Gary Schoolnik

All Publications

The competitive cost of antibiotic resistance in Mycobacterium tuberculosis SCIENCE Gagneux, S., Long, C. D., Small, P. M., Van, T., Schoolnik, G. K., Bohannan, B. J. 2006; 312 (5782): 1944-1946
Abstract

Mathematical models predict that the future of the multidrug-resistant tuberculosis epidemic will depend on the fitness cost of drug resistance. We show that in laboratory-derived mutants of Mycobacterium tuberculosis, rifampin resistance is universally associated with a competitive fitness cost and that this cost is determined by the specific resistance mutation and strain genetic background. In contrast, we demonstrate that prolonged patient treatment can result in multidrug-resistant strains with no fitness defect and that strains with low- or no-cost resistance mutations are also the most frequent among clinical isolates.

View details for DOI 10.1126/science.1124410

View details for Web of Science ID 000238848100057

View details for PubMedID 16809538
Expression profiling of host pathogen interactions: How Mycobacterium tuberculosis and the macrophage adapt to one another MICROBES AND INFECTION Schnappinger, D., Schoolnik, G. K., Ehrt, S. 2006; 8 (4): 1132-1140
Abstract

It has recently become feasible to quantify all mRNAs encoded by the genomes of bacterial pathogens and their eukaryotic host cells and to apply this approach to study the interaction of Mycobacterium tuberculosis with its primary host cell, the macrophage. These studies helped to identify regulatory circuits which mediate adaptation of the M. tuberculosis transcriptome to intraphagosomal environments and stimulated hypotheses for the function of these circuits in human tuberculosis. The macrophage transcriptome reacts to infections with the induction of a pathogen-unspecific expression program as well as the induction of pathogen-specific expression signatures, both of which contribute to the immunologic activation of the infected cell. M. tuberculosis induced changes in the macrophage transcriptome are mediated by Toll-like receptor dependent and Toll-like receptor independent signal transduction pathways. This response is shaped by macrophage produced reactive nitrogen and oxygen molecules and affected by viability and virulence of the pathogen.

View details for DOI 10.1016/j.micinf.2005.10.027

View details for Web of Science ID 000237754100021

View details for PubMedID 16517202
Identification and characterization of RbmA, a novel protein required for the development of rugose colony morphology and biofilm structure in Vibrio cholerae JOURNAL OF BACTERIOLOGY Fong, J. C., Karplus, K., SCHOOLNIK, G. K., Yildiz, F. H. 2006; 188 (3): 1049-1059
Abstract

Phase variation between smooth and rugose colony variants of Vibrio cholerae is predicted to be important for the pathogen's survival in its natural aquatic ecosystems. The rugose variant forms corrugated colonies, exhibits increased levels of resistance to osmotic, acid, and oxidative stresses, and has an enhanced capacity to form biofilms. Many of these phenotypes are mediated in part by increased production of an exopolysaccharide termed VPS. In this study, we compared total protein profiles of the smooth and rugose variants using two-dimensional gel electrophoresis and identified one protein that is present at a higher level in the rugose variant. A mutation in the gene encoding this protein, which does not have any known homologs in the protein databases, causes cells to form biofilms that are more fragile and sensitive to sodium dodecyl sulfate than wild-type biofilms. The results indicate that the gene, termed rbmA (rugosity and biofilm structure modulator A), is required for rugose colony formation and biofilm structure integrity in V. cholerae. Transcription of rbmA is positively regulated by the response regulator VpsR but not VpsT.

View details for DOI 10.1128/JB.188.3.1049-1059.2006

View details for Web of Science ID 000234840800025

View details for PubMedID 16428409
Evidence supporting predicted metabolic pathways for Vibrio cholerae: gene expression data and clinical tests NUCLEIC ACIDS RESEARCH Shi, J., Romero, P. R., Schoolnik, G. K., Spormann, A. M., Karp, P. D. 2006; 34 (8): 2438-2444
Abstract

Vibrio cholerae, the etiological agent of the diarrheal illness cholera, can kill an infected adult in 24 h. V.cholerae lives as an autochthonous microbe in estuaries, rivers and coastal waters. A better understanding of its metabolic pathways will assist the development of more effective treatments and will provide a deeper understanding of how this bacterium persists in natural aquatic habitats. Using the completed V.cholerae genome sequence and PathoLogic software, we created VchoCyc, a pathway-genome database that predicted 171 likely metabolic pathways in the bacterium. We report here experimental evidence supporting the computationally predicted pathways. The evidence comes from microarray gene expression studies of V.cholerae in the stools of three cholera patients [D. S. Merrell, S. M. Butler, F. Qadri, N. A. Dolganov, A. Alam, M. B. Cohen, S. B. Calderwood, G. K. Schoolnik and A. Camilli (2002) Nature, 417, 642-645.], from gene expression studies in minimal growth conditions and LB rich medium, and from clinical tests that identify V.cholerae. Expression data provide evidence supporting 92 (53%) of the 171 pathways. The clinical tests provide evidence supporting seven pathways, with six pathways supported by both methods. VchoCyc provides biologists with a useful tool for analyzing this organism's metabolic and genomic information, which could lead to potential insights into new anti-bacterial agents. VchoCyc is available in the BioCyc database collection (http://BioCyc.org).

View details for DOI 10.1093/nar/gkl310

View details for Web of Science ID 000237697000037

View details for PubMedID 16682451
Chitin induces natural competence in Vibrio cholerae SCIENCE Meibom, K. L., Blokesch, M., Dolganov, N. A., Wu, C. Y., Schoolnik, G. K. 2005; 310 (5755): 1824-1827
Abstract

The mosaic-structured Vibrio cholerae genome points to the importance of horizontal gene transfer (HGT) in the evolution of this human pathogen. We showed that V. cholerae can acquire new genetic material by natural transformation during growth on chitin, a biopolymer that is abundant in aquatic habitats (e.g., from crustacean exoskeletons), where it lives as an autochthonous microbe. Transformation competence was found to require a type IV pilus assembly complex, a putative DNA binding protein, and three convergent regulatory cascades, which are activated by chitin, increasing cell density, and nutrient limitation, a decline in growth rate, or stress.

View details for DOI 10.1126/science.1120096

View details for Web of Science ID 000234093600049

View details for PubMedID 16357262
Ancestral antibiotic resistance in Mycobacterium tuberculosis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Morris, R. P., Nguyen, L., Gatfield, J., Visconti, K., Nguyen, K., Schnappinger, D., Ehrt, S., Liu, Y., Heifets, L., Pieters, J., SCHOOLNIK, G., Thompson, C. J. 2005; 102 (34): 12200-12205
Abstract

Chemotherapeutic options to treat tuberculosis are severely restricted by the intrinsic resistance of Mycobacterium tuberculosis to the majority of clinically applied antibiotics. Such resistance is partially provided by the low permeability of their unique cell envelope. Here we describe a complementary system that coordinates resistance to drugs that have penetrated the envelope, allowing mycobacteria to tolerate diverse classes of antibiotics that inhibit cytoplasmic targets. This system depends on whiB7, a gene that pathogenic Mycobacterium shares with Streptomyces, a phylogenetically related genus known as the source of diverse antibiotics. In M. tuberculosis, whiB7 is induced by subinhibitory concentrations of antibiotics (erythromycin, tetracycline, and streptomycin) and whiB7 null mutants (Streptomyces and Mycobacterium) are hypersusceptible to antibiotics in vitro. M. tuberculosis is also antibiotic sensitive within a monocyte model system. In addition to antibiotics, whiB7 is induced by exposure to fatty acids that pathogenic Mycobacterium species may accumulate internally or encounter within eukaryotic hosts during infection. Gene expression profiling analyses demonstrate that whiB7 transcription determines drug resistance by activating expression of a regulon including genes involved in ribosomal protection and antibiotic efflux. Components of the whiB7 system may serve as attractive targets for the identification of inhibitors that render M. tuberculosis or multidrug-resistant derivatives more antibiotic-sensitive.

View details for DOI 10.1073/pnas.0505446102

View details for Web of Science ID 000231476500044

View details for PubMedID 16103351
Molecular analysis of rugosity in a Vibrio cholerae O1 El Tor phase variant MOLECULAR MICROBIOLOGY Yildiz, F. H., Liu, X. S., Heydorn, A., SCHOOLNIK, G. K. 2004; 53 (2): 497-515
Abstract

Reversible phase variation between the rugose and smooth colony variants is predicted to be important for the survival of Vibrio cholerae in natural aquatic habitats. Microarray expression profiling studies of the rugose and smooth variants of the same strain led to the identification of 124 differentially regulated genes. Further expression profiling experiments showed how these genes are regulated by the VpsR and HapR transcription factors, which, respectively, positively and negatively regulate production of VPS(El Tor), a rugose-associated extracellular polysaccharide. The study of mutants of rpoN and rpoS demonstrated the effects of these alternative sigma factors on phase variation-specific gene expression. Bioinformatics analysis of these expression data shows that 'rugosity' and 'smoothness' are determined by a complex hierarchy of positive and negative regulators, which also affect the biofilm, surface hydrophobicity and motility phenotypes of the organism.

View details for DOI 10.1111/j.1365-2958.2004.04154.x

View details for Web of Science ID 000222423000014

View details for PubMedID 15228530
Phage offer a real alternative NATURE BIOTECHNOLOGY Schoolnik, G. K., Summers, W. C., D Watson, J. 2004; 22 (5): 505-506
View details for DOI 10.1038/nbt0504-505

View details for Web of Science ID 000221159700008

View details for PubMedID 15122279
The Vibrio cholerae chitin utilization program PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Meibom, K. L., Li, X. B., Nielsen, A. T., Wu, C. Y., Roseman, S., Schoolnik, G. K. 2004; 101 (8): 2524-2529
Abstract

Chitin, an insoluble polymer of GlcNAc, is an abundant source of carbon, nitrogen, and energy for marine microorganisms. Microarray expression profiling and mutational studies of Vibrio cholerae growing on a natural chitin surface, or with the soluble chitin oligosaccharides (GlcNAc)(2-6), GlcNAc, or the glucosamine dimer (GlcN)2 identified three sets of differentially regulated genes. We show that (i) ChiS, a sensor histidine kinase, regulates expression of the (GlcNAc)(2-6) gene set, including a (GlcNAc)2 catabolic operon, two extracellular chitinases, a chitoporin, and a PilA-containing type IV pilus, designated ChiRP (chitin-regulated pilus) that confers a significant growth advantage to V. cholerae on a chitin surface; (ii) GlcNAc causes the coordinate expression of genes involved with chitin chemotaxis and adherence and with the transport and assimilation of GlcNAc; (iii) (GlcN)2 induces genes required for the transport and catabolism of nonacetylated chitin residues; and (iv) the constitutively expressed MSHA pilus facilitates adhesion to the chitin surface independent of surface chemistry. Collectively, these results provide a global portrait of a complex, multistage V. cholerae program for the efficient utilization of chitin.

View details for DOI 10.1073/pnas.0308707101

View details for Web of Science ID 000220140400053

View details for PubMedID 14983042
Mycobacterium tuberculosis gene expression during adaptation to stationary phase and low-oxygen dormancy TUBERCULOSIS Voskuil, M. I., Visconti, K. C., SCHOOLNIK, G. K. 2004; 84 (3-4): 218-227
Abstract

The innate mechanisms used by Mycobacterium tuberculosis to persist during periods of non-proliferation are central to understanding the physiology of the bacilli during latent disease. We have used whole genome expression profiling to expose adaptive mechanisms initiated by M. tuberculosis in two common models of M. tuberculosis non-proliferation. The first of these models was a standard growth curve in which gene expression changes were followed from exponential growth through the transition to stationary phase. In the second model, we followed the adaptive process of M. tuberculosis during transition from aerobic growth to a state of anaerobic non-replicating persistence. The most striking finding from these experiments was the strong induction of the entire DosR "dormancy" regulon over approximately 20 days during the long transition to an anaerobic state. This is contrasted by the muted overall response to aerated stationary phase with only a partial dormancy regulon response. From the results presented here we conclude that the respiration-limited environment of the oxygen-depleted NRP model recreates at least one fundamental factor for which the genome of M. tuberculosis encodes a decisive adaptive program.

