24 aa apart. This "regular" cysteine arrangement may provide a scaffolding of linker elements stabilized by disulfide bridges. When Cag7 homologues from different strains are compared, differences were found almost exclusively in the repeat regions, resulting from deletion and/or insertion of repeating units. These observations suggest that the anomalous repetitive structure of the sequence plays an important role in the conformation of Cag7 gene product and potentially in the function of the pathogenicity island. Other facets of the Cag7 sequence show significant charge clusters, high multiplet count, and extremes of amino acid usage.

    View details for Web of Science ID 000080842200079

    View details for PubMedID 10359830

  • The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hersh, D., Monack, D. M., Smith, M. R., Ghori, N., FALKOW, S., Zychlinsky, A. 1999; 96 (5): 2396-2401

    Abstract

    Recently, Salmonella spp. were shown to induce apoptosis in infected macrophages. The mechanism responsible for this process is unknown. In this report, we establish that the Inv-Spa type III secretion apparatus target invasin SipB is necessary and sufficient for the induction of apoptosis. Purified SipB microinjected into macrophages led to cell death. Binding studies show that SipB associates with the proapoptotic protease caspase-1. This interaction results in the activation of caspase-1, as seen in its proteolytic maturation and the processing of its substrate interleukin-1beta. Caspase-1 activity is essential for the cytotoxicity. Functional inhibition of caspase-1 activity by acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone blocks macrophage cytotoxicity, and macrophages lacking caspase-1 are not susceptible to Salmonella-induced apoptosis. Taken together, the data demonstrate that SipB functions as an analog of the Shigella invasin IpaB.

    View details for Web of Science ID 000078956600106

    View details for PubMedID 10051653

  • Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata GENETICS Cormack, B. P., FALKOW, S. 1999; 151 (3): 979-987

    Abstract

    The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination.

    View details for Web of Science ID 000078966100007

    View details for PubMedID 10049916

  • Bacterial epithelial cell cross talk DEFENSE OF MUCOSAL SURFACES: PATHOGENESIS, IMMUNITY AND VACCINES Raupach, B., Mecsas, J., Heczko, U., FALKOW, S., Finlay, B. B. 1999; 236: 137-161

    View details for Web of Science ID 000077639500008

    View details for PubMedID 9893359

  • Yersinia-induced apoptosis in vivo aids in the establishment of a systemic infection of mice JOURNAL OF EXPERIMENTAL MEDICINE Monack, D. M., Mecsas, J., Bouley, D., FALKOW, S. 1998; 188 (11): 2127-2137

    Abstract

    Pathogenic Yersinia cause a systemic infection in mice that is dependent on the presence of a large plasmid encoding a number of secreted virulence proteins called Yops. We previously demonstrated that a plasmid-encoded Yop, YopJ, was essential for inducing apoptosis in cultured macrophages. Here we report that YopJ is a virulence factor in mice and is important for the establishment of a systemic infection. The oral LD50 for a yopJ mutant Yersinia pseudotuberculosis increases 64-fold compared with wild-type. Although the yopJ mutant strain is able to reach the spleen of infected mice, the mutant strain seldom reaches the same high bacterial load that is seen with wild-type Yersinia strain and begins to be cleared from infected spleens on day 4 after infection. Furthermore, when in competition with wild-type Yersinia in a mixed infection, the yopJ mutant strain is deficient for spread from the Peyer's patches to other lymphoid tissue. We also show that wild-type Yersinia induces apoptosis in vivo of Mac-1(+) cells from infected mesenteric lymph nodes or spleens, as measured by quantitative flow cytometry of TUNEL (Tdt-mediated dUTP-biotin nick-end labeling)-positive cells. The levels of Mac-1(+), TUNEL+ cells from tissue infected with the yopJ mutant strain were equivalent to the levels detected in cells from uninfected tissue. YopJ is necessary for the suppression of TNF-alpha production seen in macrophages infected with wild-type Yersinia, based on previous in vitro studies (Palmer, L.E., S. Hobbie, J.E. Galan, and J.B. Bliska. 1998. Mol. Microbiol. 27:953-965). We conclude here that YopJ plays a role in the establishment of a systemic infection by inducing apoptosis and that this is consistent with the ability to suppress the production of the proinflammatory cytokine tumor necrosis factor alpha.

    View details for Web of Science ID 000077484700016

    View details for PubMedID 9841926

  • Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival MOLECULAR MICROBIOLOGY Cirillo, D. M., Valdivia, R. H., Monack, D. M., FALKOW, S. 1998; 30 (1): 175-188

    Abstract

    Salmonella pathogenicity island 2 (SPI-2) encodes a putative type III secretion system necessary for systemic infection in animals. We have investigated the transcriptional organization and regulation of SPI-2 by creating gfp fusions throughout the entire gene cluster. These gfp fusions demonstrated that SPI-2 genes encoding structural, regulatory and previously uncharacterized putative secreted proteins are preferentially expressed in the intracellular environment of the host macrophage. Furthermore, the transcription of these genes within host cells was dependent on the two-component regulatory system SsrA/SsrB and an acidic phagosomal environment. Most SPI-2 mutants failed to replicate to the same level as wild-type strains in murine macrophages and human epithelial cells. In orally infected mice, SPI-2 mutants colonized the Peyer's patches but did not progress to the mesenteric lymph nodes. We conclude that SPI-2 genes are specifically expressed upon entry into mammalian cells and are required for intracellular growth in host cells in vivo and in vitro.

    View details for Web of Science ID 000076538500015

    View details for PubMedID 9786194

  • Constitutive and inducible green fluorescent protein expression in Bartonella henselae INFECTION AND IMMUNITY Lee, A. K., FALKOW, S. 1998; 66 (8): 3964-3967

    Abstract

    The green fluorescent protein (GFP) gene was expressed on a plasmid in B. henselae, and GFP-expressing bacteria were visualized by fluorescence microscopy. HEp-2 cells infected with GFP-expressing bacteria were separated from uninfected cells with a fluorescence activated cell sorter. Promoter fusions of B. henselae chromosomal DNA to gfp were examined by flow cytometry, and a B. henselae groEL promoter fusion which induced expression at 37 degreesC was isolated.

    View details for Web of Science ID 000074973700064

    View details for PubMedID 9673287

  • Who speaks for the microbes? EMERGING INFECTIOUS DISEASES FALKOW, S. 1998; 4 (3): 495-497

    View details for Web of Science ID 000075433800042

    View details for PubMedID 9716983

  • The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells MOLECULAR MICROBIOLOGY Mecsas, J., Raupach, B., FALKOW, S. 1998; 28 (6): 1269-1281

    Abstract

    Yersinia virulence is dependent on the expression of plasmid-encoded secreted proteins called Yops. After bacterial adherence to receptors on the mammalian cell membrane, several Yops are transported by a type III secretion pathway into the host cell cytoplasm. Two Yops, YopH and YopE, prevent macrophages from phagocytosing Yersinia by disrupting the host cell cytoskeleton and signal transduction pathways. In contrast to this active inhibition of phagocytosis by Yersinia, other pathogens such as Salmonella, Shigella, Listeria and Edwardsiella actively promote their entry into mammalian cells by binding to specific host surface receptors and exploiting existing cell cytoskeletal and signalling pathways. We have tested whether Yersinia Yops can prevent the uptake of these diverse invasive pathogens. We first infected epithelial cells with Yersinia to permit delivery of Yops and subsequently with an invasive pathogen. We then measured the level of bacterial invasion. Preinfection with Yersinia inhibited invasion of Edwardsiella, Shigella and Listeria, but not Salmonella. Furthermore, we found that either YopE or YopH prevented Listeria invasion, whereas only YopE prevented Edwardsiella and Shigella invasion. We correlated the inhibitory effect of the Yops with the inhibitory action of the cell-signalling inhibitors Wortmannin, LY294002 and NDGA, and concluded that the four invasive pathogenic species enter epithelial cells using at least three distinct host cell pathways. We also speculate that YopE affects the rho pathway.

    View details for Web of Science ID 000074446800020

    View details for PubMedID 9680215

  • Flow cytometry and bacterial pathogenesis CURRENT OPINION IN MICROBIOLOGY Valdivia, R. H., FALKOW, S. 1998; 1 (3): 359-363

    Abstract

    Our understanding of microbial adaptations to diverse and threatening environments is limited by the assumption that the behavior of individual bacteria can be accurately determined by measuring the behavior of populations. Recent advances in gene expression reporter systems, fluorescence microscopy and flow cytometry allow microbiologists to explore the complex interactions between bacteria and their environment with single cell resolution. The application of these technologies has been particularly useful in systems, such as host-pathogen interactions, where genetic analysis is often cumbersome. Recently, flow cytometry is increasingly being applied to study host-pathogen interactions.

    View details for Web of Science ID 000075765200015

    View details for PubMedID 10066495

  • Fluorescence-based isolation of bacterial genes expressed within host cells SCIENCE Valdivia, R. H., FALKOW, S. 1997; 277 (5334): 2007-2011

    Abstract

    A selection strategy was devised to identify bacterial genes preferentially expressed when a bacterium associates with its host cell. Fourteen Salmonella typhimurium genes, which were under the control of at least four independent regulatory circuits, were identified to be selectively induced in host macrophages. Four genes encode virulence factors, including a component of a type III secretory apparatus. This selection methodology should be generally applicable to the identification of genes from pathogenic organisms that are induced upon association with host cells or tissues.

    View details for Web of Science ID A1997XX84900049

    View details for PubMedID 9302299

  • Yersinia signals macrophages to undergo apoptosis and YopJ is necessary for this cell death PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Monack, D. M., Mecsas, J., Ghori, N., FALKOW, S. 1997; 94 (19): 10385-10390

    Abstract

    Pathogenic Yersinia spp. carry a large common plasmid that encodes a number of essential virulence determinants. Included in these factors are the Yersinia-secreted proteins called Yops. We analyzed the consequences of wild-type and mutant strains of Yersinia pseudotuberculosis interactions with the macrophage cell line RAW264. 7 and murine bone marrow-derived macrophages. Wild-type Y. pseudotuberculosis kills approximately 70% of infected RAW264.7 macrophages and marrow-derived macrophages after an 8-h infection. We show that the cell death mediated by Y. pseudotuberculosis is apoptosis. Mutant Y. pseudotuberculosis that do not make any Yop proteins no longer cause host cell death. Attachment to host cells via invasin or YadA is necessary for the cell death phenotype. Several Yop mutant strains that fail to express one or more Yop proteins were engineered and then characterized for their ability to cause host cell death. A mutant with a polar insertion in YpkA Ser/Thr kinase that does not express YpkA or YopJ is no longer able to cause apoptosis. In contrast, a mutant no longer making YopE or YopH (a tyrosine phosphatase) induces apoptosis in macrophages similar to wild type. When yopJ is added in trans to the ypkAyopJ mutant, the ability of this strain to signal programmed cell death in macrophages is restored. Thus, YopJ is necessary for inducing apoptosis. The ability of Y. pseudotuberculosis to promote apoptosis of macrophages in cell culture suggests that this process is important for the establishment of infection in the host and for evasion of the host immune response.

    View details for Web of Science ID A1997XX39900070

    View details for PubMedID 9294220

  • Probing bacterial gene expression within host cells TRENDS IN MICROBIOLOGY Valdivia, R. H., FALKOW, S. 1997; 5 (9): 360-363

    Abstract

    The study of bacterial gene expression in the host environment is critical to our understanding of the disease process. New research tools, such as luciferase and green fluorescent protein, provide the means to measure bacterial responses to the intracellular environment with minimal perturbations and with single-cell resolution.

    View details for Web of Science ID A1997XU80100008

    View details for PubMedID 9294892

  • An Edwardsiella tarda strain containing a mutation in a gene with homology to shlB and hpmB is defective for entry into epithelial cells in culture INFECTION AND IMMUNITY Strauss, E. J., Ghori, N., FALKOW, S. 1997; 65 (9): 3924-3932

    Abstract

    Edwardsiella tarda is an enteric pathogen that causes diarrhea, wound infections, and death due to septicemia. This species is capable of invading human epithelial cell lines, and we have now been able to follow the entry and replication of E. tarda within tissue culture host cells. E. tarda escapes from the endocytic vacuole within minutes of entry and then replicates within the cytoplasm. Unlike other well-studied bacteria that replicate and reside in the cytoplasm, we never observed this organism moving directly from cell to cell; instead the bacteria spread by lysing the plasma membrane after several rounds of replication. Efforts to study the interactions of E. tarda with tissue culture cells are complicated by the presence of a potent cytotoxin that the bacterium produces. Using transposon mutagenesis, we isolated a noncytotoxic strain of E. tarda. This mutant is also defective for hemolysin production. The dual phenotype of this strain is consistent with the hypothesis that cytotoxicity is due to the previously characterized E. tarda hemolysin activity. The nonhemolytic strain is also unable to enter HEp-2 cells. The disrupted gene has sequence similarity to members of a family of genes required for transport and activation of the hemolysin genes, shlA and hpmA. A cosmid bearing 40 kb of E. tarda DNA, including wild-type copies of the E. tarda homologs of the transporter-activator protein and the hemolysin itself, confers hemolytic, cytotoxic, and invasive abilities upon normally nonhemolytic, noncytotoxic, and noninvasive strains of Escherichia coli. Sequence data indicate that the genes required for hemolytic activity are linked to a transposable element, suggesting that they arose in the E. tarda genome by horizontal transfer.

    View details for Web of Science ID A1997XT42000061

    View details for PubMedID 9284172

  • A crtB homolog essential for photochromogenicity in Mycobacterium marinum: Isolation, characterization, and gene disruption via homologous recombination JOURNAL OF BACTERIOLOGY Ramakrishnan, L., Tran, H. T., Federspiel, N. A., FALKOW, S. 1997; 179 (18): 5862-5868

    Abstract

    A gene essential for light-induced pigment production was isolated from the photochromogen Mycobacterium marinum by heterologous complementation of an M. marinum cosmid library in the nonchromogen Mycobacterium smegmatis. This gene is part of an operon and homologous to the Streptomyces griseus and Myxococcus xanthus crtB genes encoding phytoene synthase. Gene replacement at this locus was achieved via homologous recombination, demonstrating that its expression is essential for photochromogenicity. The ease of targeted gene disruption in this pathogenic Mycobacterium allows for the dissection of the molecular basis of mycobacterial pathogenesis.

    View details for Web of Science ID A1997XV69900029

    View details for PubMedID 9294446

  • Perspectives series: host/pathogen interactions. Invasion and intracellular sorting of bacteria: searching for bacterial genes expressed during host/pathogen interactions. journal of clinical investigation Falkow, S. 1997; 100 (2): 239-243

    View details for PubMedID 9218498

  • Induction of host signal transduction pathways by Helicobacter pylori PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Segal, E. D., Lange, C., Covacci, A., Tompkins, L. S., FALKOW, S. 1997; 94 (14): 7595-7599

    Abstract

    Adherence of Helicobacter pylori to cultured gastric epithelial cells is associated with several cellular events, including the tyrosine phosphorylation of a 145-kDa host protein; the reorganization of the host cell actin and associated cellular proteins, like vasodilator-stimulated phosphoprotein, adjacent to the attached bacterial cell; and the subsequent release of the cytokine, interleukin 8 (IL-8). H. pylori isolated from patients with ulcer disease and gastric cancer contain a DNA insertion, the cag pathogenicity island (PAI), that is not present in bacteria isolated from individuals with asymptomatic infection. Mutations in a number of PAI genes abolish tyrosine phosphorylation and IL-8 synthesis but not the cytoskeletal rearrangements. Kinase inhibition studies suggest there are two distinct pathways operative in stimulating IL-8 release from host cells and one of these H. pylori pathways is independent of the tyrosine phosphorylation step.

    View details for Web of Science ID A1997XJ87600085

    View details for PubMedID 9207137

  • From microbial genomics to meta-genomics DRUG DEVELOPMENT RESEARCH Covacci, A., Kennedy, G. C., Cormack, B., Rappuoli, R., FALKOW, S. 1997; 41 (3-4): 180-192
  • Microbial pathogenesis: Genomics and beyond SCIENCE Strauss, E. J., FALKOW, S. 1997; 276 (5313): 707-712

    Abstract

    The growing number of complete microbial genome sequences provides a powerful tool for studying the biology of microorganisms. In combination with assays for function, genomic-based approaches can facilitate efficient and directed research strategies to elucidate mechanisms of bacterial pathogenicity. As genomic information accrues, the challenge remains to construct a picture of the biology that accurately reflects how individual genes collaborate to create the complex world of microbial specialization.