View details for DOI 10.1016/j.tube.2004.02.003

View details for Web of Science ID 000222557800010

View details for PubMedID 15207491
Regulation of the Mycobacterium tuberculosis PE/PPE genes TUBERCULOSIS Voskuil, M. I., Schnappinger, D., Rutherford, R., Liu, Y., SCHOOLNIK, G. K. 2004; 84 (3-4): 256-262
Abstract

The genome of Mycobacterium tuberculosis encodes approximately 170 members of the unique mycobacterial PE and PPE gene families. Evidence suggests members of these families are surface-associated cell wall proteins that may provide a diverse antigenic profile and affect immunity. To determine if the expression patterns of PE/PPE genes are consistent with a role in antigenic variability, we analyzed microarray data from 132 experimental conditions for expression of PE/PPE genes. Whole genome expression patterns show that the PE/PPE genes are regulated in a variable and largely independent manner. Gene expression profiling of 15 unique conditions identified differential regulation of 128 of the 169 PE/PPE genes. Expression of the PE/PPE genes appears to be controlled by a variety of independent mechanisms. These data indicate that differential expression of the PE/PPE genes has the potential to provide a dynamic antigenic profile during the course of changing microenvironments within the host.

View details for DOI 10.1016/j.tube.2003.12.014

View details for Web of Science ID 000222557800014

View details for PubMedID 15207495
Structural and topographical studies of the type IV bundle-forming pilus assembly complex of enteropathogenic Escherichia coli JOURNAL OF BACTERIOLOGY Hwang, J. W., Bieber, D., Ramer, S. W., Wu, C. Y., SCHOOLNIK, G. K. 2003; 185 (22): 6695-6701
Abstract

The type IV bundle-forming pili (BFP) of enteropathogenic Escherichia coli (EPEC) are required for virulence in orally challenged human volunteers and for the localized adherence and autoaggregation in vitro phenotypes. BFP filament biogenesis and function are encoded by the 14-gene bfp operon. The BFP assembly complex, containing a BfpB-His6 fusion protein, was chemically cross-linked in situ, and the complex was then purified from BFP-expressing EPEC by a combination of nickel- and BfpB antibody-based affinity chromatography. Characterization of the isolated complex by immunoblotting using BFP protein-specific antibodies showed that at least 10 of the 14 proteins specified by the bfp operon physically interact to form an oligomeric complex. Proteins localized to the outer membrane, inner membrane, and periplasm are within this complex, thus demonstrating that the complex spans the periplasmic space. A combination of immunofluorescence and immuno-gold thin-section transmission electron microscopy studies localized this complex to one pole of the cell.

View details for DOI 10.1128/JB.185.22.6695-6701.2003

View details for Web of Science ID 000186436600024

View details for PubMedID 14594844
MyD88 primes macrophages for full-scale activation by interferon-gamma yet mediates few responses to Mycobacterium tuberculosis JOURNAL OF EXPERIMENTAL MEDICINE Shi, S. A., Nathan, C., Schnappinger, D., Drenkow, J., Fuortes, M., Block, E., Ding, A. H., Gingeras, T. R., SCHOOLNIK, G., Akira, S., Takeda, K., Ehrt, S. 2003; 198 (7): 987-997
Abstract

Macrophages are activated from a resting state by a combination of cytokines and microbial products. Microbes are often sensed through Toll-like receptors signaling through MyD88. We used large-scale microarrays in multiple replicate experiments followed by stringent statistical analysis to compare gene expression in wild-type (WT) and MyD88-/- macrophages. We confirmed key results by quantitative reverse transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Surprisingly, many genes, such as inducible nitric oxide synthase, IRG-1, IP-10, MIG, RANTES, and interleukin 6 were induced by interferon (IFN)-gamma from 5- to 100-fold less extensively in MyD88-/- macrophages than in WT macrophages. Thus, widespread, full-scale activation of macrophages by IFN-gamma requires MyD88. Analysis of the mechanism revealed that MyD88 mediates a process of self-priming by which resting macrophages produce a low level of tumor necrosis factor. This and other factors lead to basal activation of nuclear factor kappaB, which synergizes with IFN-gamma for gene induction. In contrast, infection by live, virulent Mycobacterium tuberculosis (Mtb) activated macrophages largely through MyD88-independent pathways, and macrophages did not need MyD88 to kill Mtb in vitro. Thus, MyD88 plays a dynamic role in resting macrophages that supports IFN-gamma-dependent activation, whereas macrophages can respond to a complex microbial stimulus, the tubercle bacillus, chiefly by other routes.

View details for DOI 10.1084/jem.20030603

View details for Web of Science ID 000185860400002

View details for PubMedID 14517275
Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chang, T. L., Chang, C. H., Simpson, D. A., Xu, Q., Martin, P. K., Lagenaur, L. A., SCHOOLNIK, G. K., Ho, D. D., Hillier, S. L., Holodniy, M., Lewicki, J. A., Lee, P. P. 2003; 100 (20): 11672-11677
Abstract

The predominant mode of HIV transmission worldwide is via heterosexual contact, with the cervico-vaginal mucosa being the main portal of entry in women. The cervico-vaginal mucosa is naturally colonized with commensal bacteria, primarily lactobacilli. To address the urgent need for female-controlled approaches to block the heterosexual transmission of HIV, we have engineered natural human vaginal isolates of Lactobacillus jensenii to secrete two-domain CD4 (2D CD4) proteins. The secreted 2D CD4 recognized a conformation-dependent anti-CD4 antibody and bound HIV type 1 (HIV-1) gp120, suggesting that the expressed proteins adopted a native conformation. Single-cycle infection assays using HIV-1HxB2 carrying a luciferase reporter gene demonstrated that Lactobacillus-derived 2D CD4 inhibited HIV-1 entry into target cells in a dose-dependent manner. Importantly, coincubation of the engineered bacteria with recombinant HIV-1HxB2 reporter virus led to a significant decrease in virus infectivity of HeLa cells expressing CD4-CXCR4-CCR5. Engineered lactobacilli also caused a modest, but statistically significant, decrease in infectivity of a primary isolate, HIV-1JR-FL. This represents an important first step toward the development of engineered commensal bacteria within the vaginal microflora to inhibit heterosexual transmission of HIV.

View details for DOI 10.1073/pnas.1934747100

View details for Web of Science ID 000185685700089

View details for PubMedID 12972635
Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: Insights into the phagosomal environment JOURNAL OF EXPERIMENTAL MEDICINE Schnappinger, D., Ehrt, S., Voskuil, M. I., Liu, Y., Mangan, J. A., Monahan, I. M., Dolganov, G., Efron, B., Butcher, P. D., Nathan, C., Schoolnik, G. K. 2003; 198 (5): 693-704
Abstract

Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2-deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon gamma- and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and beta-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of sigmaE-dependent, sodium dodecyl sulfate-regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogen's cell envelope.

View details for Web of Science ID 000185155500002

View details for PubMedID 12953091
Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program JOURNAL OF EXPERIMENTAL MEDICINE Voskuil, M. I., Schnappinger, D., Visconti, K. C., Harrell, M. I., Dolganov, G. M., Sherman, D. R., Schoolnik, G. K. 2003; 198 (5): 705-713
Abstract

An estimated two billion persons are latently infected with Mycobacterium tuberculosis. The host factors that initiate and maintain this latent state and the mechanisms by which M. tuberculosis survives within latent lesions are compelling but unanswered questions. One such host factor may be nitric oxide (NO), a product of activated macrophages that exhibits antimycobacterial properties. Evidence for the possible significance of NO comes from murine models of tuberculosis showing progressive infection in animals unable to produce the inducible isoform of NO synthase and in animals treated with a NO synthase inhibitor. Here, we show that O2 and low, nontoxic concentrations of NO competitively modulate the expression of a 48-gene regulon, which is expressed in vivo and prepares bacilli for survival during long periods of in vitro dormancy. NO was found to reversibly inhibit aerobic respiration and growth. A heme-containing enzyme, possibly the terminal oxidase in the respiratory pathway, likely senses and integrates NO and O2 levels and signals the regulon. These data lead to a model postulating that, within granulomas, inhibition of respiration by NO production and O2 limitation constrains M. tuberculosis replication rates in persons with latent tuberculosis.

View details for Web of Science ID 000185155500003

View details for PubMedID 12953092
Global stage-specific gene regulation during the developmental cycle of Chlamydia trachomatis JOURNAL OF BACTERIOLOGY Nicholson, T. L., Olinger, L., Chong, K., SCHOOLNIK, G., Stephens, R. S. 2003; 185 (10): 3179-3189
Abstract

Distinct morphological changes associated with the complex development cycle of the obligate intracellular bacterial pathogen Chlamydia trachomatis have been historically well characterized by microscopy. A number of temporally regulated genes have been characterized previously, suggesting that the chlamydial developmental cycle is regulated at the transcriptional level. This hypothesis was tested by microarray analysis in which the entire C. trachomatis genome was analyzed, providing a comprehensive assessment of global gene regulation throughout the chlamydial developmental cycle. Seven temporally cohesive gene clusters were identified, with 22% (189 genes) of the genome differentially expressed during the developmental cycle. The correlation of these gene clusters with hallmark morphological events of the chlamydial developmental cycle suggests three global stage-specific networks of gene regulation.

View details for DOI 10.1128/JB.185.10.3179-3189.2003

View details for Web of Science ID 000182686900024

View details for PubMedID 12730178
Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis MOLECULAR MICROBIOLOGY Park, H. D., Guinn, K. M., Harrell, M. I., Liao, R., Voskuil, M. I., Tompa, M., SCHOOLNIK, G. K., Sherman, D. R. 2003; 48 (3): 833-843
Abstract

Unlike many pathogens that are overtly harmful to their hosts, Mycobacterium tuberculosis can persist for years within humans in a clinically latent state. Latency is often linked to hypoxic conditions within the host. Among M. tuberculosis genes induced by hypoxia is a putative transcription factor, Rv3133c/DosR. We performed targeted disruption of this locus followed by transcriptome analysis of wild-type and mutant bacilli. Nearly all the genes powerfully regulated by hypoxia require Rv3133c/DosR for their induction. Computer analysis identified a consensus motif, a variant of which is located upstream of nearly all M. tuberculosis genes rapidly induced by hypoxia. Further, Rv3133c/DosR binds to the two copies of this motif upstream of the hypoxic response gene alpha-crystallin. Mutations within the binding sites abolish both Rv3133c/DosR binding as well as hypoxic induction of a downstream reporter gene. Also, mutation experiments with Rv3133c/DosR confirmed sequence-based predictions that the C-terminus is responsible for DNA binding and that the aspartate at position 54 is essential for function. Together, these results demonstrate that Rv3133c/DosR is a transcription factor of the two-component response regulator class, and that it is the primary mediator of a hypoxic signal within M. tuberculosis.

View details for Web of Science ID 000182191700020

View details for PubMedID 12694625
Microarray expression profiling: capturing a genome-wide portrait of the transcriptome MOLECULAR MICROBIOLOGY Conway, T., SCHOOLNIK, G. K. 2003; 47 (4): 879-889
Abstract

The bacterial transcriptome is a dynamic entity that reflects the organism's immediate, ongoing and genome-wide response to its environment. Microarray expression profiling provides a comprehensive portrait of the transcriptional world enabling us to view the organism as a 'system' that is more than the sum of its parts. The vigilance of microorganisms to environmental change, the alacrity of the transcriptional response, the short half-life of bacterial mRNA and the genome-scale nature of the investigation collectively explain the power of this method. These same features pose the most significant experimental design and execution issues which, unless surmounted, predictably generate a distorted image of the transcriptome. Conversely, the expression profile of a properly conceived and conducted microarray experiment can be used for hypothesis testing: disclosure of the metabolic and biosynthetic pathways that underlie adaptation of the organism to chang-ing conditions of growth; the identification of co-ordinately regulated genes; the regulatory circuits and signal transduction systems that mediate the adaptive response; and temporal features of developmental programmes. The study of bacterial pathogenesis by microarray expression profiling poses special challenges and opportunities. Although the technical hurdles are many, obtaining expression profiles of an organism growing in tissue will probably reveal strategies for growth and survival in the host's microenvironment. Identifying these colonization strategies and their cognate expression patterns involves a 'deconstruction' process that combines bioinformatics analysis and in vitro DNA array experimentation.

View details for Web of Science ID 000180851600002

View details for PubMedID 12581346
ideR, an essential gene in Mycobacterium tuberculosis: Role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response INFECTION AND IMMUNITY Rodriguez, G. M., Voskuil, M. I., Gold, B., SCHOOLNIK, G. K., Smith, I. 2002; 70 (7): 3371-3381
Abstract

The mycobacterial IdeR protein is a metal-dependent regulator of the DtxR (diphtheria toxin repressor) family. In the presence of iron, it binds to a specific DNA sequence in the promoter regions of the genes that it regulates, thus controlling their transcription. In this study, we provide evidence that ideR is an essential gene in Mycobacterium tuberculosis. ideR cannot normally be disrupted in this mycobacterium in the absence of a second functional copy of the gene. However, a rare ideR mutant was obtained in which the lethal effects of ideR inactivation were alleviated by a second-site suppressor mutation and which exhibited restricted iron assimilation capacity. Studies of this strain and a derivative in which IdeR expression was restored allowed us to identify phenotypic effects resulting from ideR inactivation. Using DNA microarrays, the iron-dependent transcriptional profiles of the wild-type, ideR mutant, and ideR-complemented mutant strains were analyzed, and the genes regulated by iron and IdeR were identified. These genes encode a variety of proteins, including putative transporters, proteins involved in siderophore synthesis and iron storage, members of the PE/PPE family, a membrane protein involved in virulence, transcriptional regulators, and enzymes involved in lipid metabolism.

View details for DOI 10.1128/IAI.70.7.3371-3381.2002

View details for Web of Science ID 000176302600009

View details for PubMedID 12065475
Role of the extracytoplasmic-function sigma Factor sigma(H) in Mycobacterium tuberculosis global gene expression MOLECULAR MICROBIOLOGY Manganelli, R., Voskuil, M. I., SCHOOLNIK, G. K., Dubnau, E., Gomez, M., Smith, I. 2002; 45 (2): 365-374
Abstract

Like other bacterial species, Mycobacterium tuberculosis has multiple sigma (sigma) factors encoded in its genome. In previously published work, we and others have shown that mutations in some of these transcriptional activators render M. tuberculosis sensitive to various environmental stresses and, in some cases, cause attenuated virulence phenotypes. In this paper, we characterize a M. tuberculosis mutant lacking the ECF sigma factor sigma(H). This mutant was more sensitive than the wild type to heat shock and to various oxidative stresses, but did not show decreased ability to grow inside macrophages. Using quantitative reverse transcription-PCR and microarray technology, we have started to define the sigma(H) regulon and its involvement in the global regulation of the response to heat shock and the thiol-specific oxidizing agent diamide. We identified 48 genes whose expression increased after exposure of M. tuberculosis to diamide; out of these, 39 were not induced in the sigH mutant, showing their direct or indirect dependence on sigma(H). Some of these genes encode proteins whose predicted function is related to thiol metabolism, such as thioredoxin, thioredoxin reductase and enzymes involved in cysteine and molybdopterine biosynthesis. Other genes under sigma(H) control encode transcriptional regulators such as sigB, sigE, and sigH itself.

View details for Web of Science ID 000176907100008

View details for PubMedID 12123450
The type IV pilus assembly complex: Biogenic interactions among the bundle-forming pilus proteins of enteropathogenic, Escherichia coli JOURNAL OF BACTERIOLOGY Ramer, S. W., Schoolnik, G. K., Wu, C. Y., Hwang, J. W., Schmidt, S. A., Bieber, D. 2002; 184 (13): 3457-3465
Abstract

Production of type IV bundle-forming pili (BFP) by enteropathogenic Escherichia coli (EPEC) requires the protein products of 12 genes of the 14-gene bfp operon. Antisera against each of these proteins were used to demonstrate that in-frame deletion of individual genes within the operon reduces the abundance of other bfp operon-encoded proteins. This result was demonstrated not to be due to downstream polar effects of the mutations but rather was taken as evidence for protein-protein interactions and their role in the stabilization of the BFP assembly complex. These data, combined with the results of cell compartment localization studies, suggest that pilus formation requires the presence of a topographically discrete assembly complex that is composed of BFP proteins in stoichiometric amounts. The assembly complex appears to consist of an inner membrane component containing three processed, pilin-like proteins, BfpI, -J, and -K, that localize with BfpE, -L, and -A (the major pilin subunit); an outer membrane, secretin-like component, BfpB and -G; and a periplasmic component composed of BfpU. Of these, only BfpL consistently localizes with both the inner and outer membranes and thus, together with BfpU, may articulate between the Bfp proteins in the inner membrane and outer membrane compartments.

View details for DOI 10.1128/JB.184.13.3457-3465.2002

View details for Web of Science ID 000176237400006

View details for PubMedID 12057939
Host-induced epidemic spread of the cholera bacterium NATURE Merrell, D. S., Butler, S. M., Qadri, F., Dolganov, N. A., Alam, A., Cohen, M. B., Calderwood, S. B., SCHOOLNIK, G. K., Camilli, A. 2002; 417 (6889): 642-645
Abstract

The factors that enhance the transmission of pathogens during epidemic spread are ill defined. Water-borne spread of the diarrhoeal disease cholera occurs rapidly in nature, whereas infection of human volunteers with bacteria grown in vitro is difficult in the absence of stomach acid buffering. It is unclear, however, whether stomach acidity is a principal factor contributing to epidemic spread. Here we report that characterization of Vibrio cholerae from human stools supports a model whereby human colonization creates a hyperinfectious bacterial state that is maintained after dissemination and that may contribute to epidemic spread of cholera. Transcriptional profiling of V. cholerae from stool samples revealed a unique physiological and behavioural state characterized by high expression levels of genes required for nutrient acquisition and motility, and low expression levels of genes required for bacterial chemotaxis.

View details for Web of Science ID 000176001200046

View details for PubMedID 12050664
Functional and comparative genomics of pathogenic bacteria CURRENT OPINION IN MICROBIOLOGY Schoolnik, G. K. 2002; 5 (1): 20-26
Abstract

Microarray expression profiling and the development of data-mining tools and new statistical instruments affords an unprecedented opportunity for the genome-scale study of bacterial pathogenicity. Expression profiles obtained from bacteria grown in media simulating host microenvironments yield a portrait of interacting metabolic pathways and multistage developmental programs and disclose regulatory networks. The analysis of closely related strains and species by microarray-based comparative genomics provides a measure of genetic variability within natural populations and identifies crucial differences between pathogen and commensal. In the near future, the combined use of bacterial and host microarrays to study the same infected tissue will reveal the host-pathogen dialogue in a gene-by-gene and site- and time-specific manner. This review discusses the use of microarray-based expression profiling to identify genes of pathogenic bacteria that are differentially regulated in response to host-specific signals. Additionally, the review describes the application of microarray methods to disclose differences in gene content between taxonomically related strains that vary with respect to pathogenic phenotype.

View details for Web of Science ID 000173791000002

View details for PubMedID 11834364
Microarray analysis of bacterial pathogenicity ADVANCES IN MICROBIAL PHYSIOLOGY, VOL 46 Schoolnik, G. K. 2002; 46: 1-45
Abstract

The DNA microarray, a surface that contains an ordered arrangement of each identified open reading frame of a sequenced genome, is the engine of functional genomics. Its output, the expression profile, provides a genome wide snap-shot of the transcriptome. Refined by array-specific statistical instruments and data-mined by clustering algorithms and metabolic pathway databases, the expression profile discloses, at the transcriptional level, how the microbe adapts to new conditions of growth--the regulatory networks that govern the adaptive response and the metabolic and biosynthetic pathways that effect the new phenotype. Adaptation to host microenvironments underlies the capacity of infectious agents to persist in and damage host tissues. While monitoring the whole genome transcriptional response of bacterial pathogens within infected tissues has not been achieved, it is likely that the complex, tissue-specific response is but the sum of individual responses of the bacteria to specific physicochemical features that characterize the host milieu. These are amenable to experimentation in vitro and whole-genome expression studies of this kind have defined the transcriptional response to iron starvation, low oxygen, acid pH, quorum-sensing pheromones and reactive oxygen intermediates. These have disclosed new information about even well-studied processes and provide a portrait of the adapting bacterium as a 'system', rather than the product of a few genes or even a few regulons. Amongst the regulated genes that compose this adaptive system are transcription factors. Expression profiling experiments of transcription factor mutants delineate the corresponding regulatory cascade. The genetic basis for pathogenicity can also be studied by using microarray-based comparative genomics to characterize and quantify the extent of genetic variability within natural populations at the gene level of resolution. Also identified are differences between pathogen and commensal that point to possible virulence determinants or disclose evolutionary history. The host vigorously engages the pathogen; expression studies using host genome microarrays and bacterially infected cell cultures show that the initial host reaction is dominated by the innate immune response. However, within the complex expression profile of the host cell are components mediated by pathogen-specific determinants. In the future, the combined use of bacterial and host microarrays to study the same infected tissue will reveal the dialogue between pathogen and host in a gene-by-gene and site- and time-specific manner. Translating this conversation will not be easy and will probably require a combination of powerful bioinformatic tools and traditional experimental approaches--and considerable effort and time.

View details for Web of Science ID 000176466600001

View details for PubMedID 12073651
Reprogramming of the macrophage transcriptome in response to interferon-gamma and Mycobacterium tuberculosis: Signaling roles of nitric oxide synthase-2 and phagocyte oxidase JOURNAL OF EXPERIMENTAL MEDICINE Ehrt, S., Schnappinger, D., Bekiranov, S., Drenkow, J., Shi, S. P., Gingeras, T. R., Gaasterland, T., SCHOOLNIK, G., Nathan, C. 2001; 194 (8): 1123-1139
Abstract

Macrophage activation determines the outcome of infection by Mycobacterium tuberculosis (Mtb). Interferon-gamma (IFN-gamma) activates macrophages by driving Janus tyrosine kinase (JAK)/signal transducer and activator of transcription-dependent induction of transcription and PKR-dependent suppression of translation. Microarray-based experiments reported here enlarge this picture. Exposure to IFN-gamma and/or Mtb led to altered expression of 25% of the monitored genome in macrophages. The number of genes suppressed by IFN-gamma exceeded the number of genes induced, and much of the suppression was transcriptional. Five times as many genes related to immunity and inflammation were induced than suppressed. Mtb mimicked or synergized with IFN-gamma more than antagonized its actions. Phagocytosis of nonviable Mtb or polystyrene beads affected many genes, but the transcriptional signature of macrophages infected with viable Mtb was distinct. Studies involving macrophages deficient in inducible nitric oxide synthase and/or phagocyte oxidase revealed that these two antimicrobial enzymes help orchestrate the profound transcriptional remodeling that underlies macrophage activation.