    View details for Web of Science ID A1997WW90000036

    View details for PubMedID 9115190

  • Did the inheritance of a pathogenicity island modify the virulence of Helicobacter pylori? TRENDS IN MICROBIOLOGY Covacci, A., FALKOW, S., Berg, D. E., Rappuoli, R. 1997; 5 (5): 205-208

    Abstract

    Strains of Helicobacter pylori from patients with peptic ulcer disease and gastric cancer contain a 40-kb fragment of DNA that is not present in isolates from carriers with asymptomatic infections. The discovery of the cag pathogenicity island suggests that virulence has evolved by quantum leaps through the inheritance of one or more DNA insertions.

    View details for Web of Science ID A1997WY69500015

    View details for PubMedID 9160510

  • The unique trafficking pattern of Salmonella typhimurium-containing phagosomes in murine macrophages is independent of the mechanism of bacterial entry INFECTION AND IMMUNITY Rathman, M., Barker, L. P., FALKOW, S. 1997; 65 (4): 1475-1485

    Abstract

    Although it has been known for some time that Salmonella typhimurium is able to survive and even replicate in the normally bactericidal environment of the macrophage phagosome, the mechanisms by which this organism accomplishes this feat remain obscure. In this study, a murine macrophage cell line and confocal immunofluorescence microscopy were used to more thoroughly define the specific nature of phagosomes containing latex beads or wild-type S. typhimurium (viable or heat-killed organisms). Live S. typhimurium organisms were observed to reside in phagosomes that diverge from the degradative pathway of the macrophage. These compartments contain lysosomal glycoproteins and lysosomal acid phosphatase, endocytic markers delivered to vacuoles by mannose 6-phosphate receptor-independent mechanisms, but are devoid of the mannose 6-phosphate receptor and cathepsin L. In contrast, phagosomes containing latex beads or heat-killed organisms appeared to be processed along the degradative pathway of the host cell; these compartments colocalized not only with lysosomal glycoproteins and lysosomal acid phosphatases but also with mannose 6-phosphate receptors and cathepsin L. The uniqueness of the phagosome containing viable S. typhimurium was confirmed by the observation that these compartments, in comparison to phagosomes containing latex beads, do not readily interact with incoming endocytic traffic. Finally, we show that an isogenic, noninvasive mutant of S. typhimurium, BJ66, ends up in an intracellular compartment identical to the wild-type S. typhimurium-containing phagosome. Thus, modifications of the Salmonella-containing compartment occur independently of the mechanism of bacterial entry.

    View details for Web of Science ID A1997WQ29800048

    View details for PubMedID 9119490

  • Mycobacterium marinum causes both long-term subclinical infection and acute disease in the leopard frog (Rana pipiens) INFECTION AND IMMUNITY Ramakrishnan, L., Valdivia, R. H., McKerrow, J. H., FALKOW, S. 1997; 65 (2): 767-773

    Abstract

    Mycobacterium marinum grows at an optimal temperature of 33 degrees C, far lower than that for M. tuberculosis. Consequently, M. marinum infection of mammals is restricted largely to the cooler surfaces of the body, such as the extremities, but it causes a systemic infection in a large number of poikilothermic animals. Here, we describe a laboratory animal model for M. marinum disease in the leopard frog (Rana pipiens), a natural host species. M. marinum causes a chronic granulomatous, nonlethal disease in immunocompetent frogs. Immunosuppression of the frogs with hydrocortisone results in an acute, fulminant, lethal disease. This animal model, in which a spectrum of tuberculosis-like disease can be produced, will be useful for the dissection of the genetic basis of mycobacterial pathogenesis.

    View details for Web of Science ID A1997WE90000059

    View details for PubMedID 9009340

  • Yeast-enhanced green fluorescent protein (yEGFP): A reporter of gene expression in Candida albicans MICROBIOLOGY-SGM Cormack, B. P., Bertram, G., Egerton, M., Gow, N. A., FALKOW, S., Brown, A. J. 1997; 143: 303-311

    Abstract

    The green fluorescent protein (GFP) of Aequorea victoria has been developed here as a reporter for gene expression and protein localization in Candida albicans. When wild-type (wt) GFP was expressed in C. albicans, it was not possible to detect fluorescence or a translation product for the wt protein. Since this was probably due in part to the presence of the non-canonical CTG serine codon in the Aequorea sequence, this codon was changed to the leucine codon TTG. C. albicans cells expressing this construct contained GFP mRNA but were non-fluorescent and contained no detectable translation product. Hence a codon-optimized GFP gene was constructed in which all of the 239 amino acids are encoded by optimal codons for C. albicans. In this gene were also incorporated two previously identified mutations in the chromophore that increase GFP fluorescence. C. albicans cells expressing this yeast-enhanced GFP gene (yEGFP3) are fluorescent and contain GFP protein. yEGFP3 can be used as a versatile reporter of gene expression in C. albicans and Saccharomyces cerevisiae and the optimized GFP described here should have broad applications in these and other fungal species.

    View details for Web of Science ID A1997WH22500005

    View details for PubMedID 9043107

  • Probing the intracellular life of bacteria. Harvey lectures FALKOW, S. 1997; 93: 65-74

    View details for PubMedID 10941419

  • Bacterial genetics by flow cytometry: Rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction MOLECULAR MICROBIOLOGY Valdivia, R. H., FALKOW, S. 1996; 22 (2): 367-378

    Abstract

    The ability of Salmonella typhimurium to survive and replicate within murine macrophages is dependent on a low phagosomal pH. This requirement for an acidic vacuole suggests that low pH is an important environmental stimulus for the transcription of genes necessary for intracellular survival. To study the behaviour of acid-inducible genes in response to the phagosomal environment, we have applied a novel enrichment strategy, termed differential fluorescence induction (DFI), to screen an S. typhimurium library for promoters that are upregulated at pH 4.5. DFI utilizes a fluorescence-enhanced green fluorescent protein (GFP) and a fluorescence-activated cell sorter (FACS) to perform genetic selection. In the presence of an inducing stimulus, such as low pH, a FACS is used to sort highly fluorescent bacterial clones bearing random promoters fused to the mutant GFP protein (GFPmut). This population is then amplified at neutral pH and the least fluorescent population is sorted. Sequential sorts for fluorescent and non-fluorescent bacteria in the presence or absence of inducing conditions rapidly enriches for promoter fusions that are regulated by the inducing stimulus. We have identified eight acid-inducible promoters and quantified their expression in response to pH 4.5 and to the phagosome milieu. These acid-inducible promoters exhibited extensive homology to promoter regions of genes encoding for cell-surface-maintenance enzymes, stress proteins, and generalized efflux pumps. Only a subset of these promoters was induced in macrophages with kinetics and levels of expression that do not necessarily correlate with in vitro pH-shock induction. This suggests that while low pH is a relevant inducer of intracellular gene expression, additional stimuli in the macrophage can modulate the expression of acid-inducible genes.

    View details for Web of Science ID A1996VP09700016

    View details for PubMedID 8930920

  • Salmonella typhimurium invasion induces apoptosis in infected macrophages PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Monack, D. M., Raupach, B., Hromockyj, A. E., FALKOW, S. 1996; 93 (18): 9833-9838

    Abstract

    Invasive Salmonella typhimurium induces dramatic cytoskeletal changes on the membrane surface of mammalian epithelial cells and RAW264.7 macrophages as part of its entry mechanism. Noninvasive S. typhimurium strains are unable to induce this membrane ruffling. Invasive S. typhimurium strains invade RAW264.7 macrophages in 2 h with 7- to 10-fold higher levels than noninvasive strains. Invasive S. typhimurium and Salmonella typhi, independent of their ability to replicate intracellularly, are cytotoxic to RAW264.7 macrophages and, to a greater degree, to murine bone marrow-derived macrophages. Here, we show that the macrophage cytotoxicity mediated by invasive Salmonella is apoptosis, as shown by nuclear morphology, cytoplasmic vacuolization, and host cell DNA fragmentation. S. typhimurium that enter cells causing ruffles but are mutant for subsequent intracellular replication also initiate host cell apoptosis. Mutant S. typhimurium that are incapable of inducing host cell membrane ruffling fail to induce apoptosis. The activation state of the macrophage plays a significant role in the response of macrophages to Salmonella invasion, perhaps indicating that the signal or receptor for initiating programmed cell death is upregulated in activated macrophages. The ability of Salmonella to promote apoptosis may be important for the initiation of infection, bacterial survival, and escape of the host immune response.

    View details for Web of Science ID A1996VF61400093

    View details for PubMedID 8790417

  • Acidification of phagosomes containing Salmonella typhimurium in murine macrophages INFECTION AND IMMUNITY Rathman, M., Sjaastad, M. D., FALKOW, S. 1996; 64 (7): 2765-2773

    Abstract

    Salmonella species are facultative intracellular pathogens. Following entry into mammalian host cells, they reside in membrane-bound vacuoles, resist killing, and replicate. In this work, we investigated the importance of phagosomal pH in the ability of Salmonella typhimurium to survive and replicate within macrophages. Intraphagosomal pH was measured in situ by recording the fluorescence intensity of a pH-sensitive probe, DM-NERF dextran. The majority of vacuoles containing S. typhimurium (live, heat killed, or formalin fixed) acidified from pH > or = 6.0 to between pH 4.0 and 5.0 within 60 min after formation. In contrast, Mycobacterium avium-containing vacuoles failed to acidify even at later time points. Acidification of S. typhimurium-containing vacuoles was completely blocked by treatment of host cells with bafilomycin A, a specific inhibitor of vacuolar proton-ATPases. Bafilomycin inhibition of vacuolar acidification from the onset of infection significantly decreased the survival of S. typhimurium in macrophages. Furthermore, bafilomycin treatment at 2, 4, 8, or even 12 h postinfection decreased the percentage of recoverable bacteria by up to 20-fold. Loss of bacterial viability was seen with several other reagents which, like bafilomycin, raise the pH of phagosomal compartments but are not directly lethal to the bacteria or host cells. Thus, we conclude that Salmonella-containing phagosomes acidify soon after formation and hypothesize that an acidic environment is necessary for survival and replication of the bacteria within the macrophage.

    View details for Web of Science ID A1996UT65700053

    View details for PubMedID 8698506

  • Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Segal, E. D., FALKOW, S., Tompkins, L. S. 1996; 93 (3): 1259-1264

    Abstract

    The consequences of Helicobacter pylori attachment to human gastric cells were examined by transmission electron microscopy and immunofluorescence microscopy. H. pylori attachment resulted in (i) effacement of microvilli at the site of attachment, (ii) cytoskeletal rearrangement directly beneath the bacterium, and (iii) cup/pedestal formation at the site of attachment. Double-immunofluorescence studies revealed that the cytoskeletal components actin, alpha-actinin, and talin are involved in the process. Immunoblot analysis showed that binding of H. pylori to AGS cells induced tyrosine phosphorylation of two host cell proteins of 145 and 105 kDa. These results indicate that attachment of H. pylori to gastric epithelial cells resembles that of enteropathogenic Escherichia coli. Coccoid H. pylori, which are thought to be terminally differentiated bacterial forms, are capable of binding and inducing cellular changes of the same sort as spiral H. pylori, including tyrosine phosphorylation of host proteins.

    View details for Web of Science ID A1996TU64000056

    View details for PubMedID 8577751

  • BARTONELLA-HENSELAE AND BARTONELLA-QUINTANA ADHERENCE TO AND ENTRY INTO CULTURED HUMAN EPITHELIAL-CELLS INFECTION AND IMMUNITY Batterman, H. J., Peek, J. A., Loutit, J. S., FALKOW, S., Tompkins, L. S. 1995; 63 (11): 4553-4556

    Abstract

    Bartonella henselae expresses pili phenotypically similar to type 4 pili. B. henselae pilus expression undergoes phase variation with multiple passages. Low-passage-number, piliated B. henselae adhered to and invaded HEp-2 cells to a greater extent than did multiply passaged B. henselae with reduced pilus expression. Pili may be a pathogenic determinant for Bartonella species.

    View details for Web of Science ID A1995TB40000056

    View details for PubMedID 7591104

  • THE NEW PATH TO PREVENTING ULCERS SCIENCE Tompkins, L. S., FALKOW, S. 1995; 267 (5204): 1621-1622

    View details for Web of Science ID A1995QM39700028

    View details for PubMedID 7886448

  • IDENTIFICATION AND CHARACTERIZATION OF A SALMONELLA-TYPHIMURIUM OXYGEN-REGULATED GENE REQUIRED FOR BACTERIAL INTERNALIZATION INFECTION AND IMMUNITY Jones, B. D., FALKOW, S. 1994; 62 (9): 3745-3752

    Abstract

    Growth of Salmonella typhimurium in a low-oxygen environment induces the ability of these bacteria to enter mammalian cells. We have carried out a search for invasion genes that are expressed under low-oxygen conditions by using Tn5lacZY transcriptional fusions. Several noninvasive oxygen-regulated lacZY insertion strains have been identified. The invasion defect in one of these noninvasive S. typhimurium strains, BJ66, has been complemented by introduction of a cosmid (pBDJ125) from an S. typhimurium SL1344 gene bank. A 1.9-kb EcoRV DNA fragment subcloned from this cosmid, containing a single open reading frame (orgA), restores the ability of BJ66 to invade mammalian cells. Comparative searches of the GenBank and EMBL sequence data banks with the nucleotide sequence of the gene and deduced amino acid sequence of the protein reveal no significant similarities. Interestingly, hybridization of an orgA gene probe with a P22 chromosomal mapping library demonstrated that the orgA gene maps to a region on the chromosome between 57.5 and 60 min where other Salmonella invasion genes have been mapped. Other enteroinvasive bacteria (Shigella flexneri, Escherichia coli, Yersinia spp., and Listeria monocytogenes) lack sequences which cross hybridize to the probe. We have compared the virulence of S. typhimurium SL1344 and an isogenic orgA mutant in a mouse model of typhoid fever. The orgA mutant was as virulent as the wild-type strain was when inoculated intraperitoneally but is significantly reduced (> 60-fold) in its ability to cause disease by an oral route of infection.

    View details for Web of Science ID A1994PC83600020

    View details for PubMedID 8063389

  • MYCOBACTERIUM-MARINUM PERSISTS IN CULTURED-MAMMALIAN-CELLS IN A TEMPERATURE-RESTRICTED FASHION INFECTION AND IMMUNITY Ramakrishnan, L., FALKOW, S. 1994; 62 (8): 3222-3229

    Abstract

    We have explored the relatively rapidly growing animal and human pathogen Mycobacterium marinum as an experimental model for mycobacterial pathogenesis. M. marinum, which has a lower temperature for optimal growth than does Mycobacterium tuberculosis, has a much shorter generation time and can be safely studied in ordinary laboratory facilities and examined in multiple animal infection models. We have established an in vitro assay for its interaction with eukaryotic cells and shown that it persists in these cells in a temperature-specific fashion that correlates with its ability to cause disease in vivo at lower temperatures. Additionally, preliminary evidence that M. marinum causes a chronic disease with some features resembling tuberculosis in frogs of the species Rana pipiens is presented.

    View details for Web of Science ID A1994NY87200025

    View details for PubMedID 8039892

  • GROWTH OF LEGIONELLA-PNEUMOPHILA IN ACANTHAMOEBA-CASTELLANII ENHANCES INVASION INFECTION AND IMMUNITY Cirillo, J. D., FALKOW, S., Tompkins, L. S. 1994; 62 (8): 3254-3261

    Abstract

    Legionella pneumophila is considered to be a facultative intracellular parasite. Therefore, the ability of these bacteria to enter, i.e., invade, eukaryotic cells is expected to be a key pathogenic determinant. We compared the invasive ability of bacteria grown under standard laboratory conditions with that of bacteria grown in Acanthamoeba castellanii, one of the protozoan species that serves as a natural host for L. pneumophila in the environment. Amoeba-grown L. pneumophila cells were found to be at least 100-fold more invasive for epithelial cells and 10-fold more invasive for macrophages and A. castellanii than were L. pneumophila cells grown on agar. Comparison of agar- and amoeba-grown L. pneumophila cells by light and electron microscopy demonstrated dramatic differences in the morphology and structure of the bacteria. Analyses of protein expression in the two strains of bacteria suggest that these phenotypic differences may be due to the expression of new proteins in amoeba-grown L. pneumophila cells. In addition, the amoeba-grown bacteria were found to enter macrophages via coiling phagocytosis at a higher frequency than agar-grown bacteria did. Replication of L. pneumophila in protozoans present in domestic water supplies may be necessary to produce bacteria that are competent to enter mammalian cells and produce human disease.