View details for Web of Science ID 000171734100009

View details for PubMedID 11602641
Structure-function analysis of BfpB, a secretin-like protein encoded by the bundle-forming-pilus operon of enteropathogenic Escherichia coli JOURNAL OF BACTERIOLOGY Schmidt, S. A., Bieber, D., Ramer, S. W., Hwang, J. W., Wu, C. Y., Schoolnik, G. 2001; 183 (16): 4848-4859
Abstract

Production of type IV bundle-forming pili by enteropathogenic Escherichia coli (EPEC) requires BfpB, an outer-membrane lipoprotein and member of the secretin protein superfamily. BfpB was found to compose a ring-shaped, high-molecular-weight outer-membrane complex that is stable in 4% sodium dodecyl sulfate at temperatures of < or = 65 degrees C. Chemical cross-linking and immunoprecipitation experiments disclosed that the BfpB multimeric complex interacts with BfpG, and mutational studies showed that BfpG is required for the formation and/or stability of the multimer but not for the outer-membrane localization of BfpB. Formation of the BfpB multimer also does not require BfpA, the repeating subunit of the pilus filament. Functional studies of the BfpB-BfpG complex revealed that its presence confers vancomycin sensitivity, indicating that it may form an incompletely gated channel through the outer membrane. BfpB expression is also associated with accumulation of EPEC proteins in growth medium, suggesting that it may support both pilus biogenesis and protein secretion.

View details for Web of Science ID 000170118600020

View details for PubMedID 11466288
The Mycobacterium tuberculosis ECF sigma factor sigma(E): role in global gene expression and survival in macrophages MOLECULAR MICROBIOLOGY Manganelli, R., Voskuil, M. I., SCHOOLNIK, G. K., Smith, I. 2001; 41 (2): 423-437
Abstract

In previously published work, we identified three Mycobacterium tuberculosis sigma (sigma) factor genes responding to heat shock (sigB, sigE and sigH). Two of them (sigB and sigE) also responded to SDS exposure. As these responses to stress suggested that the sigma factors encoded by these genes could be involved in pathogenicity, we are studying their role in physiology and virulence. In this work, we characterize a sigE mutant of M. tuberculosis H37Rv. The sigE mutant strain was more sensitive than the wild-type strain to heat shock, SDS and various oxidative stresses. It was also defective in the ability to grow inside both human and murine unactivated macrophages and was more sensitive than the wild-type strain to the killing activity of activated murine macrophages. Using microarray technology and quantitative reverse transcription-polymerase chain reaction (RT-PCR), we started to define the sigmaE regulon of M. tuberculosis and its involvement in the global regulation of the stress induced by SDS. We showed the requirement for a functional sigE gene for full expression of sigB and for its induction after SDS exposure but not after heat shock. We also identified several genes that are no longer induced when sigmaE is absent. These genes encode proteins belonging to different classes including transcriptional regulators, enzymes involved in fatty acid degradation and classical heat shock proteins.

View details for Web of Science ID 000170566200011

View details for PubMedID 11489128
Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha-crystallin PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sherman, D. R., Voskuil, M., Schnappinger, D., Liao, R. L., Harrell, M. I., SCHOOLNIK, G. K. 2001; 98 (13): 7534-7539
Abstract

Unlike many pathogens that are overtly toxic to their hosts, the primary virulence determinant of Mycobacterium tuberculosis appears to be its ability to persist for years or decades within humans in a clinically latent state. Since early in the 20th century latency has been linked to hypoxic conditions within the host, but the response of M. tuberculosis to a hypoxic signal remains poorly characterized. The M. tuberculosis alpha-crystallin (acr) gene is powerfully and rapidly induced at reduced oxygen tensions, providing us with a means to identify regulators of the hypoxic response. Using a whole genome microarray, we identified >100 genes whose expression is rapidly altered by defined hypoxic conditions. Numerous genes involved in biosynthesis and aerobic metabolism are repressed, whereas a high proportion of the induced genes have no known function. Among the induced genes is an apparent operon that includes the putative two-component response regulator pair Rv3133c/Rv3132c. When we interrupted expression of this operon by targeted disruption of the upstream gene Rv3134c, the hypoxic regulation of acr was eliminated. These results suggest a possible role for Rv3132c/3133c/3134c in mycobacterial latency.

View details for Web of Science ID 000169456600097

View details for PubMedID 11416222
VpsR, a member of the response regulators of the two-component regulatory systems, is required for expression of vps biosynthesis genes and EPSETr-associated phenotypes in Vibrio cholerae O1 El Tor JOURNAL OF BACTERIOLOGY Yildiz, F. H., Dolganov, N. A., SCHOOLNIK, G. K. 2001; 183 (5): 1716-1726
Abstract

The rugose colonial variant of Vibrio cholerae O1 El Tor produces an exopolysaccharide (EPS(ETr)) that enables the organism to form a biofilm and to resist oxidative stress and the bactericidal action of chlorine. Transposon mutagenesis of the rugose variant led to the identification of vpsR, which codes for a homologue of the NtrC subclass of response regulators. Targeted disruption of vpsR in the rugose colony genetic background yielded a nonreverting smooth-colony morphotype that produced no detectable EPS(ETr) and did not form an architecturally mature biofilm. Analysis of two genes, vpsA and vpsL, within the vps cluster of EPS(ETr) biosynthesis genes revealed that their expression is induced above basal levels in the rugose variant, compared to the smooth colonial variant, and requires vpsR. These results show that VpsR functions as a positive regulator of vpsA and vpsL and thus acts to positively regulate EPS(ETr) production and biofilm formation.

View details for Web of Science ID 000166943600025

View details for PubMedID 11160103
Whole genome DNA microarray expression analysis of biofilm development by Vibrio cholerae O1 El Tor MICROBIAL GROWTH IN BIOFILMS, PT A SCHOOLNIK, G. K., Voskuil, M. I., Schnappinger, D., Yildiz, F. H., Meibom, K., Dolganov, N. A., Wilson, M. A., Chong, K. H. 2001; 336: 3-18
View details for Web of Science ID 000169760800001

View details for PubMedID 11398407
The accelerating convergence of genomics and microbiology. Schoolnik, G. K. 2001: REPORTS4009-?
View details for PubMedID 11305936
DNA microarrays and combinatorial chemical libraries: tools for the drug discovery pipeline Barry, C. E., Wilson, M., LEE, R., SCHOOLNIK, G. K. INT UNION AGAINST TUBERCULOSIS LUNG DISEASE (I U A T L D). 2000: S189-S193
View details for Web of Science ID 000165821700017

View details for PubMedID 11144552
Extracytoplasmic proteins of Mycobacterium tuberculosis - mature secreted proteins often start with aspartic acid and proline MICROBIOLOGY-SGM Wiker, H. G., Wilson, M. A., SCHOOLNIK, G. K. 2000; 146: 1525-1533
Abstract

A surrogate expression system, based on fusions to the phoA bacterial reporter gene, was used to identify Mycobacterium tuberculosis genes that encode exported proteins and the promoter regions required for their expression in the heterologous host Mycobacterium smegmatis. To assess these results in the context of the complete M. tuberculosis genome sequence, the corresponding genes were identified and computational algorithms were employed to identify signal peptide (SP), transmembrane domain and membrane lipoprotein attachment motifs. This information was used to predict the subset of M. tuberculosis genes that encode exported proteins. Of the 34 genes identified by the phoA method, 22 were classified to encode potential soluble secreted proteins. Among these, 14 genes may encode novel secreted proteins. Six of the remaining 12 genes were predicted to encode membrane lipoproteins and an additional six to encode integral membrane proteins. Published observations of proteins proven to be secreted into M. tuberculosis culture filtrates were reviewed to further characterize the mycobacterial SP motif. It was concluded that mycobacterial SPs are comparable in size to Gram-positive SPs, but certain features are different. In particular, arginine was the predominant N-terminally positively charged amino acid in contrast to lysine in the Gram-positives. The hydrophobic transmembrane segment of the SP was dominated by alanine, in contrast to leucine. At the C-terminal end of the SPs, the (-3, -1) rule (AXA motif) holds, with alanine as the dominant amino acid in both positions, being most dominant in the (-1) position. A high proportion of mature sequences start with aspartic acid in the (+1) position and proline in the (+2) position - the DP motif. The authors propose that the DP sequence serves as a sorting signal, following translocation and cleavage by signal peptidase I. Alternatively, the DP motif may be part of the recognition site for the signal peptidase.

View details for Web of Science ID 000088174000004

View details for PubMedID 10878117
The complete genome sequence of Vibrio cholerae: a tale of two chromosomes and of two lifestyles. Genome biology Schoolnik, G. K., Yildiz, F. H. 2000; 1 (3): REVIEWS1016-?
Abstract

Vibrio cholerae O1 has figured prominently in the history of infectious diseases as a cause of periodic global epidemics, an affliction of refugees in areas of social strife and as the disease first subjected to modern epidemiological analysis during the classic investigations of John Snow in mid-19th century London [1]. Thus, publication of the entire genome sequence of V. cholerae O1 (biotype El Tor) in Nature [2] by a consortium of investigators from The Institute for Genomic Research, the University of Maryland and Harvard Medical School is properly regarded as an historic event that will trigger a paradigm shift in the study of this organism.

View details for PubMedID 11178241
Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wilson, W., DeRisi, J., Kristensen, H. H., Imboden, P., Rane, S., Brown, P. O., SCHOOLNIK, G. K. 1999; 96 (22): 12833-12838
Abstract

Tuberculosis is a chronic infectious disease that is transmitted by cough-propelled droplets that carry the etiologic bacterium, Mycobacterium tuberculosis. Although currently available drugs kill most isolates of M. tuberculosis, strains resistant to each of these have emerged, and multiply resistant strains are increasingly widespread. The growing problem of drug resistance combined with a global incidence of seven million new cases per year underscore the urgent need for new antituberculosis therapies. The recent publication of the complete sequence of the M. tuberculosis genome has made possible, for the first time, a comprehensive genomic approach to the biology of this organism and to the drug discovery process. We used a DNA microarray containing 97% of the ORFs predicted from this sequence to monitor changes in M. tuberculosis gene expression in response to the antituberculous drug isoniazid. Here we show that isoniazid induced several genes that encode proteins physiologically relevant to the drug's mode of action, including an operonic cluster of five genes encoding type II fatty acid synthase enzymes and fbpC, which encodes trehalose dimycolyl transferase. Other genes, not apparently within directly affected biosynthetic pathways, also were induced. These genes, efpA, fadE23, fadE24, and ahpC, likely mediate processes that are linked to the toxic consequences of the drug. Insights gained from this approach may define new drug targets and suggest new methods for identifying compounds that inhibit those targets.

View details for Web of Science ID 000083373000113

View details for PubMedID 10536008
Complete DNA sequence and structural analysis of the enteropathogenic Escherichia coli adherence factor plasmid INFECTION AND IMMUNITY Tobe, T., Hayashi, T., Han, C. G., SCHOOLNIK, G. K., Ohtsubo, E., Sasakawa, C. 1999; 67 (10): 5455-5462
Abstract

The complete nucleotide sequence and organization of the enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid of EPEC strain B171 (O111:NM) were determined. The EAF plasmid encodes two known virulence-related operons, the bfp operon, which is composed of genes necessary for biosynthesis of bundle-forming pili, and the bfpTVW (perABC) operon, composed of regulatory genes required for bfp transcription and also for transcriptional activation of the eae gene in the LEE pathogenicity island on the EPEC chromosome. The 69-kb EAF plasmid, henceforth designated pB171, contains, besides the bfp and bfpTVW (perABC) operons, potential virulence-associated genes, plasmid replication and maintenance genes, and many insertion sequence elements. Of the newly identified open reading frames (ORFs), two which comprise a single operon had the potential to encode proteins with high similarity to a C-terminal region of ToxB whose coding sequence is located on pO157, a large plasmid harbored by enterohemorrhagic E. coli. Another ORF, located between the bfp and bfpTVW operons, showed high similarity with trcA, a bfpT-regulated chaperone-like protein gene of EPEC. Two sites were found to be putative replication regions: one similar to RepFIIA of p307 or F, and the other similar to RepFIB of R100 (NR1). In addition, we identified a third region that contains plasmid maintenance genes. Insertion elements were scattered throughout the plasmid, indicating the mosaic nature of the EAF plasmid and suggesting evolutionary events by which virulence genes may have been obtained.

View details for Web of Science ID 000082813500063

View details for PubMedID 10496929
A novel chromosomal locus of enteropathogenic Escherichia coli (EPEC), which encodes a bfpT-regulated chaperone-like protein, TrcA, involved in microcolony formation by EPEC MOLECULAR MICROBIOLOGY Tobe, T., Tatsuno, I., Katayama, E., Wu, C. Y., SCHOOLNIK, G. K., Sasakawa, C. 1999; 33 (4): 741-752
Abstract

The bfpTVW operon, also known as the per operon, of enteropathogenic Escherichia coli (EPEC) is required for the transcriptional activation of the bfp operon, which encodes the major subunit and assembly machinery of bundle-forming pili (BFP). An immobilized T7-tagged BfpT fusion protein that binds specifically to upstream promoter sequences of bfpA and eae was used to 'fish out' from a promoter library other EPEC chromosomal fragments that are bound by the BfpT protein. After screening for promoters exhibiting bfpTVW-dependent expression, one was identified that was positively regulated by bfpTVW and that is not present in the chromosomes of two non-virulent E. coli laboratory strains, DH5alpha and HB101. Further analysis of this positively regulated promoter in EPEC showed that it resided within a 4.9 kb sequence that is not present in E. coli K12. This locus, located downstream of the potB gene, was found to contain four open reading frames (ORFs): bfpTVW-activated promoter was localized upstream of ORF1. An ORF1 knockout mutant produced less of the BFP structural subunit (BfpA) and formed smaller than normal adherent microcolonies on cultured epithelial cells; however, this mutation did not affect bfp transcription. An ORF1-His6 fusion protein specifically bound the preprocessed and mature forms of the BfpA protein and thus appears to stabilize the former within the cytoplasmic compartment. ORF1 therefore is a newly isolated EPEC chromosomal gene that encodes a chaperone-like protein involved in the production of BFP. Hence, ORF1 was designated trcA (bfpT-regulated chaperone-like protein gene). The TrcA protein also specifically bound 39 kDa and 90 kDa proteins that are expressed by EPEC but not by E. coli K12. The 90 kDa protein was revealed to be intimin, a protein product of the eae gene, which is required for the EPEC attaching/effacing phenotype, suggesting a direct interaction of TrcA with intimin in the cytoplasmic compartment.

View details for Web of Science ID 000082478000008

View details for PubMedID 10447884
Comparative genomics of BCG vaccines by whole-genome DNA microarray SCIENCE Behr, M. A., Wilson, M. A., Gill, W. P., Salamon, H., SCHOOLNIK, G. K., Rane, S., Small, P. M. 1999; 284 (5419): 1520-1523
Abstract

Bacille Calmette-Guérin (BCG) vaccines are live attenuated strains of Mycobacterium bovis administered to prevent tuberculosis. To better understand the differences between M. tuberculosis, M. bovis, and the various BCG daughter strains, their genomic compositions were studied by performing comparative hybridization experiments on a DNA microarray. Regions deleted from BCG vaccines relative to the virulent M. tuberculosis H37Rv reference strain were confirmed by sequencing across the missing segment of the H37Rv genome. Eleven regions (encompassing 91 open reading frames) of H37Rv were found that were absent from one or more virulent strains of M. bovis. Five additional regions representing 38 open reading frames were present in M. bovis but absent from some or all BCG strains; this is evidence for the ongoing evolution of BCG strains since their original derivation. A precise understanding of the genetic differences between closely related Mycobacteria suggests rational approaches to the design of improved diagnostics and vaccines.

View details for Web of Science ID 000080548100035

View details for PubMedID 10348738
Vibrio cholerae O1 El Tor: Identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation Yildiz, F. H., Schoolnik, G. K. NATL ACAD SCIENCES. 1999: 4028-4033
Abstract

The rugose colony variant of Vibrio cholerae O1, biotype El Tor, is shown to produce an exopolysaccharide, EPSETr, that confers chlorine resistance and biofilm-forming capacity. EPSETr production requires a chromosomal locus, vps, that contains sequences homologous to carbohydrate biosynthesis genes of other bacterial species. Mutations within this locus yield chlorine-sensitive, smooth colony variants that are biofilm deficient. The biofilm-forming properties of EPSETr may enable the survival of V. cholerae O1 within environmental aquatic habitats between outbreaks of human disease.

View details for Web of Science ID 000079507900122

View details for PubMedID 10097157
Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli SCIENCE Bieber, D., Ramer, S. W., Wu, C. Y., Murray, W. J., Tobe, T., Fernandez, R., Schoolnik, G. K. 1998; 280 (5372): 2114-2118
Abstract

Type IV bundle-forming pili of enteropathogenic Escherichia coli are required for the localized adherence and autoaggregation phenotypes. Whether these pili are also required for virulence was tested in volunteers by inactivating bfpA or bfpT (perA) encoding, respectively, the pilus subunit and the bfp operon transcriptional activator. Both mutants caused significantly less diarrhea. Mutation of the bfpF nucleotide-binding domain caused increased piliation, enhanced localized adherence, and abolished the twitching motility-dispersal phase of the autoaggregation phenotype. The bfpF mutant colonized the human intestine but was about 200-fold less virulent. Thus, BfpF is required for dispersal from the bacterial aggregate and for full virulence.

View details for Web of Science ID 000074435900045

View details for PubMedID 9641917
Construction and characterization of a partial Mycobacterium tuberculosis cDNA library of genes expressed at reduced oxygen tension GENE Imboden, P., SCHOOLNIK, G. K. 1998; 213 (1-2): 107-117
Abstract

To determine which bacterial genes could be expressed during tuberculosis in the human body, we have prepared and characterized a collection of cDNA clones corresponding to genes that are expressed by Mycobacterium tuberculosis during in vitro growth in 5% (v/v) oxygen. These cDNA clones were obtained by purifying total RNA from M. tuberculosis and cloning small cDNA segments into Escherichia coli followed by removal of clones containing ribosomal RNA sequences. From approx. 1700 clones, a collection of 170 clones containing non-ribosomal inserts were further characterized by PCR amplification. Inserts of more than 180bp were verified by Southern hybridization to have corresponding loci in M. tuberculosis genomic DNA and their sequence was determined. We describe the genes that have been identified using this approach. Multiple independent cDNA clones were obtained for two genes, one probably encoding a stable structural RNA and the other a homologue of ferritin. RNA levels for these two genes were monitored during growth at 20% oxygen, 5% oxygen and in the nearly anaerobic culture sediments. No difference in expression levels was found at 5% oxygen compared to 20% oxygen. RNA levels for the ferritin homologue gene were significantly lower in culture sediments. The stable structural RNA, however, showed very high expression levels independently of culture conditions.

View details for Web of Science ID 000074481000014

View details for PubMedID 9630551
Role of rpoS in stress survival and virulence of Vibrio cholerae JOURNAL OF BACTERIOLOGY Yildiz, F. H., SCHOOLNIK, G. K. 1998; 180 (4): 773-784
Abstract

Vibrio cholerae is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded by rpoS, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a V. cholerae rpoS homolog was cloned by functional complementation of an Escherichia coli mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported rpoS gene products. To determine the functional role of rpoS in V. cholerae, we inactivated rpoS by homologous recombination. V. cholerae strains lacking rpoS are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that rpoS may be required for the persistence of V. cholerae in aquatic habitats. In addition, the rpoS mutation led to reduced production or secretion of hemagglutinin/protease. However, rpoS is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.

View details for Web of Science ID 000071970500002

View details for PubMedID 9473029
The light organ symbiont Vibrio fischeri possesses two distinct secreted ADP-ribosyltransferases JOURNAL OF BACTERIOLOGY Reich, K. A., Biegel, T., SCHOOLNIK, G. K. 1997; 179 (5): 1591-1597
Abstract

We have previously described the purification, cloning, and initial characterization of a secreted ADP-ribosyltransferase, halovibrin (gene designation hvn), from the luminescent light organ symbiont Vibrio fischeri. This report describes a strategy for overexpression of halovibrin, the production and refinement of antihalo-vibrin antisera, and the molecular biological construction of a V. fischeri halovibrin null strain. Biochemical analysis of this mutant revealed that V. fischeri hvn null still possessed ADP-ribosyltransferase activity and that this activity is immunologically, genetically, and structurally distinct from the previously described enzyme. This unusual finding, of two ADP-ribosyltransferase enzymes produced by a microorganism, is complemented by the details of the purification to apparent homogeneity and in vitro regulation of this new protein, halovibrin-beta.

View details for Web of Science ID A1997WP41500022

View details for PubMedID 9045818
Immunogenicity of a fusion protein linking the beta subunit carboxyl terminal peptide (CTP) of human chorionic gonadotropin to the B subunit of Escherichia coli heat labile enterotoxin (LTB) VACCINE ROCK, E. P., Reich, K. A., Lyu, D. M., Hovi, M., Hardy, J., SCHOOLNIK, G. K., STOCKER, B. A., Stevens, V. 1996; 14 (16): 1560-1568
Abstract

Human chorionic gonadotropin (hCG) is currently under investigation as an antigenic target in both anti-cancer and anti-fertility vaccines. Formulations studied to date show promise in clinical trials for both applications yet are expensive to produce and require frequented administration in order to maintain an effective antibody titer. We have engineered a fusion protein consisting of Escherichia coli heat-labile enterotoxin subunit B (LTB) genetically linked at its C terminus via a nine amino acid linker to the 37 amino acid carboxyl terminal peptide (CTP) of the hCG beta chain. This LTB-CTP fusion protein is stably expressed in bacteria and forms pentamers of full-length protein subunits. Purified LTB-CTP protein hCG-specific antibodies in mice without additional adjuvants.

View details for Web of Science ID A1996WC53400013

View details for PubMedID 9014300
BfpB, an outer membrane lipoprotein required for the biogenesis of bundle-forming pili in enteropathogenic Escherichia coli JOURNAL OF BACTERIOLOGY Ramer, S. W., Bieber, D., SCHOOLNIK, G. K. 1996; 178 (22): 6555-6563
Abstract

The bundle-forming pili (BFP) of enteropathogenic Escherichia coli are believed to play a role in pathogenesis by causing the formation of bacterial microcolonies that bind epithelial surfaces of the small intestine. This in vivo process is mimicked in vitro by the autoaggregation and localized adherence phenotypes. Expression of BFP, a member of the type IV pilus family, requires the enteroadherence factor (EAF) plasmid, which contains bfpA, the gene that encodes the principal structural subunit of BFP. Immediately downstream of bfpA are 13 open reading frames transcribed in the same direction as bfpA; together with bfpA, these compose the bfp gene cluster. Disruption of bfpB, the second open reading frame downstream of bfpA, was performed by allelic exchange. The resulting mutant, B171-8deltaB, did not exhibit the autoaggregation or localized adherence phenotype or produce BFP filaments. Thus, BfpB is required for pilus biogenesis. However, BfpA was produced at wild-type levels and processed normally by B171-8deltaB, indicating that BfpB acts at a step in the BFP biogenic pathway after production and processing of the structural subunit. Biochemical and cell fractionation studies showed that BfpB is a 58-kDa lipoprotein that is located primarily in the outer membrane. Assays of bfpA and bfpB mRNAs and protein expression showed that both genes are cotranscribed as part of an environmentally responsive operon that is regulated by growth phase and ammonium.