    View details for Web of Science ID A1994NY87200029

    View details for PubMedID 8039895

  • SALMONELLA-TYPHIMURIUM INITIATES MURINE INFECTION BY PENETRATING AND DESTROYING THE SPECIALIZED EPITHELIAL M-CELLS OF THE PEYERS-PATCHES JOURNAL OF EXPERIMENTAL MEDICINE Jones, B. D., Ghori, N., FALKOW, S. 1994; 180 (1): 15-23

    Abstract

    Salmonella species are known to initiate infection of mammalian hosts by penetrating the intestinal epithelium of the small bowel. These bacteria preferentially interact with Peyer's patches which are collections of lymphoid follicles making up the gut-associated lymphoid tissue. We infected murine ligated intestinal loops with invasive and noninvasive Salmonella typhimurium strains for 30, 60, 120, and 180 min and examined the infected tissue by transmission electron microscopy. Within 30 min, we found that invasive S. typhimurium exclusively entered M cells found within the follicle-associated epithelium (FAE) of the Peyer's patches. Initially, interactions between invasive bacteria and enterocytes adjacent to the M cells were not found. Invasion of M cells was associated with the ability of the bacteria to invade tissue culture cells. S. typhimurium mutants, which were noninvasive for tissue culture cells, could not be found in ligated loops associated with M cells or enterocytes after incubations of 30, 60, 120, or 180 min. At 60 min, internalized invasive S. typhimurium were cytotoxic for the M cells. Destruction of an M cell formed a gap in the FAE which allowed organisms to invade enterocytes adjacent to the dead cell. Later in the infection process (120 and 180 min), the presence of bacteria beneath the FAE correlated with changes in the cytoarchitecture of the lymphoid follicle. In addition, replicating Salmonella began to enter both the apical and basolateral surfaces of enterocytes adjacent to infected M cells.

    View details for Web of Science ID A1994NR98900005

    View details for PubMedID 8006579

  • INTERPLAY BETWEEN DETERMINANTS OF CELLULAR ENTRY AND CELLULAR DISRUPTION IN THE ENTEROPATHOGENIC YERSINIA CURRENT OPINION IN INFECTIOUS DISEASES Bliska, J. B., FALKOW, S. 1994; 7 (3): 323-328
  • Microbes as tools for cell biology. Introduction. Methods in cell biology FALKOW, S. 1994; 45: xv-xvi

    View details for PubMedID 7707980

  • CLONING OF BORDETELLA-BRONCHISEPTICA UREASE GENES AND ANALYSIS OF COLONIZATION BY A UREASE-NEGATIVE MUTANT STRAIN IN A GUINEA-PIG MODEL MOLECULAR MICROBIOLOGY Monack, D. M., FALKOW, S. 1993; 10 (3): 545-553

    Abstract

    The genes encoding urease were cloned from Bordetella bronchiseptica and the 5.2 kb of DNA essential for expression analysed in a T7 RNA polymerase transcription-translation system. At least four polypeptides with predicted molecular weights of 69,000, 26,000, 12,200 and 11,000 were found. Partial DNA sequence of the gene encoding the 69,000 Da polypeptide revealed high amino acid identity to the alpha-subunit of Proteus mirabilis urease, UreC and jack bean urease. A stable, unmarked deletion was constructed in this gene to create a urease-negative mutant of B. bronchiseptica. To assess colonization in a guinea-pig model, the urease-negative strain was inoculated with the urease-positive parental strain in a mixed infection. The urease-negative strain out competed the urease-positive strain in the trachea, lungs and caecum. We demonstrate that urease is not essential for B. bronchiseptica colonization of the guinea-pig respiratory and digestive tracts.

    View details for Web of Science ID A1993MF56500010

    View details for PubMedID 7968532

  • THE EAE GENE OF CITROBACTER-FREUNDII BIOTYPE 4280 IS NECESSARY FOR COLONIZATION IN TRANSMISSIBLE MURINE COLONIC HYPERPLASIA INFECTION AND IMMUNITY Schauer, D. B., FALKOW, S. 1993; 61 (11): 4654-4661

    Abstract

    Transmissible murine colonic hyperplasia is characterized by proliferation of anchored stem cells in the mucosa of the descending colon of laboratory mice and is caused by Citrobacter freundii biotype 4280. This bacterium produces attaching and effacing lesions in the descending colon prior to the onset of gross hyperplasia. By mutational analysis, the chromosomal eae gene of C. freundii biotype 4280 was shown to be necessary for colonic colonization. Conversely, bacteria cured of a 65-kb plasmid, which was identified in C. freundii biotype 4280, were not attenuated for colonic colonization or for the induction of colonic hyperplasia.

    View details for Web of Science ID A1993ME61700017

    View details for PubMedID 8406863

  • SALMONELLA-TYPHIMURIUM INDUCES MEMBRANE RUFFLING BY A GROWTH FACTOR-RECEPTOR-INDEPENDENT MECHANISM PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jones, B. D., Paterson, H. F., Hall, A., FALKOW, S. 1993; 90 (21): 10390-10394

    Abstract

    Invasive Salmonella typhimurium induces dramatic actin rearrangements on the membrane surface of mammalian cells as part of its entry mechanism. These changes, which are best characterized as membranous ruffles, closely resemble the membrane changes that occur when a growth factor binds to its receptor. Recently, inhibition of the function of the small GTPases rac and rho in quiescent serum-starved fibroblasts was demonstrated to abolish growth factor-mediated ruffling and stress-fiber formation, respectively. In addition, actin changes induced by the oncogene ras were also shown to be regulated by rac and rho. Because Salmonella-induced actin rearrangements resemble those caused by growth factors, we investigated whether ras, rho, or rac regulates the membrane ruffling elicited by S. typhimurium. Surprisingly, inhibition of the functions of these GTPases had no effect on the ability of invasive S. typhimurium to induce membrane ruffles on a variety of tissue culture cells including Madin-Darby canine kidney cells, Swiss 3T3 fibroblasts, and Hep-2 cells. These results led us to examine the interactions of S. typhimurium with Henle-407 intestinal cells, which lack epidermal growth factor receptor on their membrane surface. We found no difference in the ability of invasive S. typhimurium to induce membrane ruffling and to enter Henle-407 cells with or without the epidermal growth factor receptor on the membrane surface. We, therefore, conclude that invasive S. typhimurium induces membrane ruffling and its own internalization by a rac-independent, growth factor-receptor-independent signaling pathway.

    View details for Web of Science ID A1993MF29600133

    View details for PubMedID 8234304

  • ISOLATION AND CHARACTERIZATION OF FLEXISPIRA-RAPPINI FROM LABORATORY MICE JOURNAL OF CLINICAL MICROBIOLOGY Schauer, D. B., Ghori, N., FALKOW, S. 1993; 31 (10): 2709-2714

    Abstract

    A bacterium with an unusual ultrastructure and possessing a fusiform protoplasmic cylinder, spiral periplasmic fibers, and bipolar tufts of sheathed flagella was identified in the intestinal mucosae of laboratory mice. The organism was cultured under microaerophilic conditions and was found to rapidly hydrolyze urea. On the basis of 16S rRNA gene sequence analysis, the organism was shown to be "Flexispira rappini." "F. rappini" is closely related to members of the genus Helicobacter and has been reported to be associated with human gastroenteritis and ovine abortion. "F. rappini" has not previously been observed in the gastrointestinal tracts of mice.

    View details for Web of Science ID A1993LY05900028

    View details for PubMedID 7504685

  • RUFFLES INDUCED BY SALMONELLA AND OTHER STIMULI DIRECT MACROPINOCYTOSIS OF BACTERIA NATURE Francis, C. L., Ryan, T. A., Jones, B. D., Smith, S. J., FALKOW, S. 1993; 364 (6438): 639-642

    Abstract

    Ruffles are specialized plasma membrane ultrastructures of mammalian cells though to be integral to growth, development and locomotion. Induced by growth factors, mitogens or oncogene expression, ruffles are sites of filamentous actin rearrangement and are temporally associated with enhanced pinocytosis. But the function of ruffles, their mechanism of induction and their role in pinocytosis are not understood. We have observed formation of structures resembling ruffles associated with the site of entry of invasive Salmonella typhimurium. Here we report that ruffles elicited by invasive Salmonella directly mediate internalization of non-invasive bacteria in a macropinocytotic fashion, a phenomenon we term 'passive entry'. Furthermore, ruffles induced in the absence of Salmonella also facilitate passive entry. We present evidence that ruffles, common to many signalling events, comprise the macropinocytotic machinery mediating pinocytosis and are subverted by Salmonella so as to enter mammalian cells.

    View details for Web of Science ID A1993LR77100056

    View details for PubMedID 8350922

  • MUTATION IN THE PLA GENE OF YERSINIA-PESTIS ALTERS THE COURSE OF THE PLAGUE BACILLUS-FLEA (SIPHONAPTERA, CERATOPHYLLIDAE) INTERACTION JOURNAL OF MEDICAL ENTOMOLOGY McDonough, K. A., Barnes, A. M., Quan, T. J., Montenieri, J., FALKOW, S. 1993; 30 (4): 772-780

    Abstract

    Yersinia pestis possesses a unique gene (pla) encoding coagulase and fibrinolysin which is implicated in the transmission of plague by fleas. This gene is encoded on the highly conserved but poorly characterized 'pesticin' plasmid pKYP1. The role of the pKYP1-encoded gene, pla, in plague transmission was addressed by feeding fleas on blood containing avirulent Y. pestis strain EV76-6 and three derivatives of this strain (K10-2, K10-3, and K10-5) carrying Tn801 insertions in pKYP1. One of these mutant strains, K10-5, contains an insertion within the pla gene that eliminates both coagulase and fibrinolysin activities, whereas strains K10-3 and K10-2 retain both pla-associated phenotypes. After feeding, it was found that flea mortality at 4 d after infection associated with strain K10-5 (26%) was significantly lower than the mortality observed with other strains (53-64%). These results suggest that expression of the pla gene product may contribute to the deleterious effects of plague bacilli on fleas that have been associated with flea blockage and plague transmission. This increased mortality is not caused simply by an increased bacterial load in fleas containing pla+ bacteria because fleas ingesting pla+ strains contained no more bacteria by flea blot hybridization analysis than did those that ingested the pla- strain K10-5. It is anticipated that further work in this area will clarify the mechanism by which pla acts and will reveal additional genetic loci in the plague bacillus which are required for transmission by fleas.

    View details for Web of Science ID A1993LL09300015

    View details for PubMedID 8360901

  • ATTACHING AND EFFACING LOCUS OF A CITROBACTER-FREUNDII BIOTYPE THAT CAUSES TRANSMISSIBLE MURINE COLONIC HYPERPLASIA INFECTION AND IMMUNITY Schauer, D. B., FALKOW, S. 1993; 61 (6): 2486-2492

    Abstract

    Citrobacter freundii biotype 4280 produces attaching and effacing (AE) lesions in the large intestine of laboratory mice and is the causative agent of transmissible murine colonic hyperplasia. AE lesions are also produced by enteropathogenic Escherichia coli in humans. Southern analysis revealed that biotype 4280, but not 20 other strains of C. freundii, contained DNA homologous to the eae (E. coli attaching and effacing) gene which is necessary for AE activity by enteropathogenic E. coli in vitro. We have cloned and determined the nucleotide sequence of the C. freundii eae homolog. Our findings suggest that the eae locus of C. freundii biotype 4280 is necessary for AE activity and has a role in the pathogenesis of transmissible murine colonic hyperplasia.

    View details for Web of Science ID A1993LE49800030

    View details for PubMedID 8500884

  • HIGH-MOLECULAR-WEIGHT PROTEINS OF NONTYPABLE HAEMOPHILUS-INFLUENZAE MEDIATE ATTACHMENT TO HUMAN EPITHELIAL-CELLS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA StGeme, J. W., FALKOW, S., Barenkamp, S. J. 1993; 90 (7): 2875-2879

    Abstract

    Nontypable Haemophilus influenzae are Gram-negative bacilli that represent a common cause of human disease. These organisms initiate infection by colonizing the upper respiratory tract. Despite the essential role of colonization, the bacterial determinants of this process remain poorly defined. We recently identified a family of surface-exposed high-molecular-weight proteins of nontypable H. influenzae that are related to filamentous hemagglutinin, a critical adherence factor of Bordetella pertussis. The genes encoding the two such high-molecular-weight proteins (HMW-1 and HMW-2) expressed by a prototypic nontypable H. influenzae strain have now been cloned and sequenced. In this study we examined the role of the HMWs in adherence to human epithelial cells. We found that loss of expression of HMW-1 by the prototypic strain and a HMW-1-like protein in a heterologous nontypable H. influenzae strain markedly decreased the capacity to adhere. The absence of expression of both HMW-1 and HMW-2 in the prototypic strain or their homologs in the second strain was associated with a further decrease in adherence. Expression of either HMW-1 or HMW-2 in nonadherent laboratory strains of Escherichia coli resulted in acquisition of the adherence phenotype. These results indicate that both HMW-1 and HMW-2 and the homologous proteins from a second strain can mediate attachment. We speculate that these proteins and the related proteins in other nontypable H. influenzae isolates are important colonization factors.

    View details for Web of Science ID A1993KV97500067

    View details for PubMedID 8464902

  • THE ROLE OF HOST TYROSINE PHOSPHORYLATION IN BACTERIAL PATHOGENESIS TRENDS IN GENETICS Bliska, J. B., FALKOW, S. 1993; 9 (3): 85-89

    Abstract

    Recent studies on the pathogenic mechanisms of several bacterial genera, including the Yersinia, Salmonella and Escherichia, have revealed novel strategies of infection that involve the signal transduction processes of eukaryotic cells. These model systems are reviewed here, with emphasis on the role of tyrosine phosphorylation in these bacterial-host cell interactions.

    View details for Web of Science ID A1993KN89500008

    View details for PubMedID 8488567

  • THE YERSINIA TYROSINE PHOSPHATASE - SPECIFICITY OF A BACTERIAL VIRULENCE DETERMINANT FOR PHOSPHOPROTEINS IN THE J774A.1 MACROPHAGE JOURNAL OF EXPERIMENTAL MEDICINE Bliska, J. B., Clemens, J. C., Dixon, J. E., FALKOW, S. 1992; 176 (6): 1625-1630

    Abstract

    YopH is a plasmid-encoded protein tyrosine phosphatase (PTPase) secreted by pathogenic Yersinia. Although the enzyme likely acts to dephosphorylate eukaryotic proteins during Yersinia infection of the mammalian host, the targets of YopH have not been identified. We infected the murine macrophage-like cell line J774A.1 with Yersinia pseudotuberculosis and investigated the specificity of YopH and YopHC403A, a catalytically inactive mutant derivative, for eukaryotic phosphoproteins. Upon infection, YopH specifically and rapidly dephosphorylated a macrophage protein of 120 kD. The 120-kD protein and a previously detected 55-kD substrate of YopH coprecipitated with YopHC403A. Coprecipitation of these proteins required tyrosine phosphorylation and could be competitively inhibited with excess phosphotyrosine. The 120- and 55-kD proteins that coprecipitate with YopHC403A exhibited the in vitro activity of protein tyrosine kinases (PTKases), suggesting that YopH dephosphorylates activated tyrosine kinases in vivo.

    View details for Web of Science ID A1992KA48100017

    View details for PubMedID 1281213

  • MORPHOLOGICAL AND CYTOSKELETAL CHANGES IN EPITHELIAL-CELLS OCCUR IMMEDIATELY UPON INTERACTION WITH SALMONELLA-TYPHIMURIUM GROWN UNDER LOW-OXYGEN CONDITIONS MOLECULAR MICROBIOLOGY Francis, C. L., Starnbach, M. N., FALKOW, S. 1992; 6 (21): 3077-3087

    Abstract

    Salmonella typhimurium grown under oxygen-limiting conditions were found to enter into, elicit actin filament rearrangement in, and effect morphological changes upon HEp-2 cells within 15 min after infection. Video microscopy revealed that host cell morphological changes associated with entry began within 1 min of productive adherence. Polarized Caco-2 cell morphology was affected 40 s after infection with low-oxygen-grown S. typhimurium. Stationary-phase S. typhimurium did not elicit these phenomena within this time-period even when adherence was enhanced with the afimbial adhesin, AFA-I. Thus, environmental cues regulate S. typhimurium invasion factors, allowing for immediate entry into host cells. Additionally, actin filament rearrangement and morphological changes in the eukaryotic host cell are essential for entry and occur within minutes of infection.