View details for Web of Science ID A1996VR93100018

View details for PubMedID 8932312
Cloning and characterization of bfpTVW, genes required for the transcriptional activation of bfpA in enteropathogenic Escherichia coli MOLECULAR MICROBIOLOGY Tobe, T., SCHOOLNIK, G. K., SOHEL, I., Bustamante, V. H., Puente, J. L. 1996; 21 (5): 963-975
Abstract

Expression of the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is regulated at the transcriptional level by growth phase, temperature, calcium and ammonium. Genes required for the transcriptional activation of bfpA were localized to a 1.8 kb fragment of the enteroadherent factor (EAF) plasmid of EPEC that is separated from the bfp operon by 6 kb. Within this fragment three identically oriented and closely spaced open reading frames (ORFs) were identified and designated bfpT, bfpV and bfpW. bfpT is predicted to encode a 31.8 kDa protein that shares homology with the AraC family of transcriptional regulators, including the presence of a conserved C-terminal DNA-binding helix-turn-helix motif. Insertional inactivation of bfpT led to the loss of bfpA transcription, BfpA protein production and the localized adherence (LA) phenotype; this mutant phenotype could be complemented by introduction of bfpTVW and, on separate plasmids, bfpT + bfpW. However, introduction of bfpT + bfpV, bfpV alone, bfpW alone, or bfpV + bfpW did not enable recovery of the wild-type phenotype. Maximal efficiency of bfpA transcription required all three genes, but bfpV and bfpW each enhanced transcription providing bfpT was also present. A series of deletions of the bfpA upstream promoter region was prepared; with respect to the bfpA transcription start site, sequence between nucleotides -94 and -55 was found to bind bfpT. BfpT also bound a DNA fragment containing the eaeA promoter region on the EPEC chromosome. From these results we conclude that bfpTV W causes transcriptional activation of bfpA, and possibly eaeA, by a trans-acting mechanism that may co-ordinately regulate the expression of EPEC virulence determinants.

View details for Web of Science ID A1996VH13100008

View details for PubMedID 8885267
Enteropathogenic Escherichia coli: Identification of a gene cluster coding for bundle-forming pilus morphogenesis JOURNAL OF BACTERIOLOGY SOHEL, I., Puente, J. L., Ramer, S. W., Bieber, D., Wu, C. Y., SCHOOLNIK, G. K. 1996; 178 (9): 2613-2628
Abstract

Sequence flanking the bfpA locus on the enteroadherent factor plasmid of the enteropathogenic Escherichia coli (EPEC) strain B171-8 (O111:NM) was obtained to identify genes that might be required for bundle-forming pilus (BFP) biosynthesis. Deletion experiments led to the identification of a contiguous cluster of at least 12 open reading frames, including bfpA, that could direct the synthesis of a morphologically normal BFP filament. Within the bfp gene cluster, we identified open reading frames that share homology with other type IV pilus accessory genes and with genes required for transformation competence and protein secretion. Immediately upstream of the bfp gene cluster, we identified a potential replication origin including genes that are predicted to encode proteins homologous with replicase and resolvase. Restriction fragment length polymorphism analysis of DNA from six additional EPEC serotypes showed that the organization of the bfp gene cluster and its juxtaposition with a potential plasmid origin of replication are highly conserved features of the EPEC biotype.

View details for Web of Science ID A1996UJ12200020

View details for PubMedID 8626330
The bundle-forming pili of enteropathogenic Escherichia coli: Transcriptional regulation by environmental signals MOLECULAR MICROBIOLOGY Puente, J. L., Bieber, D., Ramer, S. W., Murray, W., SCHOOLNIK, G. K. 1996; 20 (1): 87-100
Abstract

The bundle-forming pili (BFP) of enteropathogenic Escherichia coil (EPEC) are required for the development of circumscribed colonies of bacteria attached to the surfaces of cultured epithelial cells, a process termed the localized adherence (LA) phenotype. Similar lesions are evident in jejunal biopsies from EPEC-infected children. BFP production is not constitutive, but instead occurs upon transfer of bacteria from nutrient broth to tissue culture media, indicating that the expression of BFP may be environmentally regulated. To learn more about how BFP protein expression is induced during epithelial-cell adherence, bfpA-cat transcriptional fusions and northern blot analyses were employed to monitor bfpA expression as a function of environmental signals and growth kinetics. bfpA expression was found to be regulated at the transcriptional level, and to require a separate locus on the EPEC adherence factor (EAF) plasmid. Expression occurred selectively during exponential-growth phase and was greatest between 35 and 37 degrees C, and in the presence of calcium. Ammonium (20 mM) significantly reduced bfpA mRNA and protein expression and the development of the LA phenotype. Analysis of the bfpA upstream sequence and identification of the transcription initiation site revealed a conventional sigma (70)-dependent promoter and an AT-rich tract that might affect promoter activity. Taken together, these findings further support the pathogenic role of BFP by explaining how BFP production would commence in the small intestine and terminate in the colon and in external habitats.

View details for Web of Science ID A1996UG23800010

View details for PubMedID 8861207
Halovibrin, secreted from the light organ symbiont Vibrio fischeri, is a member of a new class of ADP-ribosyltransferases JOURNAL OF BACTERIOLOGY Reich, K. A., SCHOOLNIK, G. K. 1996; 178 (1): 209-215
Abstract

The purification, cloning, and deduced amino acid sequence of an ADP-ribosyltransferase secreted from the marine bacterium Vibrio fischeri (V. fischeri ADP-r) is described. This enzyme was purified from culture supernatant, and partial amino acid sequence obtained from the purified protein was used to design a degenerate oligonucleotide probe that was used to clone a cross-hybridizing DNA fragment from V. fischeri genomic DNA. Recombinant Escherichia coli clones harboring this fragment possessed ADP-ribosyltransferase activity. The DNA fragment was sequenced, and deletion analysis localized the ADP-ribosyltransferase activity to one of the three possible open reading frames in the fragment; the deduced amino acid sequence from this open reading frame matched the amino acid sequence obtained from the purified protein. V. fischeri ADP-r has no significant homology (DNA or amino acid) with other known ADP-ribosyltransferases. This enzyme appears to require neither proteolytic cleavage nor a reducing agent for enzymatic activity. The cloned gene is expressed but not secreted in E. coli; however, it is secreted from a heterologous marine Vibrio species. We have named this enzyme halovibrin.

View details for Web of Science ID A1996TM78900029

View details for PubMedID 8550419
DELIVERY OF CLASS-I AND CLASS-II MHC-RESTRICTED T-CELL EPITOPES OF LISTERIOLYSIN OF LISTERIA-MONOCYTOGENES BY ATTENUATED SALMONELLA VACCINE Verma, N. K., Ziegler, H. K., Wilson, M., Khan, M., Safley, S., STOCKER, B. A., SCHOOLNIK, G. K. 1995; 13 (2): 142-150
Abstract

Using a Salmonella vaccine-Listeria infection model of intracellular infection, we studied the capacity of an attenuated strain of Salmonella carrying T-cell epitopes of listeriolysin (LLO) of L. monocytogenes to elicit epitope-specific T-cell responses. Class II (LLO 215-226) or class I (LLO 91-99) MHC-restricted T-cell epitopes of LLO were inserted within a central, hypervariable domain of the flagellin protein of an attenuated delta aroA Salmonella dublin strain. T cells from Listeria-immunized mice were activated by lysates or heat-killed preparations of Salmonella construct expressing the LLO 215-226 epitope, indicating that LLO 215-226 is processed and presented to T cells when offered to antigen-presenting cells as part of a flagellin-epitope fusion protein. The chimeric flagellin genes were integrated into the chromosome of the flagellin-negative S. dublin strain to obtain stable expression of the epitopes. Immunization with the living, chromosomally integrated Salmonella construct carrying LLO 215-226 epitope as part of the flagellin protein generated T cells reactive with the corresponding LLO peptide, indicating that this chimera can stimulate a class-specific immune response in vitro. The effect of flanking residues on the processing and presentation of MHC class I LLO 91-99 epitope was studied using Salmonella vaccine strains that express chimeric flagellins containing one of three LLO 91-99 inserts: 91-99 (normal flagellin amino acids as flanking residues); KK91-99KK (Lys-Lys flanking residues); and AAA91-99AAA (Ala-Ala-Ala flanking residues).(ABSTRACT TRUNCATED AT 250 WORDS)

View details for Web of Science ID A1995QK47300002

View details for PubMedID 7625107
INDUCTION OF A CELLULAR IMMUNE-RESPONSE TO A DEFINED T-CELL EPITOPE AS AN INSERT IN THE FLAGELLIN OF A LIVE VACCINE STRAIN OF SALMONELLA VACCINE Verma, N. K., Ziegler, H. K., STOCKER, B. A., SCHOOLNIK, G. K. 1995; 13 (3): 235-244
Abstract

Attenuated strains of Salmonella have been used as vaccines to deliver heterologous antigens mainly to generate a humoral immune response. However, little is known about their ability to induce a cell-mediated immune response to the T-cell epitopes of another infectious agent or how optimally to deliver these epitopes to the host immune system. In order to study this question, a well defined MHC class II-restricted epitope (residues 88-103) from moth cytochrome C (MCC) was inserted into the central hypervariable domain of the flagellin of an attenuated strain of Salmonella dublin. The resulting flagellin was exported to the bacterial surface and polymerized into flagellar filaments that contained multiple copies of the MCC epitope. When flanked by Lys-Lys cathepsin B cleavage sites to facilitate its proteolytic release within the endosomal compartment of antigen-presenting cells, the MCC-chimeric flagellin epitope was efficiently processed in vitro by mouse peritoneal macrophages and presented to 2B4 T-hybridoma cells (specific for the MCC epitope 88-103). Stable expression of the epitope and a higher immune response was obtained in H-2k mice by integrating the chimeric flagellin gene into the chromosome of the vaccine strain. Bacteria with MCC-chimeric flagellins that were expressed from a stable chromosomal locus and flanked by cathepsin B cleavage sites were cleared more rapidly from the livers and spleens of transgenic mice with T-cell receptor (TCR) alpha and beta chains specific for the MCC epitope than were bacteria lacking the epitope. Antigen processing and presentation of class II-restricted epitopes expressed as chimeric proteins by attenuated bacterial vaccine vectors may be facilitated by the presence of endosomal protease cleavage sites on each side of the epitope and by chromosomal integration of the coding sequence.

View details for Web of Science ID A1995QL82900001

View details for PubMedID 7543230
TEMPORAL TRENDS AND TRANSMISSION PATTERNS DURING THE EMERGENCE OF MULTIDRUG-RESISTANT TUBERCULOSIS IN NEW-YORK-CITY - A MOLECULAR EPIDEMIOLOGIC ASSESSMENT JOURNAL OF INFECTIOUS DISEASES Shafer, R. W., Small, P. M., Larkin, C., Singh, S. P., Kelly, P., Sierra, M. F., SCHOOLNIK, G., Chirgwin, K. D. 1995; 171 (1): 170-176
Abstract

To ascertain the role of human immunodeficiency virus (HIV) and Mycobacterium tuberculosis transmission on multidrug-resistant (MDR) tuberculosis (TB) emergence in New York City, medical records, drug susceptibilities, and restriction fragment length polymorphisms (RFLPs) of TB cases at a city hospital between two 9-month periods (1987-1988 and 1990-1991) were reviewed. The proportion of TB patients with MDRTB increased from 10% (27/267) to 17% (38/222; P = .03). Among MDRTB patients of known HIV status, the proportion with HIV increased from 16% (3/19) to 58% (22/38; P = .006). HIV-infected MDRTB patients were more likely than the seronegative ones to have initial MDRTB (88% vs. 56%; P = .03). Among 56 MDR cases with RFLP results, 12 had unique patterns; 44 belonged to one of six clusters. During 1990-1991, 27 (75%) of 36 MDRTB patients were infected with strains isolated from HIV-seronegative patients during 1987-1988. The increase in MDRTB caused by transmission from immunocompetent to immunocompromised persons underscores the urgency of TB control in populations with increasing HIV prevalence.

View details for Web of Science ID A1995PZ87300024

View details for PubMedID 7798657
FLAGELLAR SEROTYPES OF SALMONELLA-TYPHI IN INDONESIA - RELATIONSHIPS AMONG MOTILITY, INVASIVENESS, AND CLINICAL ILLNESS JOURNAL OF INFECTIOUS DISEASES Grossman, D. A., WITHAM, N. D., Burr, D. H., Lesmana, M., Rubin, F. A., SCHOOLNIK, G. K., Parsonnet, J. 1995; 171 (1): 212-216
Abstract

While the H1-d flagellar serotype of Salmonella typhi has been found worldwide, the H1-j serotype occurs only in Indonesia. A cross-sectional survey in Indonesia compared epidemiologic, clinical, and pathogenetic characteristics of these two serotypes. S. typhi isolates were collected from patients with acute typhoid fever in four Indonesian cities. Flagellar serotype was determined by polymerase chain reaction amplification of the fliC locus of the flg gene. Of 321 isolates, 51 (15.9%) were H1-j. Patients with H1-j infection were older than those with H1-d (P < .001). Among 30 patients with known clinical outcomes, H1-j infection was associated with milder clinical illness than H1-d (P = .06). In vitro, H1-j isolates were both less motile on semi-solid agar plates (P = .004) and less invasive of HEp-2 cells (P = .002) than H1-d isolates. The association of decreased severity of illness with decreased motility and invasiveness suggests that flagellar properties are a component of S. typhi's virulence.

View details for Web of Science ID A1995PZ87300033

View details for PubMedID 7798666
INDUCTION OF HEAT-STABLE ENTEROTOXIN RECEPTOR ACTIVITY BY A HUMAN ALU REPEAT JOURNAL OF BIOLOGICAL CHEMISTRY Almenoff, J. S., Jurka, J., SCHOOLNIK, G. K. 1994; 269 (24): 16610-16617
Abstract

The heat-stable enterotoxins (ST) elaborated by enterotoxigenic Escherichia coli are a family of small cysteine-rich peptides that bind to specific epithelial receptors in the mammalian intestine, causing a secretory diarrhea. The expression of ST receptors is tightly regulated; they are found primarily in intestine, and their expression is developmentally modulated. One receptor for ST has been cloned, and its cDNA encodes a approximately 120-kDa particulate guanylyl cyclase (guanylyl cyclase-C). Recent studies suggest that there are additional ST receptors that are not homologous to guanylyl cyclase-C. We used an expression cloning strategy to identify intestinal mRNAs that lead to expression of ST receptor activity in transfected cells. Using an ST-specific affinity panning system, we identified a novel 1891-base pair cDNA that does not encode a receptor protein, but instead, consists primarily of untranslated sequence. This cDNA induced receptor activity in both COS and 293 embryonic kidney cells. Northern analysis of the T84 human intestinal cell line, from which this cDNA was cloned, suggests that it is part of a 7.8-kilobase mRNA transcript. This transcript was also identified in human small intestine and colon, as well as in several extra-intestinal tissues. Functional analysis of subcloned fragments reveals that ST binding activity is induced by a 457-base pair human Alu repetitive sequence within the cDNA and that the phenotype is independent of orientation. These findings suggest that a human Alu element induces expression of a unique ST receptor by a transacting mechanism. An unrelated Alu-rich genomic clone did not confer ST binding, suggesting that there may be structural and functional specificity within individual Alu sequences.

View details for Web of Science ID A1994NR29600017

View details for PubMedID 8206979
THE EPIDEMIOLOGY OF TUBERCULOSIS IN SAN-FRANCISCO - A POPULATION-BASED STUDY USING CONVENTIONAL AND MOLECULAR METHODS NEW ENGLAND JOURNAL OF MEDICINE Small, P. M., Hopewell, P. C., Singh, S. P., Paz, A., Parsonnet, J., Ruston, D. C., Schecter, G. F., Daley, C. L., SCHOOLNIK, G. K. 1994; 330 (24): 1703-1709
Abstract

The epidemiology of tuberculosis in urban populations is changing. Combining conventional epidemiologic techniques with DNA fingerprinting of Mycobacterium tuberculosis can improve the understanding of how tuberculosis is transmitted.We used restriction-fragment-length polymorphism (RFLP) analysis to study M. tuberculosis isolates from all patients reported to the tuberculosis registry in San Francisco during 1991 and 1992. These results were interpreted along with clinical, demographic, and epidemiologic data. Patients infected with the same strains were identified according to their RFLP patterns, and patients with identical patterns were grouped in clusters. Risk factors for being in a cluster were analyzed.Of 473 patients studied, 191 appeared to have active tuberculosis as a result of recent infection. Tracing of patients' contacts with the use of conventional methods identified links among only 10 percent of these patients. DNA fingerprinting, however, identified 44 clusters, 20 of which consisted of only 2 persons and the largest of which consisted of 30 persons. In patients under 60 years of age, Hispanic ethnicity (odds ratio, 3.3; P = 0.02), black race (odds ratio, 2.3; P = 0.02), birth in the United States (odds ratio, 5.8; P < 0.001), and a diagnosis of the acquired immunodeficiency syndrome (odds ratio, 1.8; P = 0.04) were independently associated with being in a cluster. Further study of patients in clusters confirmed that poorly compliant patients with infectious tuberculosis have a substantial adverse effect on the control of this disease.Despite an efficient tuberculosis-control program, nearly a third of new cases of tuberculosis in San Francisco are the result of recent infection. Few of these instances of transmission are identified by conventional contact tracing.

View details for Web of Science ID A1994NR10900002

View details for PubMedID 7910661
THE LIGHT ORGAN SYMBIONT VIBRIO-FISCHERI POSSESSES A HOMOLOG OF THE VIBRIO-CHOLERAE TRANSMEMBRANE TRANSCRIPTIONAL ACTIVATOR TOXR JOURNAL OF BACTERIOLOGY Reich, K. A., SCHOOLNIK, G. K. 1994; 176 (10): 3085-3088
Abstract

A cross-hybridizing DNA fragment to Vibrio cholerae toxR was cloned from the nonpathogenic light organ symbiont Vibrio fischeri, and three proteins homologous to V. cholerae ToxR, ToxS, and HtpG were deduced from its DNA sequence. V. fischeri ToxR was found to activate a V. cholerae ToxR-regulated promoter, and an antiserum raised against the amino-terminal domain of V. cholerae ToxR cross-reacts V. fischeri ToxR.

View details for Web of Science ID A1994NL83800041

View details for PubMedID 8188612
PURIFICATION OF SOMATIC PILI BACTERIAL PATHOGENESIS, PT B SCHOOLNIK, G. K. 1994; 236: 271-282
View details for Web of Science ID A1994BA88S00019

View details for PubMedID 7968615
CHARACTERIZATION OF FIMBRIAE PRODUCED BY ENTEROPATHOGENIC ESCHERICHIA-COLI JOURNAL OF BACTERIOLOGY Giron, J. A., HO, A. S., SCHOOLNIK, G. K. 1993; 175 (22): 7391-7403
Abstract

Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J. A. Girón, A. S. Y. Ho, and G. K. Schoolnik, Science 254:710-713, 1991). Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid. In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains. Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E. coli and F1845 of diffuse-adhering E. coli, respectively. The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E. coli, which suggests a different receptor specificity. Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin. Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane. We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC.

View details for Web of Science ID A1993MG71100033

View details for PubMedID 7901197
VARIABILITY OF HUMAN PAPILLOMAVIRUS DNA TESTING IN A LONGITUDINAL COHORT OF YOUNG-WOMEN OBSTETRICS AND GYNECOLOGY Moscicki, A. B., Palefsky, J., Smith, G., SIBOSHSKI, S., SCHOOLNIK, G. 1993; 82 (4): 578-585
Abstract

To determine the variability in human papillomavirus (HPV) DNA testing of the cervix in young women found positive for HPV DNA by dot blot hybridization on routine examination.Young women who were found to be HPV DNA-positive on routine screening using an RNA-DNA dot blot hybridization method were asked to return for repeat HPV DNA sampling, cytology, colposcopic examination, and biopsy if indicated. Those who had no histologic evidence of cervical dysplasia were asked to return every 4 months for cytology and HPV DNA testing using standardized RNA-DNA hybridization and polymerase chain reaction techniques.The women were followed for a mean of 27.6 months (range 13-40) with a mean of six visits (range four to ten). One-third of the women remained consistently or intermittently HPV DNA-positive by RNA-DNA dot blot hybridization, and almost 50% of the women remained consistently or intermittently positive using polymerase chain reaction techniques. Women were more likely to be positive by polymerase chain reaction than by RNA-DNA hybridization at both 1 year and after 2 years of follow-up (P < .05). However, rates for persistent positive tests by either method were similar at 1 and 2 years of follow-up. Forty percent of the subjects had new or different types than the original HPV type appear during follow-up. All five women who had evidence of spontaneous regression of cytologic abnormalities became HPV DNA-negative by both methods.Our data suggest that a portion of women infected with HPV appear to eliminate the infection over a relatively short period and are at low or no risk of developing disease. Persistent DNA negativity was also found in those women undergoing spontaneous regression. However, a substantial proportion of women remained intermittently positive by RNA-DNA hybridization and polymerase chain reaction. This finding suggests that the virus remains latent in some individuals and may undergo reactivation, defined by sufficient replication to allow detection by means less sensitive than polymerase chain reaction.

View details for Web of Science ID A1993LY72700021

View details for PubMedID 8397358
INTIMIN AND THE INTIMATE ATTACHMENT OF BACTERIA TO HUMAN-CELLS JOURNAL OF CLINICAL INVESTIGATION SCHOOLNIK, G. K. 1993; 92 (3): 1117-1118
View details for Web of Science ID A1993LX77000003

View details for PubMedID 8376575
MOLECULAR STRAIN TYPING OF MYCOBACTERIUM-TUBERCULOSIS TO CONFIRM CROSS-CONTAMINATION IN THE MYCOBACTERIOLOGY LABORATORY AND MODIFICATION OF PROCEDURES TO MINIMIZE OCCURRENCE OF FALSE-POSITIVE CULTURES JOURNAL OF CLINICAL MICROBIOLOGY Small, P. M., McClenny, N. B., Singh, S. P., SCHOOLNIK, G. K., Tompkins, L. S., Mickelsen, P. A. 1993; 31 (7): 1677-1682
Abstract

Molecular strain typing by restriction fragment length polymorphism analysis was used to demonstrate that two clusters of Mycobacterium tuberculosis cultures involving six patients resulted from cross-contamination in the mycobacteriology laboratory. Contaminated cultures were processed by the decontamination procedure and were read on the BACTEC instrument following acid-fast bacillus smear-positive specimens from patients with active tuberculosis. Investigation of these episodes suggested opportunities for modification of laboratory procedures to minimize cross-contamination and confirmed the adverse medical and public health consequences of false-positive cultures. Strain-typing results were used in decisions regarding patient care, including the curtailment of unnecessary treatment in one patient. Molecular strain typing appears to be a valuable means of identifying false-positive cultures of M. tuberculosis in selected settings.