    View details for Web of Science ID A1992JW88800001

    View details for PubMedID 1360615

  • IDENTIFICATION OF UNCULTURED MICROORGANISMS - EXPANDING THE SPECTRUM OF CHARACTERIZED MICROBIAL PATHOGENS INFECTIOUS AGENTS AND DISEASE-REVIEWS ISSUES AND COMMENTARY Relman, D. A., FALKOW, S. 1992; 1 (5): 245-253

    Abstract

    The combination of enzymatic nucleic acid amplification techniques with 16S rRNA-based molecular phylogeny has brought about a new approach to the identification of microbial pathogens that can not be cultivated in the laboratory. The applications of this experimental approach to bacillary angiomatosis and to Whipple's disease have revealed the presence of two previously uncharacterized organisms. These results suggest the existence of a far greater microbial diversity among human pathogens than has been so far appreciated with culture-dependent methods. PCR-based studies of aquatic environmental microbial communities have already reached similar conclusions. As a result, new and provocative questions are raised concerning the association of amplified 16S rRNA sequences with diseased tissue. The answers must await the results of further investigations and the expansion of sequence data bases.

    View details for Web of Science ID A1992KD52000003

    View details for PubMedID 1285351

  • IDENTIFICATION OF THE UNCULTURED BACILLUS OF WHIPPLES DISEASE NEW ENGLAND JOURNAL OF MEDICINE Relman, D. A., Schmidt, T. M., MacDermott, R. P., FALKOW, S. 1992; 327 (5): 293-301

    Abstract

    Whipple's disease is a systemic disorder known for 85 years to be associated with an uncultured, and therefore unidentified, bacillus.We used a molecular genetic approach to identify this organism. The bacterial 16S ribosomal RNA (rRNA) sequence was amplified directly from tissues of five unrelated patients with Whipple's disease by means of the polymerase chain reaction, first with broad-range primers and then with specific primers. We determined and analyzed the nucleotide sequence of the amplification products.A unique 1321-base bacterial 16S rRNA sequence was amplified from duodenal tissue of one patient. This sequence indicated the presence of a previously uncharacterized organism. We then detected this sequence in tissues from all 5 patients with Whipple's disease, but in none of those from 10 patients without the disorder. According to phylogenetic analysis, this bacterium is a gram-positive actinomycete that is not closely related to any known genus.We have identified the uncultured bacillus associated with Whipple's disease. The phylogenetic relations of this bacterium, its distinct morphologic characteristics, and the unusual features of the disease are sufficient grounds for naming this bacillus Tropheryma whippelii gen. nov. sp. nov. Our findings also provide a basis for a specific diagnostic test for this organism.

    View details for Web of Science ID A1992JF65500001

    View details for PubMedID 1377787

  • INVASION BY SALMONELLA-TYPHIMURIUM IS AFFECTED BY THE DIRECTION OF FLAGELLAR ROTATION INFECTION AND IMMUNITY Jones, B. D., Lee, C. A., FALKOW, S. 1992; 60 (6): 2475-2480

    Abstract

    When grown aerobically, Salmonella typhimurium exhibits a low level of entry into tissue culture cells. We have isolated an S. typhimurium Tn10 mutant which, when grown under aerobic conditions, efficiently invades HEp-2 cells. Sequencing of S. typhimurium DNA adjacent to the site of the Tn10 element showed that the insertion disrupted transcription of the aspartate receptor gene, tar. Polar effects of the transposon on downstream genes also eliminated chemotaxis. Isogenic nonchemotactic (Che-), as well as nonmotile (Mot-) and nonflagellated (Fla-), S. typhimurium strains were examined for their ability to invade HEp-2 cells. "Smooth" swimming Che- mutants (cheA, cheW, cheR, and cheY) were found to possess increased invasiveness for cultured mammalian cells. In contrast, a "tumbly" cheB mutant and Mot- (flagellated) strain were found to have decreased levels of tissue culture invasiveness. A Fla- strain was found to be as invasive as the wild-type strain if centrifugation was used to facilitate contact with the monolayer surface. In addition, the observed hyperinvasiveness of the smooth swimming tar::Tn10 mutant was suppressed when the strain was paralyzed by the introduction of a mot or fla mutation. A murine infection model was used to demonstrate that the mutant invasive phenotypes were also observed in vivo. These data are most consistent with the idea that the rotation and physical orientation of flagella around the bacteria affect the ability of salmonellae to enter host cells.

    View details for Web of Science ID A1992HX42100049

    View details for PubMedID 1587617

  • BACTERIAL-RESISTANCE TO COMPLEMENT KILLING MEDIATED BY THE AIL PROTEIN OF YERSINIA-ENTEROCOLITICA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bliska, J. B., FALKOW, S. 1992; 89 (8): 3561-3565

    Abstract

    Ail is a 17-kDa outer membrane Yersinia protein that mediates bacterial attachment to, and invasion of, cultured epithelial cells. We report here an alternative role for Ail in the pathogenesis of Yersinia infection. We found that Escherichia coli HB101 harboring the 4-kilobase recombinant ail clone pVM102 were highly resistant to killing in up to 50% normal human serum. A 674-base-pair fragment of DNA from pVM102, which encodes the ail gene, was inserted into pUC18 and shown to promote full resistance to complement killing in E. coli HB101. Cellular attachment and resistance to complement killing in a plasmid-cured inv- strain of Yersinia enterocolitica (0:8) was correlated with the thermoinduced expression of Ail at 37 degrees C. Insertional inactivation of ail in Y. enterocolitica resulted in loss of both thermoinduced bacterial properties. Cellular attachment and serum resistance were restored by complementation of the defect by plasmid-encoded ail. Complementation of cell attachment activity required bacterial growth at 37 degrees C, indicating that an additional thermoinduced factor is required for this Ail function. In addition, these studies reveal that functional homology exists between Ail and the structurally related protein Rck, which promotes resistance to complement killing in Salmonella typhimurium.

    View details for Web of Science ID A1992HP04300083

    View details for PubMedID 1565652

  • IDENTIFICATION OF A SALMONELLA-TYPHIMURIUM INVASION LOCUS BY SELECTION FOR HYPERINVASIVE MUTANTS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lee, C. A., Jones, B. D., FALKOW, S. 1992; 89 (5): 1847-1851

    Abstract

    Salmonella typhimurium penetrate intestinal epithelial cells during infection. In vitro studies reveal that the availability of oxygen during bacterial growth decreases their capacity to adhere to and enter cultured epithelial cells. To identify S. typhimurium genes involved in epithelial cell entry, mutants were selected that entered HEp-2 cells when grown under repressing, aerobic culture conditions. Two types of transposons were used to generate bacterial mutations--transposons that disrupt genes (Tn10 and Tn5) and one transposon (Tn5B50) that, in addition to disrupting genes, can cause constitutive expression of genes from the neo promoter at one end of the transposon. Three classes of mutations were found that increased the ability of aerobically grown S. typhimurium to enter HEp-2 cells. One class of mutations disrupts the che operons and results in a nonchemotactic phenotype. The second class of mutations revealed that defects in rho, which encodes an essential transcription termination factor, result in hyperinvasiveness. The third class of mutations was obtained only from mutagenesis with Tn5B50, suggesting that their increased invasiveness is due to constitutive expression of a gene(s) from the exogenous neo promoter. Analysis of this third class of mutations identified a S. typhimurium locus hil (hyperinvasion locus), which is essential for bacterial entry into epithelial cells. The results suggest that hil encodes an invasion factor or an activator of invasion factor expression. hil maps between srl and mutS near minute 59.5 of the S. typhimurium chromosome, a region adjacent to other loci that have been identified as required for S. typhimurium invasiveness and virulence.

    View details for Web of Science ID A1992HG68100068

    View details for PubMedID 1311853

  • THE INTERACTION OF BACTERIA WITH MAMMALIAN-CELLS ANNUAL REVIEW OF CELL BIOLOGY FALKOW, S., ISBERG, R. R., Portnoy, D. A. 1992; 8: 333-363

    View details for Web of Science ID A1992JZ40200012

    View details for PubMedID 1476803

  • THE INVASIN PROTEIN OF YERSINIA-ENTEROCOLITICA - INTERNALIZATION OF INVASIN-BEARING BACTERIA BY EUKARYOTIC CELLS IS ASSOCIATED WITH REORGANIZATION OF THE CYTOSKELETON JOURNAL OF CELL BIOLOGY YOUNG, V. B., FALKOW, S., SCHOOLNIK, G. K. 1992; 116 (1): 197-207

    Abstract

    Yersinia enterocolitica, a facultative intracellular pathogen of mammals, readily enters (i.e., invades) cultured eukaryotic cells, a process that can be conferred by the cloned inv locus of the species. We have studied the mechanism by which the product of inv, a microbial outer membrane protein termed "invasin," mediates the internalization of bacteria by HEp-2 cells and chicken embryo fibroblasts. Invasin-bearing bacteria initially bound the filopodia and the leading edges of cultured cells. Multiple points of contact between the bacterial surface and the surface of the cell ensued and led to the internalization of the bacterium within an endocytic vacuole; the same multi-step process could be induced by an inert particle coated with invasin-containing membranes. Both adherence and internalization were blocked by an antisera directed against the beta 1 integrin cell-adherence molecule. Ultrastructural studies of detergent-insoluble cytoskeletons from infected cells and immunofluorescence microscopy of phalloidin-labeled cells showed alterations in the structure of the cytoskeleton during the internalization process including the accumulation of polymerized actin around entering bacteria. Bacterial entry was prevented by cytochalasin D indicating that the internalization process requires actin microfilament function. Possible linkages between beta 1 containing integrins and the cytoskeleton were examined during the internalization process through the use of protein-specific antibodies and immunofluorescence microscopy. Like actin, the actin-associated proteins filamin, talin and the beta 1 integrin subunit were also found to accumulate around entering bacteria. These findings suggest that the invasin-mediated internalization process is associated with cytoskeletal reorganization.

    View details for Web of Science ID A1992GY95900017

    View details for PubMedID 1730744

  • CHARACTERIZATION OF INTERACTIONS OF ENTEROPATHOGENIC ESCHERICHIA-COLI O127-H6 WITH MAMMALIAN-CELLS INVITRO JOURNAL OF INFECTIOUS DISEASES Francis, C. L., Jerse, A. E., KAPER, J. B., FALKOW, S. 1991; 164 (4): 693-703

    Abstract

    Previous studies have identified two bacterial factors involved in enteropathogenic Escherichia coli (EPEC) infection. A plasmid-mediated EPEC adherence factor (EAF) is responsible for initial and localized adherence. A chromosomally encoded E. coli attachment and effacement factor (eae) is involved in effacement of the eukaryotic cell surface and characteristic "pedestal" formation. By using isogenic strains deficient in either EAF, eae, or both, the process of EPEC adherence and entry in vitro was examined. While EAF proved necessary and sufficient for efficient bacterial association with HEp-2 cells, both EAF and eae were required for efficient effacement of and entry into these cells and other cultured cell lines. Invasion mediated by eae was markedly inhibited by cytochalasin D and colchicine. Afimbrial adhesion or type I pili from uropathogenic strains of E. coli substituted for EAF in EAF-Eae+ strains to provide initial adherence to HEp-2 cells and to facilitate actin condensation.

    View details for Web of Science ID A1991GF80500009

    View details for PubMedID 1680136

  • SURFACE-STRUCTURES AND ADHERENCE PROPERTIES OF DIVERSE STRAINS OF HAEMOPHILUS-INFLUENZAE BIOGROUP AEGYPTIUS INFECTION AND IMMUNITY StGeme, J. W., Gilsdorf, J. R., FALKOW, S. 1991; 59 (10): 3366-3371

    Abstract

    Haemophilus influenzae biogroup aegyptius is an important cause of conjunctivitis and has recently been associated with Brazilian purpuric fever (BPF), a fulminant systemic disease of children. To gain insight into the bacterial factors involved in the pathogenesis of this disease, we investigated the surface structures and adherence properties of eight different strains of H. influenzae biogroup aegyptius, including both BPF and non-BPF isolates. All eight strains were able to express long peritrichous pili similar to those observed in H. influenzae. As in H. influenzae, piliation correlated with colony binding of human erythrocytes. However, two strains were capable of hemagglutination in the absence of pili; in these strains, hemagglutination was insensitive to protease treatment, suggesting a nonproteinaceous hemagglutinin. All strains possessed short, thin, surface fibers distinct from long pili and demonstrated efficient attachment to cultured human conjunctival cells. Attachment to conjunctival cells occurred independently of long pili or a capacity for hemagglutination.

    View details for Web of Science ID A1991GH07600003

    View details for PubMedID 1680103

  • STRUCTURAL AND GENETIC-ANALYSIS OF THE BVG-LOCUS IN BORDETELLA SPECIES MOLECULAR MICROBIOLOGY Arico, B., Scarlato, V., Monack, D. M., FALKOW, S., Rappuoli, R. 1991; 5 (10): 2481-2491

    Abstract

    The bvg locus contains two genes, bvgA and bvgS, which control the expression of the virulence-associated genes in Bordetella species by a system similar to the two-component systems used by a variety of bacterial species to respond to environmental stimuli. We determined the nucleotide sequence of the bvg loci of Bordetella parapertussis and Bordetella bronchiseptica and compared them with the previously determined sequence of Bordetella pertussis. The nucleotide and amino acid sequences of the bvg loci of these species are well conserved in those regions coding for the protein domains which have putative kinase and DNA-binding activities. In marked contrast, the region of BvgS that codes for the protein domain with putative sensor activity shows a high degree of variability. In total, we find 198 base-pair changes in the bvg loci of B. parapertussis and B. bronchiseptica relative to the bvg locus of B. pertussis. One hundred and seventy-three of these base-pair changes are identical in B. parapertussis and B. bronchiseptica. This confirms our previous observation that B. parapertussis and B. bronchiseptica are more related to each other than to B. pertussis. We have mapped the mutations that cause phase changes in B. bronchiseptica and we have found that in three cases these are due to spontaneous deletions in the bvgS gene. The wild-type bvg locus present on a multicopy plasmid cannot complement avirulent derivatives of B. bronchiseptica to wild-type levels, but it can do so when the bvgA gene on the plasmid is inactivated. This suggests that hyperexpression of bvgA down-regulates the bvg system.

    View details for Web of Science ID A1991GL95400020

    View details for PubMedID 1791760

  • BACTERIAL ENTRY INTO EUKARYOTIC CELLS CELL FALKOW, S. 1991; 65 (7): 1099-1102

    View details for Web of Science ID A1991FU89900003

    View details for PubMedID 1905978

  • THE AGENT OF BACILLARY ANGIOMATOSIS - REPLY NEW ENGLAND JOURNAL OF MEDICINE Relman, D. A., Loutit, J. S., Schmidt, T. M., FALKOW, S., Tompkins, L. S. 1991; 324 (21): 1513-1513
  • LOSS OF CAPSULE EXPRESSION BY HAEMOPHILUS-INFLUENZAE TYPE-B RESULTS IN ENHANCED ADHERENCE TO AND INVASION OF HUMAN-CELLS INFECTION AND IMMUNITY StGeme, J. W., FALKOW, S. 1991; 59 (4): 1325-1333

    Abstract

    Haemophilus influenzae type b is a common cause of systemic bacterial disease in children, and the serotype b capsule is a major determinant of virulence. Nevertheless, as a consequence of the genetic configuration of the capb locus, type b strains become capsule deficient at a high frequency. To investigate the potential biological relevance of the predisposition to capsule loss, we compared the adherent and invasive abilities of several strains of H. influenzae type b and their isogenic capsule-deficient mutants by using cultured human epithelial cells. In all cases the capsule-deficient mutant demonstrated significantly greater adherence and invasion than the encapsulated parent. Transformation of one capsule-deficient mutant to restore encapsulation resulted in a marked decrease in adherence and invasion. All strains were capable of adherence and invasion by a pilus-independent mechanism. We conclude that capsule loss by H. influenzae type b results in enhanced in vitro adherence and invasion, properties that may be relevant to colonization of the nasopharynx and persistence within the respiratory tract. These observations suggest an explanation for the evolution of the capb locus as directly repeated segments of DNA with a consequent predisposition to recombination resulting in capsule loss.

    View details for Web of Science ID A1991FD91500017

    View details for PubMedID 1672302

  • IDENTIFICATION OF BORDETELLA-PERTUSSIS REGULATORY SEQUENCES REQUIRED FOR TRANSCRIPTIONAL ACTIVATION OF THE FHAB GENE AND AUTOREGULATION OF THE BVGAS OPERON JOURNAL OF BACTERIOLOGY Roy, C. R., FALKOW, S. 1991; 173 (7): 2385-2392

    Abstract

    Transcription of numerous virulence genes in Bordetella pertussis is positively regulated by the products of the bvgAS genes. In this study a series of lacZYA fusions containing deletions in either the fhaB or bvgA promoter regions was used to identify cis-acting regulatory regions required for bvg activation of these two genes. Gel retardation and DNase I protection analyses have shown that specific protein-DNA interactions occur at these regulatory regions and that these interactions require the transcriptional activator protein BvgA. The regulatory regions found upstream of fhaB and bvgA which are involved in protein binding both contain the sequence TTTCCTA. This sequence is part of an inverted repeat upstream of fhaB and a direct repeat upstream of bvgA. Homologous repeats are not apparent upstream of other bvg-activated genes, such as ptx and cyaA. These data suggest that the mechanism for transcriptional regulation of the other bvg-activated genes is complex and may require regulatory factors in addition to the bvgAS gene products.

    View details for Web of Science ID A1991FE18400032

    View details for PubMedID 2007557

  • TYROSINE PHOSPHATE HYDROLYSIS OF HOST PROTEINS BY AN ESSENTIAL YERSINIA-VIRULENCE DETERMINANT PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bliska, J. B., Guan, K. L., Dixon, J. E., FALKOW, S. 1991; 88 (4): 1187-1191

    Abstract

    The plasmid-encoded YopH protein is a protein-tyrosine phosphatase (PTPase; EC 3.1.3.48) that is essential for Yersinia virulence. We have investigated the molecular basis for the role of PTPase activity in Yersinia pathogenesis. Allelic recombination was employed to introduce a defined mutation into the yopH plasmid gene. Conversion of the essential Cys-403 to Ala in the catalytic domain of the protein abolished YopH PTPase activity and significantly reduced the virulence of Yersinia pseudotuberculosis in a murine infection model. 32P-labeled phosphotyrosine-containing proteins were immunoprecipitated from extracts of Y. pseudotuberculosis-infected cell monolayers and analyzed by SDS/PAGE to assess the impact of YopH on host protein phosphorylation. Major proteins of 200, 120, and 60 kDa were dephosphorylated in macrophages associated with wild-type Y. pseudotuberculosis. Selective removal of phosphate from the 120- and 60-kDa proteins was shown to be specific to the YopH PTPase activity. Phagocytosis of the bacteria was not required for this dephosphorylation activity, suggesting that YopH is functionally expressed by extracellular bacteria. These observations indicate that the essential function of YopH in Yersinia pathogenesis is host-protein dephosphorylation.

    View details for Web of Science ID A1991EY61700022

    View details for PubMedID 1705028

  • THE AGENT OF BACILLARY ANGIOMATOSIS - AN APPROACH TO THE IDENTIFICATION OF UNCULTURED PATHOGENS NEW ENGLAND JOURNAL OF MEDICINE Relman, D. A., Loutit, J. S., Schmidt, T. M., FALKOW, S., Tompkins, L. S. 1990; 323 (23): 1573-1580

    Abstract

    Bacillary angiomatosis is an infectious disease causing proliferation of small blood vessels in the skin and visceral organs of patients with human immunodeficiency virus infection and other immunocompromised hosts. The agent is often visualized in tissue sections of lesions with Warthin-Starry staining, but the bacillus has not been successfully cultured or identified. This bacillus may also cause cat scratch disease.In attempting to identify this organism, we used the polymerase chain reaction. We used oligonucleotide primers complementary to the 16S ribosomal RNA genes of eubacteria to amplify 16S ribosomal gene fragments directly from tissue samples of bacillary angiomatosis. The DNA sequence of these fragments was determined and analyzed for phylogenetic relatedness to other known organisms. Normal tissues were studied in parallel.Tissue from three unrelated patients with bacillary angiomatosis yielded a unique 16S gene sequence. A sequence obtained from a fourth patient with bacillary angiomatosis differed from the sequence found in the other three patients at only 4 of 241 base positions. No related 16S gene fragment was detected in the normal tissues. These 16S sequences associated with bacillary angiomatosis belong to a previously uncharacterized microorganism, most closely related to Rochalimaea quintana.The cause of bacillary angiomatosis is a previously uncharacterized rickettsia-like organism, closely related to R. quintana. This method for the identification of an uncultured pathogen may be applicable to other infectious diseases of unknown cause.

    View details for Web of Science ID A1990EK69600001

    View details for PubMedID 2233945

  • HAEMOPHILUS-INFLUENZAE ADHERES TO AND ENTERS CULTURED HUMAN EPITHELIAL-CELLS INFECTION AND IMMUNITY StGeme, J. W., FALKOW, S. 1990; 58 (12): 4036-4044

    Abstract

    Haemophilus influenzae is a common commensal organism of the human respiratory tract that initiates infection by colonizing the nasopharyngeal epithelium. In some individuals, colonization is followed by localized respiratory tract or systemic disease. To gain insight into the mechanisms by which H. influenzae attaches to and persists within the nasopharynx, we examined the interactions between a nonpiliated clinical isolate of H. influenzae and human epithelial cells. We noted substantial adherence that occurred independently of pili and required viable bacteria capable of de novo protein synthesis. Comparison of profiles of outer membrane proteins synthesized during incubation with epithelial cells for adherent and nonadherent bacteria identified several candidate adhesin molecules. In addition, a small number of adherent bacteria were capable of entering epithelial cells in a process that was inhibited by cytochalasin D and colchicine. The suggestion from our studies is that one or more of several newly synthesized nonpilus bacterial proteins are required for maximal in vitro adherence and invasion. We speculate that H. influenzae entry into epithelial cells may provide a mechanism for evasion of host defenses, thereby allowing persistence in the nasopharynx.

    View details for Web of Science ID A1990EK78100033

    View details for PubMedID 2254028

  • SEQUENCE, LOCALIZATION AND FUNCTION OF THE INVASIN PROTEIN OF YERSINIA-ENTEROCOLITICA MOLECULAR MICROBIOLOGY YOUNG, V. B., MILLER, V. L., FALKOW, S., SCHOOLNIK, G. K. 1990; 4 (7): 1119-1128

    Abstract

    The inv locus of Yersinia enterocolitica is sufficient to convert a non-invasive Escherichia coli K12 strain into a microorganism that is able to penetrate cultured mammalian cells. The nucleotide sequence of inv reveals an open reading frame corresponding to an 835-amino-acid protein that is homologous to the invasin protein from Yersinia pseudotuberculosis. A polyclonal antiserum elicited by a synthetic peptide corresponding to the C-terminal 88 amino acids of this open reading frame detected a unique 100 kD protein in cell lysates of Y. enterocolitica strain 8081 c and in an E. coli strain harbouring the cloned inv gene. This protein localized to the outer membranes of both microorganisms and was cleaved by low concentrations of extracellular trypsin. HEp-2 cells were shown to attach to surfaces coated with bacterial outer membranes containing invasin and this attachment was destroyed by treatment of the membranes with trypsin. Thus it appears that the invasin protein from Y. enterocolitica is able to mediate both attachment to and entry of cultured epithelial cells.

    View details for Web of Science ID A1990DQ78000009

    View details for PubMedID 2233250

  • RECOGNITION OF A BACTERIAL ADHESIN BY AN INTEGRIN - MACROPHAGE CR3 (ALPHA-M-BETA-2, CD11B CD18) BINDS FILAMENTOUS HEMAGGLUTININ OF BORDETELLA-PERTUSSIS CELL Relman, D., Tuomanen, E., FALKOW, S., Golenbock, D. T., Saukkonen, K., Wright, S. D. 1990; 61 (7): 1375-1382

    Abstract

    During the course of whooping cough, Bordetella pertussis interacts with alveolar macrophages and other leukocytes on the respiratory epithelium. We report here mechanisms by which these bacteria adhere to human macrophages in vitro. Whole bacteria adhere by means of two proteins, filamentous hemagglutinin (FHA) and pertussis toxin, either of which is sufficient to mediate adherence. FHA interacts with two classes of molecules on macrophages, galactose-containing glycoconjugates and the integrin CR3 (alpha M beta 2, CD11b/CD18). The interaction between CR3 and FHA involves recognition of the Arg-Gly-Asp (RGD) sequence at positions 1097-1099 in FHA. This study demonstrates that bacterial adherence can be based on the interaction of a bacterial adhesin RGD sequence with an integrin and that bacterial adhesins can have multiple binding sites characteristic of eukaryotic extracellular matrix proteins.

    View details for Web of Science ID A1990DM15600025

    View details for PubMedID 2364431

  • THE ABILITY OF SALMONELLA TO ENTER MAMMALIAN-CELLS IS AFFECTED BY BACTERIAL-GROWTH STATE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lee, C. A., FALKOW, S. 1990; 87 (11): 4304-4308

    Abstract

    We have examined the effect of different growth conditions on the ability of Salmonella to interact with Madin-Darby canine kidney cells. Two growth conditions that affect the expression of Salmonella adherence and invasiveness have been identified. First, bacteria lose their invasiveness in the stationary phase of growth. Second, bacteria growing in oxygen-limited growth conditions are induced for adherence and invasiveness, whereas those growing aerobically are relatively nonadherent and noninvasive. Salmonella from cultures aerated with gas mixtures containing 0% or 1% oxygen were 6- to 70-fold more adherent and invasive than those from cultures aerated with a gas mixture containing 20% oxygen. The Salmonella typhimurium oxrA gene that is required for the anaerobic induction of many proteins is not involved in the regulation of Salmonella invasiveness. We speculate that oxygen limitation might be an environmental cue that triggers the expression of Salmonella invasiveness within the intestinal lumen and other tissues.

    View details for Web of Science ID A1990DG09200059

    View details for PubMedID 2349239

  • DETECTION OF SHIGELLA IN FECES USING DNA AMPLIFICATION JOURNAL OF INFECTIOUS DISEASES Frankel, G., Riley, L., Giron, J. A., VALMASSOI, J., Friedmann, A., Strockbine, N., FALKOW, S., SCHOOLNIK, G. K. 1990; 161 (6): 1252-1256

    Abstract

    A rapid diagnostic method employing a polymerase chain reaction procedure (PCR) was used to identify Shigella and enteroinvasive Escherichia coli. This procedure amplified a region of the invasive-associated locus (ial) from a crude DNA extract of feces. A synthetic 21-base oligonucleotide corresponding to the ial gene sequence was shown to specifically hybridize only with enteroinvasive E. coli (EIEC) strains and Shigella species. Upon PCR amplification, a 320-base pair fragment was generated in DNA extracted from feces reconstituted with EIEC or Shigella flexneri but not in DNA from 70 normal stools lacking these organisms and could be readily detected by the ial probe. For identifying Shigella and EIEC, the PCR assay was 10(5)- and 10(2)-fold more sensitive than standard biochemical tests and the macrocolony hybridization assay, respectively. These findings demonstrate a novel methodology for rapid, sensitive, and culture-independent diagnosis of diarrhea caused by these pathogens and underscores the utility of PCR in the diagnostic laboratory.

    View details for Web of Science ID A1990DG05100032

    View details for PubMedID 2189008

  • GENETIC-CHARACTERIZATION OF BORDETELLA-PERTUSSIS FILAMENTOUS HEMAGGLUTININ - A PROTEIN PROCESSED FROM AN UNUSUALLY LARGE PRECURSOR MOLECULAR MICROBIOLOGY Domenighini, M., Relman, D., Capiau, C., FALKOW, S., PRUGNOLA, A., Scarlato, V., Rappuoli, R. 1990; 4 (5): 787-800

    Abstract

    The nucleotide sequence of the structural gene for filamentous haemagglutinin (FHA), fhaB, a crucial adherence factor for Bordetella pertussis, has been determined. Its 10774 nucleotides are far more than necessary to encode the 220 kD biologically active, mature polypeptide product, suggesting a role for co- or post-translational processing. Fusion proteins derived from various portions of the fhaB open reading frame (ORF) were used to generate polyclonal antisera. Western immunoblot analysis of purified FHA and Bordetella sp. whole cell extracts with these antisera indicated that the 220 kD product is encoded by the 5' portion of the ORF and that the smaller polypeptide species are breakdown products of this polypeptide. These data, as well as N-terminal amino acid sequencing of the major polypeptide species, suggest a scheme for the proteolytic processing of an FHA precursor polypeptide.

    View details for Web of Science ID A1990DF11700011

    View details for PubMedID 2388559

  • AUTOGENOUS REGULATION OF THE BORDETELLA-PERTUSSIS BVGABC OPERON PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Roy, C. R., Miller, J. F., FALKOW, S. 1990; 87 (10): 3763-3767

    Abstract

    The bvgABC operon of the bacterial pathogen Bordetella pertussis encodes a sensory transduction system that regulates the expression of several virulence genes in response to environmental stimuli. In this study we have examined the transcriptional regulation of the bvgABC operon. Transcriptional bvg::lacZYA fusions in Escherichia coli show that the bvgABC operon is autogenously activated. Autoactivation is inhibited by the same environmental stimuli that result in the lack of expression of bvg-activated genes in B. pertussis. These observations were confirmed in B. pertussis using a chromosomal chloramphenicol acetyltransferase transcriptional fusion in bvgC. Transcriptional initiation sites upstream of bvgA were mapped by primer extension analysis in E. coli and B. pertussis. Two differentially regulated bvg promoters were identified. The bvgP1 promoter is a positively autoregulated promoter located 90 base pairs upstream of bvgA. The bvgP2 promoter is located 141 base pairs upstream of bvgA and does not appear to require any positive regulatory factors for activity. These results suggest a model describing the regulatory events that take place upstream of the bvgABC operon.

    View details for Web of Science ID A1990DD87300027

    View details for PubMedID 2111016

  • NONPATHOGENIC ISOLATES OF YERSINIA-ENTEROCOLITICA DO NOT CONTAIN FUNCTIONAL INV-HOMOLOGOUS SEQUENCES INFECTION AND IMMUNITY Pierson, D. E., FALKOW, S. 1990; 58 (4): 1059-1064

    Abstract

    Previous studies have demonstrated a correlation between the ability of isolates of Yersinia enterocolitica to cause disease and to invade tissue culture cells in vitro. Two genes, inv and ail, isolated from a pathogenic strain of Y. enterocolitica have each been shown to confer this invasive phenotype upon Escherichia coli. Eighty pathogenic, invasive isolates studied by Miller et al. (Infect. Immun. 57:121-131, 1989) contained sequences homologous to both of these genes. Thirty-five nonpathogenic, noninvasive isolates similarly studied had no ail homology but carried inv-homologous sequences. We investigated inv-homologous sequences from four nonpathogenic isolates. Recombinant clones of these inv-homologous sequences did not confer the invasive phenotype upon E. coli. No RNA transcripts capable of encoding a full-length Inv protein were detected in the four noninvasive Yersinia strains. When the inv gene from a pathogenic isolate was introduced into two of these strains, the resulting transformants invaded tissue culture cells in vitro. The inv gene was transcribed in a pathogenic Yersinia isolate grown at 30 degrees C but not at all in these cells grown at 37 degrees C. The production of RNA transcripts homologous to inv in transformants was not regulated by temperature to the same degree as was seen for pathogenic isolates. We conclude that the inv gene in nonpathogenic strains of Y. enterocolitica is nonfunctional. Y. enterocolitica isolates epidemiologically linked to disease contain both a functional inv gene and a functional ail gene. Environmental isolates not associated with disease have a nonfunctional inv gene and no ail gene.

    View details for Web of Science ID A1990CV67900033

    View details for PubMedID 1690684

  • NUCLEOTIDE-SEQUENCE OF THE YERSINIA-ENTEROCOLITICA AIL GENE AND CHARACTERIZATION OF THE AIL PROTEIN PRODUCT JOURNAL OF BACTERIOLOGY MILLER, V. L., Bliska, J. B., FALKOW, S. 1990; 172 (2): 1062-1069

    Abstract

    The ability to enter (invade) eucaryotic cells is a property common to many pathogenic bacteria. Yersinia enterocolitica is a facultative intracellular pathogen whose primary site of multiplication is the reticuloendothelial system. In an effort to understand how Y. enterocolitica crosses the intestinal epithelial cell layer, we previously reported the cloning of two loci from Y. enterocolitica that individually conferred an invasive phenotype to the normally noninvasive Escherichia coli HB101. One of these loci, ail, is encoded by a region of DNA that is less than 650 base pairs. We have identified the ail gene product in maxicells as a 17-kilodalton membrane-associated protein. The Ail protein has been purified, and its N-terminal sequence has been determined. The nucleotide sequence of the ail gene revealed a single unique open reading frame of 178 amino acids. Comparison of amino acid sequences deduced from the gene and obtained by analysis of the purified protein identified the first 23 amino acids as a signal sequence. The site(s) at which transcription initiates on the ail gene was identified by primer extension analysis and shown to be identical in E. coli and Y. enterocolitica. Two small open reading frames downstream of ail were found and shown to exhibit considerable identity to the proposed IS3 transposase.

    View details for Web of Science ID A1990CL74300067

    View details for PubMedID 1688838

  • PHASE VARIANTS OF BORDETELLA-BRONCHISEPTICA ARISE BY SPONTANEOUS DELETIONS IN THE VIR LOCUS MOLECULAR MICROBIOLOGY Monack, D. M., Arico, B., Rappuoli, R., FALKOW, S. 1989; 3 (12): 1719-1728

    Abstract

    Bordetella bronchiseptica is a common respiratory tract pathogen of many mammalian species. Nucleotide sequences from the locus involved in coordinate regulation of B. pertussis virulence factors, vir, were shown to have a high degree of homology to chromosomal DNA from virulent (Vir+) and avirulent (Vir-) strains of B. bronchiseptica. Small deletions, 50 bp to 500 bp, within the vir locus were found in some of the Vir- phase variants. The vir locus and the adjacent 5' portion of the fhaB structural gene were cloned from the parental Vir+ B. bronchiseptica strain on a 23.5 kb BamHI fragment. Restriction enzyme mapping of the cloned B. bronchiseptica vir locus revealed similarities with and differences from the previously cloned B. pertussis vir locus. The cloned B. bronchiseptica vir locus complemented spontaneous Vir- variants of Bordetella pertussis and B. bronchiseptica as well as vir::Tn5 mutants of B. pertussis. Comparison of various functions of the vir loci of B. bronchiseptica and B. pertussis revealed some interesting differences in the coordinate regulation of virulence factors.

    View details for Web of Science ID A1989CH04400005

    View details for PubMedID 2560120

  • ANALYSIS OF BORDETELLA-PERTUSSIS VIRULENCE GENE-REGULATION BY USE OF TRANSCRIPTIONAL FUSIONS IN ESCHERICHIA-COLI JOURNAL OF BACTERIOLOGY Miller, J. F., Roy, C. R., FALKOW, S. 1989; 171 (11): 6345-6348

    Abstract

    The virulence regulon of Bordetella pertussis includes a trans-acting regulatory locus, bvg, that is required for expression of several virulence factors. The virulence control system also responds to environmental signals. We have reconstructed a bvg-dependent regulatory system in Escherichia coli by using bacteriophage lambda vectors carrying transcriptional fusions to lacZYA. Single-copy lacZYA fusions to the B. pertussis fhaB locus, which encodes the attachment factor filamentous hemagglutinin, were activated nearly 400-fold by pBR322 replicons carrying sequences that included bvg. In contrast, bvg had no effect on the pertussis toxin operon (ptxA-E) promoter in E. coli as measured by ptxA-lacZ expression. Environmental signals that modulate expression of virulence genes in B. pertussis had a pronounced effect on bvg-mediated activation of fhaB-lacZ. MgSO4, nicotinic acid, and low temperature resulted in decreases in beta-galactosidase activities of 175-, 115-, and 45-fold respectively. Sensory transduction and transcriptional activation were tightly coupled, and both required an intact bvg locus as determined by 5' and 3' deletions that eliminated both activities.

    View details for Web of Science ID A1989AX60400076

    View details for PubMedID 2553678

  • THE BVGA GENE OF BORDETELLA-PERTUSSIS ENCODES A TRANSCRIPTIONAL ACTIVATOR REQUIRED FOR COORDINATE REGULATION OF SEVERAL VIRULENCE GENES JOURNAL OF BACTERIOLOGY Roy, C. R., Miller, J. F., FALKOW, S. 1989; 171 (11): 6338-6344

    Abstract

    The bvg region of the respiratory pathogen Bordetella pertussis coordinately regulates the expression of several unlinked virulence determinants in response to environmental signals. The DNA sequence of the bvg region contains three genes (bvgA, bvgB, and bvgC). Transcription of a single-copy fusion consisting of the upstream region of a bvg-activated B. pertussis gene (fhaB) attached to the promoterless lac operon in Escherichia coli requires the entire bvgABC region in trans. Activation of the fhaB::lacZYA fusion is sensitive to the same environmental stimuli in E. coli that modulate the expression of bvg-activated genes in B. pertussis. Our data show that overexpression of the bvgA gene from a strong heterologous promoter results in transcriptional activation of the fhaB::lacZYA fusion even in the absence of the bvgB and bvgC products. Activation of fhaB transcription by bvgA overexpression in E. coli is no longer repressed by environmental conditions. The bvgA product has been identified by maxicell analysis as a 23-kilodalton protein. A B. pertussis mutant containing an in-frame deletion in bvgA was constructed. This mutant was nonhemolytic and no longer produced filamentous hemagglutinin and pertussis toxin. The mutation in this strain was complemented by returning the bvgA gene in trans. Transcriptional chloramphenicol acetyltransferase fusions to the fhaB and ptx promoter regions were returned to both the B. pertussis bvgA deletion mutant and its parental wild-type strain. Analysis of these strains indicated that the deletion mutant was defective in transcription of both ptx and fhaB. We conclude from these data that bvgA, bvgB, and bvgC comprise an operon encoding the components essential for coordinate regulation and sensory transduction. The BvgA protein is a transcriptional regulatory factor. The bvgB and bvgC products may be important in regulating the activity of BvgA in response to the changing environmental stimuli that B. pertussis encounters during the diseases whooping cough.

    View details for Web of Science ID A1989AX60400075

    View details for PubMedID 2553677

  • SEQUENCES REQUIRED FOR EXPRESSION OF BORDETELLA-PERTUSSIS VIRULENCE FACTORS SHARE HOMOLOGY WITH PROKARYOTIC SIGNAL TRANSDUCTION PROTEINS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Arico, B., Miller, J. F., Roy, C., Stibitz, S., Monack, D., FALKOW, S., Gross, R., Rappuoli, R. 1989; 86 (17): 6671-6675

    Abstract

    The bvg locus of Bordetella pertussis is required for coordinate regulation of several factors associated with virulence. The control system is modulated by various environmental signals, including low temperature, MgSO4, and nicotinic acid. The nucleotide sequence of the bvg region has been determined and three open reading frames, bvgA, bvgB, and bvgC, are present. Twelve-base-pair linker insertion mutations in any of these open reading frames result in a Bvg- phenotype. The predicted protein products of bvgA and bvgC share homology with a family of prokaryotic regulatory proteins that respond to environmental stimuli and are members of two-component sensory transduction systems. We propose a model in which BvgB and the N-terminal portion of BvgC are localized in the periplasm. Environmental signals are recognized, transduced to the cytoplasmic portion of BvgC, and then transmitted to BvgA, a positive regulator of transcription.

    View details for Web of Science ID A1989AP19100049

    View details for PubMedID 2549542

  • SPECIES-SPECIFIC DETECTION OF LEGIONELLA-PNEUMOPHILA IN WATER BY DNA AMPLIFICATION AND HYBRIDIZATION JOURNAL OF CLINICAL MICROBIOLOGY Starnbach, M. N., FALKOW, S., Tompkins, L. S. 1989; 27 (6): 1257-1261

    Abstract

    A sensitive detection system specific for Legionella pneumophila in water was developed. This system is based on amplification of a chromosomal DNA sequence from L. pneumophila by the polymerase chain reaction, followed by detection of the amplified product by hybridization of a radiolabeled oligodeoxynucleotide. After 35 cycles of amplification, a water sample which had been seeded with 35 CFU of L. pneumophila contained sufficient amplified DNA to be detected on dot blots. Bacteria of other genera tested did not generate positive signals under these conditions. Application of this technique to environmental water samples may help identify the natural reservoirs of nosocomial and community-acquired L. pneumophila infections.

    View details for Web of Science ID A1989U715700022

    View details for PubMedID 2754000

  • A YERSINIA-PESTIS-SPECIFIC DNA FRAGMENT ENCODES TEMPERATURE-DEPENDENT COAGULASE AND FIBRINOLYSIN-ASSOCIATED PHENOTYPES MOLECULAR MICROBIOLOGY McDonough, K. A., FALKOW, S. 1989; 3 (6): 767-775

    Abstract

    The effect of temperature on coagulase and fibrinolysin expression (Pla) by Yersinia pestis has been implicated in the transmission of plague by fleas. In an attempt to improve our understanding of this process, we have cloned, sequenced and characterized the gene encoding the Pla phenotypes in Y. pestis, and examined its temperature-dependent regulation. The coding region for this gene overlaps a 900bp Y. pestis-specific DNA fragment that we have previously shown to be capable of detecting plague bacilli in fleas. The pla gene contains a single open reading frame encoding 312 amino acids with a predicted molecular weight of 34.7 kD and a putative signal sequence of 20 amino acids. This coding region appears to be sufficient for both coagulase and fibrinolytic activities. In Y. pestis, modulation between coagulase and fibrinolytic activities is temperature-dependent: coagulase activity is most evident at temperatures below 30 degrees C but fibrinolytic activity increases with higher temperatures (greater than 30 degrees C), regardless of the temperature at which the bacteria are grown. Our results lead us to believe that this regulation occurs post-translationally. It is possible that the alternative forms of the Pla protein are essential to 'flea blockage' and subsequent transmission of the plague bacillus to animals.

    View details for Web of Science ID A1989AA80200008

    View details for PubMedID 2526282

  • EXPRESSION OF GONOCOCCAL PROTEIN-II IN ESCHERICHIA-COLI BY TRANSLATIONAL FUSION MOLECULAR MICROBIOLOGY Palmer, L., Brooks, G. F., FALKOW, S. 1989; 3 (5): 663-671

    Abstract

    A protein II (P.II) gene from Neisseria gonorrhoeae was cloned in Escherichia coli and characterized by DNA sequence analysis. As with other reported P.II sequences, this gene contains an ATG initiation codon which is out of frame with respect to the remainder of the P.II amino acid sequence. A translational fusion was constructed in E. coli which linked the P.II sequence to the signal peptide of beta-lactamase. This P.II fusion differs from the gonococcal protein only in the first seven residues at the N terminus. In E. coli, the P.II fusion product exhibits properties analogous to those of P.II in N. gonorrhoeae. The P.II fusion product is a major component of the E. coli outer membrane and it is exposed on the cell surface. The P.II fusion protein also exhibits the heat-modifiable phenotype of gonococcal P.II.

    View details for Web of Science ID A1989U818300011

    View details for PubMedID 2503682

  • USE OF A CHLAMYDIA-TRACHOMATIS DNA PROBE FOR DETECTION OF OCULAR CHLAMYDIAE JOURNAL OF CLINICAL MICROBIOLOGY Dean, D., Palmer, L., PANT, C. R., Courtright, P., FALKOW, S., OHANLEY, P. 1989; 27 (5): 1062-1067

    Abstract

    We examined the efficacy of a Chlamydia trachomatis DNA probe in detecting ocular chlamydiae by comparing it with tissue culture isolation, direct fluorescent-antibody cytology, and clinical eye exams. In a trachoma-endemic area of Nepal, 430 Nepalese villagers were examined according to the World Health Organization trachoma grading scale. Upper tarsal conjunctival specimens from each subject were obtained for DNA probing, tissue culture, and fluorescent-antibody screening. Moderate to severe intensity of inflammation was found in 85 (21%) of 430 people studied. An additional 25 (7.2%) of 345 people with low or no intensity of inflammation also had microbiologically proven infection, which may reflect asymptomatic carriage. Compared with culture, the DNA probe had a sensitivity of 86.9% and a specificity of 91%. For direct fluorescent antibody versus culture, the values were 47.8 and 96.9%, respectively. Results from this study indicate that the DNA probe for C. trachomatis might be considered a valuable epidemiologic tool in screening trachoma-endemic populations for ocular chlamydiae.

    View details for Web of Science ID A1989U210800053

    View details for PubMedID 2663912

  • FILAMENTOUS HEMAGGLUTININ OF BORDETELLA-PERTUSSIS - NUCLEOTIDE SEQUENCE AND CRUCIAL ROLE IN ADHERENCE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Relman, D. A., Domenighini, M., Tuomanen, E., Rappuoli, R., FALKOW, S. 1989; 86 (8): 2637-2641

    Abstract

    Filamentous hemagglutinin is a surface-associated adherence protein of Bordetella pertussis, which is a component of some new acellular pertussis vaccines. The nucleotide sequence of an open reading frame that encompasses the filamentous hemagglutinin structural gene, fhaB, suggests that proteolytic processing is necessary to generate the mature 220-kDa filamentous hemagglutinin product. An Arg-Gly-Asp (RGD) tripeptide is found within filamentous hemagglutinin that may be involved in its adherence properties. An internal in-frame deletion in fhaB, encompassing the RGD region, causes loss of B. pertussis-binding to ciliated eukaryotic cells, confirming a potential role for this protein in host-cell binding and infection.

    View details for Web of Science ID A1989U231400025

    View details for PubMedID 2539596

  • PHASE VARIATION IN BORDETELLA-PERTUSSIS BY FRAMESHIFT MUTATION IN A GENE FOR A NOVEL 2-COMPONENT SYSTEM NATURE Stibitz, S., AARONSON, W., Monack, D., FALKOW, S. 1989; 338 (6212): 266-269

    Abstract

    Bordetella pertussis, the aetiological agent of whooping cough, coordinately regulates the expression of many virulence-associated determinants, including filamentous haemagglutinin, pertussis toxin, adenylyl cyclase toxin, dermonecrotic toxin and haemolysin. The coordinate regulation is apparent in the repression of synthesis of these determinants in response to environmental stimuli; a phenomenon known as antigenic or phenotypic modulation. B. pertussis also varies between metastable genetic states, or phases. There is a virulent phase in which virulence-associated determinants are synthesized, and an avirulent phase in which they are not. Previous studies have shown that a genetic locus, vir, is required for expression from many virulence-associated loci, and that replacing the cloned vir locus in trans can restore the virulent phase phenotype to spontaneously occurring avirulent phase strains. Here, we show that phase variation in one series of strains is due to a frameshift mutation within an open reading frame that is predicted to code for a Vir protein product. The deduced protein sequence is similar to both components of the 'two-component' regulatory system which control gene expression in response to environmental stimuli in a range of bacterial species.

    View details for Web of Science ID A1989T690600065

    View details for PubMedID 2537932

  • COORDINATE REGULATION AND SENSORY TRANSDUCTION IN THE CONTROL OF BACTERIAL VIRULENCE SCIENCE Miller, J. F., Mekalanos, J. J., FALKOW, S. 1989; 243 (4893): 916-922

    Abstract

    Genes and operons that encode bacterial virulence factors are often subject to coordinate regulation. These regulatory systems are capable of responding to various environmental signals that may be encountered during the infectious cycle. For some pathogens, proteins that mediate sensory transduction and virulence control are similar to components of other bacterial information processing systems. Understanding the molecular mechanisms governing global regulation of pathogenicity is essential for understanding bacterial infectious diseases.

    View details for Web of Science ID A1989T264500031

    View details for PubMedID 2537530

  • EPITHELIAL-CELL SURFACES INDUCE SALMONELLA PROTEINS REQUIRED FOR BACTERIAL ADHERENCE AND INVASION SCIENCE Finlay, B. B., Heffron, F., FALKOW, S. 1989; 243 (4893): 940-943

    Abstract

    Salmonella bacteria are capable of entering (invading) and multiplying within eukaryotic cells. Stable adherence to and invasion of epithelial cells by S. choleraesuis and S. typhimurium were found to require de novo synthesis of several new bacterial proteins. This inducible event appears to be a coordinately regulated system dependent on trypsin- and neuraminidase-sensitive structures present on the epithelial cell surface. Mutants of S. choleraesuis and S. typhimurium were unable to synthesize these proteins and did not stably adhere to nor invade eukaryotic cells. Two such S. typhimurium mutants were avirulent in mice, an indication that these proteins are required for Salmonella virulence.

    View details for Web of Science ID A1989T264500038

    View details for PubMedID 2919285

  • EXPRESSION OF PERTUSSIS TOXIN CORRELATES WITH PATHOGENESIS IN BORDETELLA SPECIES JOURNAL OF INFECTIOUS DISEASES Monack, D., Munoz, J. J., Peacock, M. G., BLACK, W. J., FALKOW, S. 1989; 159 (2): 205-210

    Abstract

    Pertussis toxin is a principal determinant of virulence produced by Bordetella pertussis in the disease whooping cough and is the primary toxinogenic component of the pertussis vaccine. This toxin is not produced by the closely related species Bordetella parapertussis. Toxinogenic strains of B. parapertussis were constructed by the conjugative introduction of cloned pertussis toxin genes. These and analogous toxinogenic and nontoxinogenic strains of B. pertussis were assayed for their toxicity and reactogenic activities. Expression of active toxin by either Bordetella sp. correlated with the induction of leukocytosis, anaphylaxis, and histamine sensitivity.

    View details for Web of Science ID A1989R947300005

    View details for PubMedID 2464653

  • THE AIL LOCUS IS FOUND UNIQUELY IN YERSINIA-ENTEROCOLITICA SEROTYPES COMMONLY ASSOCIATED WITH DISEASE INFECTION AND IMMUNITY MILLER, V. L., Farmer, J. J., Hill, W. E., FALKOW, S. 1989; 57 (1): 121-131

    Abstract

    Yersinia enterocolitica is a heterogeneous group of organisms with more than 50 serotypes and several biotypes. Only a few of these serotypes cause gastrointestinal disease in otherwise healthy hosts; these serotypes are the pathogenic serotypes. Although Y. enterocolitica requires a high-molecular-weight plasmid to cause disease, chromosome-encoded determinants are required for the full expression of virulence. The ability of Yersinia spp. to invade eucaryotic cells is thought to be a virulence factor, because nonpathogenic serotypes are noninvasive in animals and in tissue culture cell models. Current evidence indicates that invasion ability is chromosome encoded. We recently reported cloning two loci, inv and ail, from Y. enterocolitica O8 strain 8081c that allow Escherichia coli to invade tissue culture cells. We investigated the link between invasion in an in vitro tissue culture invasion (TCI) model and hybridization to probes derived from the two invasion loci, inv and ail. We examined 177 Yersinia strains. Strains of serotypes and species associated with disease were TCI+, whereas strains of serotypes and species not associated with disease were TCI-. Only TCI+ strains had DNA homologous to probes derived from ail. All strains (TCI+ and TCI-) had DNA homologous to probes derived from inv, but there were certain restriction fragment-linked polymorphisms that were associated primarily with TCI+ strains. These observations held true for strains epidemiologically associated with disease. Both the inv and ail loci were found to be clearly located on the chromosome. No other genera, including other invasive organisms, had DNA homologous to inv or ail. These data support the hypothesis that the ail locus encodes a Y. enterocolitica invasion factor that may be involved in pathogenesis.

    View details for Web of Science ID A1989R447200020

    View details for PubMedID 2642465

  • PASSAGE OF SALMONELLA THROUGH POLARIZED EPITHELIAL-CELLS - ROLE OF THE HOST AND BACTERIUM JOURNAL OF CELL SCIENCE Finlay, B. B., Fry, J., ROCK, E. P., FALKOW, S. 1989: 99-107

    Abstract

    Salmonella are intracellular parasites which enter their hosts by penetrating the intestinal epithelial barrier. We examined the interaction of S. choleraesuis and S. typhimurium with Madin Darby canine kidney (MDCK) and human larynx (HEp-2) epithelial cells to characterize bacterial adherence, invasion and penetration through epithelial monolayers. Epithelial cell microfilaments were required for bacterial internalization and surrounded the bacteria as they were internalized. The bacteria entered membrane-bound vacuoles inside epithelial cells where they replicated. When polarized MDCK cell monolayers were infected, we found that Salmonella could pass through this barrier and enter medium bathing the opposite surface, although most bacteria remained within the monolayer. Synthesis of several Salmonella proteins was induced by the presence of epithelial cell surfaces, and these proteins were required for bacterial adherence and invasion. This induction was stimulated by trypsin- and neuraminidase-sensitive structures on epithelial cells.

    View details for Web of Science ID A1989AK51300009

    View details for PubMedID 2693464

  • IDENTIFICATION OF A YERSINIA-PESTIS-SPECIFIC DNA PROBE WITH POTENTIAL FOR USE IN PLAGUE SURVEILLANCE JOURNAL OF CLINICAL MICROBIOLOGY McDonough, K. A., Schwan, T. G., Thomas, R. E., FALKOW, S. 1988; 26 (12): 2515-2519

    Abstract

    A 900-base-pair DNA fragment derived from a 9.5-kilobase plasmid in Yersinia pestis hybridized specifically with Y. pestis DNA. We demonstrated the feasibility of using this DNA fragment to detect plague bacilli directly in fleas, suggesting that this Y. pestis-specific DNA probe may be used for plague surveillance in the field. Additional applications for this DNA probe may include plague diagnosis and pathogenesis research.

    View details for Web of Science ID A1988R033900010

    View details for PubMedID 3230131

  • IDENTIFICATION AND CHARACTERIZATION OF TNPHOA MUTANTS OF SALMONELLA THAT ARE UNABLE TO PASS THROUGH A POLARIZED MDCK EPITHELIAL-CELL MONOLAYER MOLECULAR MICROBIOLOGY Finlay, B. B., Starnbach, M. N., Francis, C. L., STOCKER, B. A., Chatfield, S., Dougan, G., FALKOW, S. 1988; 2 (6): 757-766

    Abstract

    Surface protein mutants of the invasive Salmonella species, S. choleraesuis, were generated using the transposon TnphoA. 626 alkaline phosphatase (PhoA+) fusion mutants were identified and screened for their ability to pass through (transcytose) polarized epithelial monolayers of Madin Darby canine kidney (MDCK) cells grown on membrane filters. Forty two mutants were unable to pass through this barrier. All of these transcytosis mutants were unable to adhere to or invade MDCK monolayers, yet these mutations were not in the genes encoding type 1 pili or mannose-resistant haemagglutination (MRHA). These transcytosis mutants could be grouped into six classes. Class 1 mutants had altered lipopolysaccharide (LPS) O side-chain structures while Class 2 mutants had defects in their LPS core. Mutants belonging to Classes 5 and 6 did not decrease the transepithelial electrical resistance of polarized MDCK cell monolayers, in contrast to the parental strain and the other mutants (Classes 1, 2, 3 and 4). Mutants belonging to Class 1 were less virulent in mice, while Class 2 (defective core) and Classes 4 and 5 (normal LPS) mutant strains were avirulent in mice. Mutants from Classes 3 and 6 were as virulent in mice as S. choleraesuis. These results suggest that the ability to pass through epithelial barriers may be an important virulence characteristic of Salmonella. These data indicate that bacterial adherence, internalization and monolayer transcytosis are closely linked events. It was also demonstrated that a mutant with decreased rates of intracellular replication still passed through the monolayer at rates similar to wild-type S. choleraesuis.

    View details for Web of Science ID A1988Q831000009

    View details for PubMedID 2850443

  • VIRULENCE FACTORS ASSOCIATED WITH SALMONELLA SPECIES MICROBIOLOGICAL SCIENCES Finlay, B. B., FALKOW, S. 1988; 5 (11): 324-328

    Abstract

    Salmonella species can cause diseases ranging in severity from acute gastroenteritis to typhoid fever. These bacteria are considered as intracellular pathogens and produce several products which are required for entry and survival in the intracellular environment, in addition to factors necessary for existence in the gastrointestinal tract and the outside environment. The virulence factors used by these bacteria to cause these diseases are complex, and only recently have we begun to characterize these factors and determine the contribution they make to Salmonella virulence.

    View details for Web of Science ID A1988R163900001

    View details for PubMedID 3079173

  • PURIFICATION, CHARACTERIZATION, AND PATHOGENICITY OF MORAXELLA-BOVIS PILI JOURNAL OF EXPERIMENTAL MEDICINE Ruehl, W. W., Marrs, C. F., Fernandez, R., FALKOW, S., SCHOOLNIK, G. K. 1988; 168 (3): 983-1002

    Abstract

    Pilins composed of the alpha or beta pilins of Moraxella bovis strain Epp63 were purified, subjected to chemical or enzymatic cleavage, and the resulting fragments sequenced by automated Edman degradation. alpha Pilin was found to be a 155-amino-acid polypeptide with a single intramolecular disulfide bridge. The beta pilin amino acid sequence substantiated the previously reported structure derived from the beta pilin gene DNA sequence, and indicated that the alpha and beta pilins of this strain are approximately 70% homologous. DNA hybridization studies of genomic DNA from the alpha- and beta-piliated variants of strain Epp63 indicated that the expression of the two pilin types was governed by an oscillating mechanism of chromosomal rearrangement. The alpha and beta pili were evaluated serologically and found to exhibit approximately 50% shared antigenicity, indicating that regions of conserved and heterologous sequence specify both type-specific and crossreacting epitopes. The pathogenicity of the alpha- and beta-piliated variants was studied by ocular inoculation of calves eyes; beta-piliated organisms were significantly more infectious than alpha-piliated organisms, indicating that beta pili confer, or are associated with, a relative advantage during the first stages of ocular infection. Preliminary analysis of other M. bovis strains suggests that each strain produces two types of pilin, and that this property may be characteristic of the species.

    View details for Web of Science ID A1988Q316200013

    View details for PubMedID 2902184

  • COMPARISON OF THE INVASION STRATEGIES USED BY SALMONELLA-CHOLERAE-SUIS, SHIGELLA-FLEXNERI AND YERSINIA-ENTEROCOLITICA TO ENTER CULTURED ANIMAL-CELLS - ENDOSOME ACIDIFICATION IS NOT REQUIRED FOR BACTERIAL INVASION OR INTRACELLULAR REPLICATION BIOCHIMIE Finlay, B. B., FALKOW, S. 1988; 70 (8): 1089-1099

    Abstract

    Strains of Escherichia, Salmonella, Shigella and Yersinia actively enter eukaryotic cells. Several techniques were used to compare and contrast the invasion mechanisms of Salmonella cholerae-suis, Yersinia enterocolitica and Shigella flexneri. Three animal cell lines (CHO, HEp-2 and MDCK) were examined for susceptibility to bacterial entry by these strains. Levels of intracellular bacteria varied widely between cell lines, but CHO cells were the most susceptible to bacterial invasion, HEp-2 invasion levels were intermediary, whereas polarized MDCK cells were invaded to a lesser extent. This illustrates that tissue culture models can be optimized to study bacterial invasion and intracellular replication. We used these tissue culture models to examine the interactions between host cells and these invasive bacteria. The use of lysosomotropic agents (methylamine and ammonium chloride), cationic ionophores (monensin) and acidification-defective CHO cell lines demonstrated that endosome acidification is not required for bacterial invasion or intracellular replication. Drugs which inhibited microfilament formation (cytochalasins B and D) prevented internalization of S. cholerae-suis, Y. enterocolitica and S. flexneri, indicating that invasion is a microfilament-dependent event. The microtubule inhibitors, colchicine, vincristine and vinblastine, did not affect bacterial internalization.

    View details for Web of Science ID A1988Q626900010

    View details for PubMedID 3147700

  • PILIN GENE PHASE VARIATION OF MORAXELLA-BOVIS IS CAUSED BY AN INVERSION OF THE PILIN GENES JOURNAL OF BACTERIOLOGY Marrs, C. F., Ruehl, W. W., SCHOOLNIK, G. K., FALKOW, S. 1988; 170 (7): 3032-3039

    Abstract

    Moraxella bovis Epp63 can express either of two different pilin proteins, called alpha and beta. We have previously cloned and sequenced the beta-pilin gene and now report that DNAs isolated from bacteria expressing alpha pilin have hybridization patterns consistently different from those of bacteria expressing beta pilin. The phase variation between alpha- and beta-pilin gene expression appears to be associated with an inversion of about 2 kilobases of DNA, whose endpoints occur within the coding region of the expressed pilin gene. Comparisons of the beta-pilin gene sequence with those of well-studied bacterial inversion systems revealed a stretch of 58% sequence similarity (21 of 36 base pairs) between the left inverted repeat of the Salmonella typhimurium flagellar hin control region and the amino-terminal portion of the beta-pilin gene.

    View details for Web of Science ID A1988P022600020

    View details for PubMedID 2898471

  • PENETRATION OF SALMONELLA THROUGH A POLARIZED MADIN-DARBY CANINE KIDNEY EPITHELIAL-CELL MONOLAYER JOURNAL OF CELL BIOLOGY Finlay, B. B., Gumbiner, B., FALKOW, S. 1988; 107 (1): 221-230

    Abstract

    Many intracellular parasites are capable of penetrating host epithelial barriers. To study this process in more detail we examined the interactions between the pathogenic bacteria Salmonella choleraesuis and polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells grown on membrane filters. Association of bacteria with the MDCK cell apical surface was an active event, requiring bacterial RNA and protein synthesis, and was blocked by low temperatures. Salmonella were internalized within a membrane-bound vacuole and exhibited penetration through, but not between MDCK cells. A maximum of 14 Salmonella per MDCK cell crossed the monolayer per hour to the basolateral surface yet the monolayer remained viable and impermeable to Escherichia coli. Apical S. choleraesuis infection resulted in an increase in paracellular permeability but the MDCK intercellular contacts were not significantly disrupted. Basolateral S. choleraesuis infection was inefficient, and only small numbers of S. choleraesuis penetrated to the apical medium.

    View details for Web of Science ID A1988P399500021

    View details for PubMedID 3292541

  • GENETIC-ANALYSIS OF A REGION OF THE BORDETELLA-PERTUSSIS CHROMOSOME ENCODING FILAMENTOUS HEMAGGLUTININ AND THE PLEIOTROPIC REGULATORY LOCUS VIR JOURNAL OF BACTERIOLOGY Stibitz, S., Weiss, A. A., FALKOW, S. 1988; 170 (7): 2904-2913

    Abstract

    The vir locus of Bordetella pertussis apparently encodes a trans-acting positive regulator that is required for the coordinate expression of genes associated with virulence: pertussis toxin, filamentous hemagglutinin (FHA), hemolysin, and adenylate cyclase toxin. DNA clones of vir and of genes required for the synthesis of some of the factors under vir control were obtained with DNA probes from the chromosomal DNA surrounding sites of Tn5 insertion mutations that inactivated those genes. Two vir clones were found which also contained genes required for the proper expression of FHA in B. pertussis. The plasmids which contained both the fha and vir genes expressed immunologically reactive FHA in Escherichia coli, as detected by colony blots, whereas plasmids which contained only fha or vir were negative in this assay. The regulation of FHA production in E. coli, as in B. pertussis, was temperature dependent and inhibited by high concentrations of either magnesium ions or nicotinic acid, indicating that the sequences cloned in E. coli contained the information required to preserve the physiological responses seen in B. pertussis. Further characterization of the vir-fha clones by Tn5 mutagenesis in E. coli and by the return of cloned sequences to B. pertussis in trans and to the B. pertussis chromosome led to the localization of the vir locus, the structural gene for FHA, and genes that are possibly required for the synthesis and export of FHA.

    View details for Web of Science ID A1988P022600003

    View details for PubMedID 2898470

  • MOLECULAR KOCHS POSTULATES APPLIED TO MICROBIAL PATHOGENICITY REVIEWS OF INFECTIOUS DISEASES FALKOW, S. 1988; 10: S274-S276

    Abstract

    Microbial genetics and molecular cloning now permit us to routinely isolate specific genes from a variety of microbial pathogens. Obviously not all genes from pathogenic microorganisms play a role in pathogenicity or virulence. Just as Koch's postulates were formulated to identify the causal relationship between an organism and a specific disease, the notion is presented here that a form of molecular Koch's postulates is needed when examining the potential role of genes and their products in the pathogenesis of infection and disease.

    View details for Web of Science ID A1988P836900002

    View details for PubMedID 3055197

  • MODEL FOR INVASION OF HUMAN-TISSUE CULTURE CELLS BY NEISSERIA-GONORRHOEAE INFECTION AND IMMUNITY Shaw, J. H., FALKOW, S. 1988; 56 (6): 1625-1632

    Abstract

    A tissue culture model has been developed for studying the ability of Neisseria gonorrhoeae to invade eucaryotic cells. The cell line HecIB, a human adenocarcinoma endometrial cell line, was found to support gonococcal invasion. The bactericidal antibiotic gentamicin was used to kill those bacteria that had not entered the HecIB cells, allowing us to quantitate internalized bacteria. Kinetic studies showed an increase in the titer of gentamicin-protected gonococci at 4 h postinfection followed by a decrease; a second increase occurred after 6 h. The state of piliation did not affect the degree of invasion when the bacteria were spun down onto the monolayer. Gonococcal invasion was inhibited when the HecIB cells were preincubated with cytochalasin D before bacterial infection. N. lactamica was used as a negative control. No internalized N. lactamica cells were observed by electron microscopy. Electron microscopy documented the intracellular location of the gonococci in HecIB cells and the eventual destruction of the invaded HecIB cells. After 24 h, clusters of gonococci encased in a matrix of cell debris were observed.

    View details for Web of Science ID A1988N527300035

    View details for PubMedID 3131248

  • EVIDENCE FOR 2 GENETIC-LOCI IN YERSINIA-ENTEROCOLITICA THAT CAN PROMOTE INVASION OF EPITHELIAL-CELLS INFECTION AND IMMUNITY MILLER, V. L., FALKOW, S. 1988; 56 (5): 1242-1248

    Abstract

    Virulent strains of Yersinia enterocolitica cause disease syndromes ranging from mild gastroenteritis to lymphadenitis and septicemia. The ability of these bacteria to invade intestinal epithelial cells to gain access to the reticuloendothelial system is thought to be an important aspect of their virulence. We report here on the cloning of two Y. enterocolitica chromosomal loci, inv and ail, each of which confers an invasive phenotype on Escherichia coli HB101. The inv locus allows a uniformly high level of invasion in several tissue culture lines and is homologous to the inv gene of Yersinia pseudotuberculosis. The second locus, ail, shows more host specificity than inv in that it allows invasion to a variable degree of some cell lines (e.g., HEp-2, HEC1B, and CHO cells) but allows no invasion of others (e.g., Madin-Darby canine kidney cells).

    View details for Web of Science ID A1988N021200037

    View details for PubMedID 2833444

  • ADP-RIBOSYLTRANSFERASE ACTIVITY OF PERTUSSIS TOXIN AND IMMUNOMODULATION BY BORDETELLA-PERTUSSIS SCIENCE BLACK, W. J., Munoz, J. J., Peacock, M. G., Schad, P. A., COWELL, J. L., BURCHALL, J. J., Lim, M., Kent, A., Steinman, L., FALKOW, S. 1988; 240 (4852): 656-659

    Abstract

    Pertussis toxin is produced by the causative agent of whooping cough, Bordetella pertussis, and is an adenosine diphosphate (ADP)-ribosyltransferase capable of covalently modifying and thereby inactivating many eukaryotic G proteins involved in cellular metabolism. The toxin is a principal determinant of virulence in whooping cough and is a primary candidate for an acellular pertussis vaccine, yet it is unclear whether the ADP-ribosyltransferase activity is required for both pathogenic and immunoprotective activities. A B. pertussis strain that produced an assembled pertussis holotoxin with only 1 percent of the ADP-ribosyltransferase activity of the native toxin was constructed and was found to be deficient in pathogenic activities associated with B. pertussis including induction of leukocytosis, potentiation of anaphylaxis, and stimulation of histamine sensitivity. Moreover, this mutant strain failed to function as an adjuvant and was less effective in protecting mice from intracerebral challenge infection. These data suggest that the ADP-ribosyltransferase activity is necessary for both pathogenicity and optimum immunoprotection. These findings bear directly on the design of a nontoxic pertussis vaccine.

    View details for Web of Science ID A1988N126500034

    View details for PubMedID 2896387

  • GENETIC-ANALYSIS OF AN ESCHERICHIA-COLI UREASE LOCUS - EVIDENCE OF DNA REARRANGEMENT JOURNAL OF BACTERIOLOGY Collins, C. M., FALKOW, S. 1988; 170 (3): 1041-1045

    Abstract

    Ureolytic Escherichia coli strains are uncommon clinical isolates. The urease phenotype in a large percentage of these isolates is unstable and lost upon storage. We examined two urease-positive uropathogenic E. coli isolates that give off urease-negative segregants and determined that the urease phenotype was chromosomally encoded. The urease phenotype was cloned from E. coli 1021 and found to be encoded on a 9.4-kilobase HindIII restriction fragment. Transposon mutagenesis indicated that at least 3.2 kilobases of this fragment were necessary for production of urease. The urease recombinant plasmid pURE coded for at least four insert-specific polypeptides as determined by maxicell analysis. Disruption of the region encoding two of these polypeptides (67 and 27 kilodaltons) abolished urease activity. Analysis by Southern hybridization of urease-positive E. coli 1021 and seven independently isolated urease-negative segregants showed that a DNA rearrangement was associated with the urease-negative phenotype.

    View details for Web of Science ID A1988M260100003

    View details for PubMedID 3277942

  • IDENTIFICATION OF REGIONS ON A 230-KILOBASE PLASMID FROM ENTEROINVASIVE ESCHERICHIA-COLI THAT ARE REQUIRED FOR ENTRY INTO HEP-2 CELLS INFECTION AND IMMUNITY Small, P. L., FALKOW, S. 1988; 56 (1): 225-229

    Abstract

    Certain strains of Escherichia coli can cause an invasive diarrheal disease in humans which clinically resembles shigellosis. These strains share with Shigella species the ability to enter and replicate within colonic epithelial cells and the ability to bind Congo red dye in vitro when grown at 37 degrees C. Like shigellae, they contain a large plasmid essential for virulence. A 230-kilobase (kb) plasmid from enteroinvasive E. coli was genetically marked with a transposon and mobilized into an E. coli K-12 background. This plasmid conferred upon E. coli K-12 the ability to enter and multiply within cultured epithelial cells, as well as the ability to bind Congo red. Expression of these phenotypes required growth at 37 degrees C. Transposon mutagenesis was used to identify regions on the 230-kb plasmid required for virulence. All transposon insertions which resulted in loss of the ability to enter epithelial cells, as well as the ability to bind Congo red dye, were mapped to a single 25-kb BamHI fragment. Subclones from this 25-kb region were tested for the ability to complement invasion in noninvasive derivatives. A subclone containing about 8 kb of the left end of the 25-kb BamHI fragment was capable of complementing noninvasive mutants with Tn5 insertions in this region and restored to these noninvasive mutants the ability to enter epithelial cells.

    View details for Web of Science ID A1988L437400038

    View details for PubMedID 2826335

  • Using knowledge of virulence factors to select or design organisms with low risk of pathogenicity. Basic life sciences FALKOW, S. 1988; 45: 121-126

    View details for PubMedID 3178634

  • The vir locus and phase-variation in Bordetella pertussis. The Tokai journal of experimental and clinical medicine Stibitz, S., AARONSON, W., Monack, D., FALKOW, S. 1988; 13: 223-226

    Abstract

    By a phenomenon known as phase-variation Bordetella pertussis is capable of changing between a virulent-phase in which multiple virulence-associated determinants are expressed, and an avirulent-phase in which the virulence-associated determinants are not expressed. Mutations in the vir locus of B. pertussis have a similar effect. We have examined the state of the vir locus in each of a series of strains derived one from the other by phase-variation. We have found that a single base-pair change is associated with the change between the virulent and avirulent phases. This single base-pair change corresponds to a frameshift mutation in the vir locus.

    View details for PubMedID 2908523

  • CONSTRUCTION AND CHARACTERIZATION OF BORDETELLA-PERTUSSIS TOXIN MUTANTS INFECTION AND IMMUNITY BLACK, W. J., FALKOW, S. 1987; 55 (10): 2465-2470

    Abstract

    Pertussis toxin is one of the major virulence determinants produced by Bordetella pertussis. The DNA encoding the structural genes for pertussis toxin was cloned in Escherichia coli, and pertussis toxin subunit S4 was expressed under the control of the tac promoter. Mutations were introduced into the cloned toxin genes, and a conjugative shuttle vector system was devised for delivering the mutations from E. coli back into B. pertussis. The mutations were introduced by allelic exchange into the chromosome of B. pertussis resulting in a series of B. pertussis strains which were isogenic except at the loci encoding the structural genes for pertussis toxin. These B. pertussis strains were utilized to study the biogenesis of pertussis toxin. Polar mutations in the S1 gene led to a lack of detectable S2 or S4 subunits in whole-cell lysates, suggesting a polycistronic arrangement for these genes. Mutations in the S5 subunit gene resulted in a truncated S1 subunit, while mutations in the S4 gene resulted in a lack of detectable S2 subunit, suggesting that physical relationships among the toxin subunits are directly reflected in the stable biogenesis of the subunits.

    View details for Web of Science ID A1987K095600024

    View details for PubMedID 2888733

  • A MOLECULAR STRATEGY FOR THE STUDY OF BACTERIAL INVASION REVIEWS OF INFECTIOUS DISEASES FALKOW, S., Small, P., Isberg, R., Hayes, S. F., Corwin, D. 1987; 9: S450-S455

    Abstract

    Bacterial populations are often clonal, and even within a bacterial species, the frequency of gene exchange and recombination is quite low. Consequently, mobile genetic elements--plasmids, bacteriophages, and transposons--have been the central factors in the evolution of pathogenic traits. One central feature of pathogenicity, the capacity to enter epithelial cells, is encoded by the bacterial chromosome of Yersinia pseudotuberculosis but by plasmid genes in enteroinvasive Escherichia coli. A single Yersinia gene, inv, which encodes a single 107,000-dalton protein, can be cloned in E. coli K12, and its presence is sufficient to permit the bacteria to enter cultured human cells. In contrast, fully 70 kilobases of a virulence plasmid from enteroinvasive E. coli must be transferred to E. coli K12 to achieve the same result. While researchers are at the early stages of understanding microbial entry into host cells, they can now investigate the molecular basis of this event in greater detail than has previously been possible.

    View details for Web of Science ID A1987K144000005

    View details for PubMedID 2825322

  • Uropathogenic Escherichia coli: molecular mechanisms of adherence. Advances in experimental medicine and biology SCHOOLNIK, G. K., O'Hanley, P., LARK, D., Normark, S., Vosti, K., FALKOW, S. 1987; 224: 53-62

    View details for PubMedID 3329813

  • DEVELOPMENT OF A TISSUE-CULTURE MODEL FOR GONOCOCCAL INVASION ANTONIE VAN LEEUWENHOEK JOURNAL OF MICROBIOLOGY Shaw, J. H., Hayes, F., Brooks, G. F., FALKOW, S. 1987; 53 (6): 485-491

    Abstract

    Neisseria gonorrhoeae invasion of the human endometrial cell line HecIB was monitored by electron microscopy. Within six hours postinfection, the gonococci have attached to the surface of some HecIB cells and are embraced by microvilli. Gonococci subsequently enter the HecIB cells in membrane bound vesicles but by eight hours, gonococci can be seen free in the cytoplasm. At twelve hours post-infection some HecIB cells are observed containing hundreds of internalized bacteria. At twenty-four hours gonococci appear in large clusters embedded in a matrix of cellular debris, which are possibly the remains of lysed infected cells. In contrast, N. lactamica is adherent to the monolayer but noninvasive.

    View details for Web of Science ID A1987M330400019

    View details for PubMedID 3130786

  • GENETIC-ANALYSIS OF CELL INVASIVENESS BY YERSINIA-PSEUDOTUBERCULOSIS ANNALES DE L INSTITUT PASTEUR-MICROBIOLOGIE ISBERG, R. R., FALKOW, S. 1986; 137A (3): 308-310

    View details for Web of Science ID A1986D036900011

    View details for PubMedID 3322173

  • MOLECULAR-BASIS OF ESCHERICHIA-COLI COLONIZATION OF THE UPPER URINARY-TRACT IN BALB/C MICE - GAL-GAL PILI IMMUNIZATION PREVENTS ESCHERICHIA-COLI PYELONEPHRITIS IN THE BALB/C MOUSE MODEL OF HUMAN PYELONEPHRITIS JOURNAL OF CLINICAL INVESTIGATION OHANLEY, P., LARK, D., FALKOW, S., SCHOOLNIK, G. 1985; 75 (2): 347-360

    Abstract

    Most human pyelonephritis Escherichia coli isolates express both mannose (MS)- and globoside (Gal-Gal)-binding pili. An ascending E. coli urinary tract infection model was established in the 16-wk-old female BALB/c mouse to compare the pathogenic significance of MS and Gal-Gal pili and their efficacy as vaccines for the prevention of pyelonephritis. The distribution and density of pilus receptor compounds in urogenital tissues and as soluble compounds in urine were determined with antibodies to the synthetic receptor analogues, alpha D-Gal(1----4) beta D-Gal and alpha D-Man(1----2) alpha D-Man. Both carbohydrates were detected in vagina, bladder, ureter, and renal pelvis epithelium and in collecting duct and tubular cells. A pilus receptor compound also was detected in urine. It competitively inhibited the binding capacity of MS pili and was found to be physically, chemically, and immunologically related to Tamm-Horsfall uromucoid. Infectivity and invasiveness were quantitatively and histologically characterized for four E. coli strains: J96, a human pyelonephritis strain that expresses both MS and Gal-Gal pili; two recombinant strains prepared from J96 chromosomal DNA encoding MS pili or Gal-Gal pili; and the nonpiliated K12 recipient. Intravesicular administration of J96 (10(6) colony-forming units [CFU]) resulted in renal colonization and invasion in each of nine mice. The Gal-Gal clone (10(6) CFU) colonized the kidneys in each of 10 mice but did not invade. In contrast, the MS clone (10(6) CFU) did not colonize renal epithelium or invade. This effect was superceded when larger doses (greater than or equal to 10(10) CFU) of the MS clone were administered in volumes that cause acute vesicoureteric reflux. The efficacy was determined of vaccines composed of pure MS or Gal-Gal pili or the lipopolysaccharide containing O somatic antigen of the challenge strain, J96. The Gal-Gal pilus vaccine blocked renal colonization in 19 of 22 mice and renal invasion in 10 of 11 mice. Gal-Gal pili may be useful immunogens for the prevention of pyelonephritis in anatomically normal urinary tracts.

    View details for Web of Science ID A1985ACA2500004

    View details for PubMedID 2857730

  • MANNOSE-SENSITIVE AND GAL-GAL BINDING ESCHERICHIA-COLI PILI FROM RECOMBINANT STRAINS - CHEMICAL, FUNCTIONAL, AND SEROLOGICAL PROPERTIES JOURNAL OF EXPERIMENTAL MEDICINE OHANLEY, P., LARK, D., Normark, S., FALKOW, S., SCHOOLNIK, G. K. 1983; 158 (5): 1713-1719

    Abstract

    Chromosomal genes encoding the MS and Gal-Gal binding properties have been cloned into separate recombinants and their respective pili characterized. Hapten inhibition of hemagglutination with synthetic carbohydrate receptor analogues and carbohydrate-adsorbed latex agglutination studies indicate that Gal-Gal and MS pili collectively exhibit the binding properties of the parent strain. MS pili migrated in SDS-PAGE with an Mr of 19 kdaltons and 17 kdaltons; the Mr of Gal-Gal pili was 17.5 kdaltons. The pili are chemically similar by amino acid composition and when the N-terminal cysteines are aligned, 8 of the 13 residues between positions 9 and 22 are homologous. Further, carboxy-terminal sequence homology was inferred from the carboxypeptidase digestion of a MS pili and the sequence of a carboxy-terminal tryptic peptide from Gal-Gal pili.

    View details for Web of Science ID A1983RQ28400025

    View details for PubMedID 6195290

Conference Proceedings


  • Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions Valdivia, R. H., Hromockyj, A. E., Monack, D., Ramakrishnan, L., FALKOW, S. ELSEVIER SCIENCE BV. 1996: 47-52

    Abstract

    The green fluorescent protein (GFP) from Aequorea victoria is a novel fluorescent marker that has potential use in the study of bacterial pathogenicity. To explore some of the potential applications of GFP to the study of host-parasite interactions, we constructed two GFP expression vectors suitable for different facultative intracellular bacterial pathogens. The first expression vector was tested in the enteric pathogens, Salmonella typhimurium and Yersinia pseudotuberculosis, and the second vector tested in Mycobacterium marinum (Mm). Both expression vectors were found to be stable and to direct high levels of GFP synthesis. Standard epifluorescence microscopy was used to detect all three bacterial pathogenic species during the early and late stages of infection of live mammalian cells. Mm expressing gfp was also visualized in infected animal tissues. gfp expression did not adversely affect bacterial survival, nor did it compromise entry into mammalian cells or their survival within macrophages. In addition, all three gfp-expressing bacterial pathogens could be detected and sorted in a flow cytometer, either alone or in association with epithelial cells or macrophages. Therefore, GFP not only provides a convenient tool to image pathogenic bacteria, but allows the quantitative measurement of bacterial association with mammalian cells.

    View details for Web of Science ID A1996UY07300008

    View details for PubMedID 8707055

  • FACS-optimized mutants of the green fluorescent protein (GFP) Cormack, B. P., Valdivia, R. H., FALKOW, S. ELSEVIER SCIENCE BV. 1996: 33-38

    Abstract

    We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.

    View details for Web of Science ID A1996UY07300006

    View details for PubMedID 8707053

  • CAPSULE LOSS BY HAEMOPHILUS-INFLUENZAE TYPE-B RESULTS IN ENHANCED ADHERENCE TO AND ENTRY INTO HUMAN-CELLS StGeme, J. W., FALKOW, S. UNIV CHICAGO PRESS. 1992: S117-S118

    View details for Web of Science ID A1992HW74500040

    View details for PubMedID 1350299

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