View details for Web of Science ID A1993LJ20100001

View details for PubMedID 8102372
LIGAND-BASED HISTOCHEMICAL-LOCALIZATION AND CAPTURE OF CELLS EXPRESSING HEAT-STABLE ENTEROTOXIN RECEPTORS MOLECULAR MICROBIOLOGY Almenoff, J. S., Williams, S. I., Scheving, L. A., Judd, A. K., SCHOOLNIK, G. K. 1993; 8 (5): 865-873
Abstract

The heat stable enterotoxins (ST) of enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by binding specific intestinal receptors. Precise histochemical localization of ST receptors could provide more information about the pathophysiology of secretory diarrhoea and the role of ST receptors in normal biology. To accomplish this, we quantitatively coupled biotin to the N-terminus of ST1b using biotin-X-X-N-hydroxysuccinimide ester. The derivatized toxin (BST) has an apparent Kd of 11.7 +/- 10 nM for rat brush border receptors. We used BST in an affinity panning cell-capture system, to validate its ability to discriminate between receptor-positive and receptor-negative cells. Cell lines expressing ST receptors (human colon carcinoma T84, and COS cells transfected with guanylyl cyclase-C (GC-C) ST receptor cDNA) were captured to streptavidin and anti-biotin-coated plates with high efficiency and specificity. This system provides a novel approach to screening cells for the presence of unique ST-binding proteins. BST was then used with streptavidin-gold to demonstrate the cellular topography of ST receptors at the light microscopic level. Villus enterocytes were intensely stained, but only a faint signal was observed in upper crypts of rat small intestine. Thus, a gradient of increasing receptor density was seen as upper crypt cells matured into villus enterocytes. Higher magnification revealed that ST receptors are concentrated at the apical aspect of villus enterocytes. Recently, guanylin, a putative endogenous ligand for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts. Thus, ST receptors are concentrated in villus enterocytes, while guanylin appears to be produced at the base of the crypts.(ABSTRACT TRUNCATED AT 250 WORDS)

View details for Web of Science ID A1993LF03600009

View details for PubMedID 8102772
EXOGENOUS REINFECTION WITH MULTIDRUG-RESISTANT MYCOBACTERIUM-TUBERCULOSIS IN PATIENTS WITH ADVANCED HIV-INFECTION NEW ENGLAND JOURNAL OF MEDICINE Small, P. M., Shafer, R. W., Hopewell, P. C., Singh, S. P., MURPHY, M. J., Desmond, E., Sierra, M. F., SCHOOLNIK, G. K. 1993; 328 (16): 1137-1144
Abstract

In the United States there have been recent outbreaks of multidrug-resistant tuberculosis. These outbreaks have primarily involved persons infected with the human immunodeficiency virus (HIV).We collected clinical information on 17 patients seen at a New York City hospital who had repeatedly positive cultures for Mycobacterium tuberculosis. Analysis of restriction-fragment--length polymorphisms (RFLPs) was performed on serial isolates of M. tuberculosis obtained from these patients.Six patients had isolates that remained drug-susceptible, and the RFLP patterns of these isolates did not change over time. Eleven patients had isolates that became resistant to antimicrobial agents. The RFLP patterns of the isolates from six of these patients remained essentially unchanged (two strains showed one additional band) despite the development of drug resistance. In five other patients, however, the RFLP patterns of the isolates changed dramatically at the time that drug resistance was detected. The change in the RFLP pattern of the isolate from one patient appeared to be the result of contamination during processing in the laboratory. In the remaining four patients, all of whom had advanced HIV disease, the clinical and microbiologic evidence was consistent with the presence of active tuberculosis caused by a new strain of M. tuberculosis.Resistance to antituberculous drugs can develop not only in the strain that caused the initial disease, but also as a result of reinfection with a new strain of M. tuberculosis that is drug-resistant. Exogenous reinfection with multidrug-resistant M. tuberculosis can occur either during therapy for the original infection or after therapy has been completed.

View details for Web of Science ID A1993KY44700001

View details for PubMedID 8096066
CLONING AND CHARACTERIZATION OF THE BUNDLE-FORMING PILIN GENE OF ENTEROPATHOGENIC ESCHERICHIA-COLI AND ITS DISTRIBUTION IN SALMONELLA SEROTYPES MOLECULAR MICROBIOLOGY SOHEL, I., Puente, J. L., Murray, W. J., VUOPIOVARKILA, J., SCHOOLNIK, G. K. 1993; 7 (4): 563-575
Abstract

bfp, the structural gene of the major repeating bundle-forming pilus (BFP) subunit, was cloned from the enteroadherent factor (EAF) plasmid of enteropathogenic Escherichia coli (EPEC) strain B171 (O111:NM). The bfp open reading frame encoded a 193-amino-acid protein; comparison of this sequence with the biochemically determined N-terminal amino acid sequence showed that the mature pilin protein is comprised of 180 amino acids, that this sequence is similar to other members of the type IV pilin family, and that it is preceded by a 13-amino-acid signal peptide. Expression of the cloned bfp structural gene in an EPEC strain that had been cured of the EAF plasmid yielded a 21,000 dalton protein that co-migrated with the BFP precursor protein. Thus, other genes, probably carried by the EAF plasmid, are required for the maturation of the bfp product and for the production of extracellular pilus filaments. Use of bfp as a hybridization probe showed that homologous sequences are present in all tested EPEC strains and in 13 of 16 tested Salmonella serotypes. Fifty per cent of these bfp probe-sensitive salmonellae exhibited the localized-adherence (LA) phenotype when incubated with tissue culture cell monolayers, a trait previously associated with EAF plasmid-containing EPEC strains. Scanning electron micrographs of a bfp probe-sensitive, LA-positive Salmonella dublin strain showed that it grows as adherent colonies on infected monolayers and that within these colonies, BFP-like fibres form inter-bacterial linkages. For EAF plasmid-containing EPEC strains and for several Salmonella serotypes, BFP expression may lead to the development of adherent colonies on epithelial surfaces early in the infective process.

View details for Web of Science ID A1993KM73700009

View details for PubMedID 8096320
INFECTION-RATES, DISEASE FREQUENCY, PILIN GENE REARRANGEMENT, AND PILIN EXPRESSION IN CALVES INOCULATED WITH MORAXELLA-BOVIS PILIN-SPECIFIC ISOGENIC VARIANTS AMERICAN JOURNAL OF VETERINARY RESEARCH Ruehl, W. W., Marrs, C. F., George, L., BANKS, S. J., SCHOOLNIK, G. K. 1993; 54 (2): 248-253
Abstract

Pili have been implicated as virulence factors that result in increased infectivity of Moraxella bovis, the causative agent of infectious bovine keratoconjunctivitis (IBK). Healthy calves' eyes were inoculated with I- or Q-piliate or nonpiliate M bovis Epp63 to compare the pathogenicity of these isogenic variants. Pathogenicity was determined by the rate of persistent M bovis infection and the prevalence of clinical IBK. Inoculation with M bovis expressing the Q pili resulted in the highest frequency of infection and IBK, whereas I-piliate M bovis elicited a lower rate and nonpiliate M bovis did not result in infection. In vivo pilin gene rearrangement and pilin-type switching were evaluated by DNA hybridization and immunoblot. Gene rearrangement and type switching varied dependently, and were observed only in eyes inoculated with Q-piliate M bovis. This study suggests that Q pili are specific for colonization of bovine corneal epithelium, whereas I pili enable maintenance of an established infection.

View details for Web of Science ID A1993KJ76400009

View details for PubMedID 8094276
COLPOSCOPIC AND HISTOLOGIC-FINDINGS AND HUMAN PAPILLOMAVIRUS (HPV) DNA TEST VARIABILITY IN YOUNG-WOMEN POSITIVE FOR HPV DNA JOURNAL OF INFECTIOUS DISEASES Moscicki, A. B., Palefsky, J. M., Gonzales, J., Smith, G., SCHOOLNIK, G. K. 1992; 166 (5): 951-957
Abstract

The purpose of this study was to determine the prevalence of human papillomavirus (HPV)-related disease in women positive for HPV DNA by using a commercially available DNA detection kit and to compare these results to HPV DNA variability observed in repeated sampling. Young women attending family planning clinics who were positive for HPV DNA on routine screening were asked to return for a repeat HPV DNA test, cytology, colposcopy, and biopsy if indicated. Of the 78 women examined, 35% had biopsy-verified low-grade dysplasia of the cervix, and 64% had evidence of HPV-related disease somewhere in the anogenital area. Fifty percent of the women had a negative test on repeat sampling, but there was no association between DNA persistence at the second visit and colposcopic/histologic findings. In conclusion, the minority of women positive for HPV DNA have latent infection. In addition, variability in detection is common and unrelated to the presence of HPV-related disease.

View details for Web of Science ID A1992JV01500001

View details for PubMedID 1328408
CLONING AND CHARACTERIZATION OF FXP, THE FLEXIBLE PILIN GENE OF AEROMONAS-HYDROPHILA MOLECULAR MICROBIOLOGY HO, A. S., SOHEL, I., SCHOOLNIK, G. K. 1992; 6 (18): 2725-2732
Abstract

The flexible pilus of Aeromonas hydrophila is a morphologically and biochemically unique organelle which binds eukaryotic cell surfaces and whose expression is induced by specific physiochemical conditions. fxp, the structural gene coding for the flexible pilus subunit, was localized on a 7.6kb plasmid of A. hydrophila strain AH26. A putative Shine-Dalgarno sequence and -10 and -35 regions were identified, a signal peptide sequence delineated, and the coding sequence compared with other bacterial sequences and found to be unique. Plasmid and chromosomal DNA was prepared from 66 other Aeromonas strains and 12 strains from other bacterial genera and examined by Southern blot hybridization using a labelled fxp oligonucleotide and the 7.6kb plasmid as probes. No hybridizing sequences were identified except in the original strain, AH26. It is proposed that fxp codes for a highly evolved organelle, possibly widely distributed in nature, but that it is carried on a genetic element that is rapidly lost from most strains upon in vitro cultivation and storage.

View details for Web of Science ID A1992JP27100019

View details for PubMedID 1360140
MONOPHASIC AND BIPHASIC SALMONELLA-TYPHI - GENETIC HOMOGENEITY AND DISTINGUISHING CHARACTERISTICS MOLECULAR MICROBIOLOGY MOSHITCH, S., Doll, L., RUBINFELD, B. Z., STOCKER, B. A., SCHOOLNIK, G. K., Gafni, Y., Frankel, G. 1992; 6 (18): 2589-2597
Abstract

Several lines of evidence indicate a relatively low genetic heterogeneity in the natural Salmonella typhi population. However, some S. typhi isolates found in Indonesia express, instead of the usual fliC-d flagellin gene, a different flagellar gene fliC-j. In addition, Indonesian strains may have a second flagellar antigen fliC-z66. We have previously suggested, on the basis of the flagellar antigen constitution, that S. typhi evolved in an isolated human population in Indonesia. In order to test this hypothesis, we have gathered S. typhi isolates from around the world and tested the genetic heterogeneity among them. In general, polymorphism was greater in isolates from the Far East, as was indicated by Southern hybridizations with rDNA and fliC DNA probes. Gene fliC-j was not found in S. typhi isolates, other than those from Indonesia. However, the one-clone origin of S. typhi was indicated by a common DNA fingerprint pattern and by the occurrence, in the 5' end region of the fliC gene, of 10 scattered nucleotides that differ from the corresponding 10 nucleotides in other fliC alleles studied. These nucleotides were present in all isolates tested but did not change the amino acid sequence of the flagellin polypeptide.

View details for Web of Science ID A1992JP27100006

View details for PubMedID 1360138
AN OUTBREAK OF TUBERCULOSIS WITH ACCELERATED PROGRESSION AMONG PERSONS INFECTED WITH THE HUMAN-IMMUNODEFICIENCY-VIRUS - AN ANALYSIS USING RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISMS NEW ENGLAND JOURNAL OF MEDICINE Daley, C. L., Small, P. M., Schecter, G. F., SCHOOLNIK, G. K., McAdam, R. A., Jacobs, W. R., Hopewell, P. C. 1992; 326 (4): 231-235
Abstract

Tuberculosis typically develops from a reactivation of latent infection. Clinical tuberculosis may also arise from a primary infection, and this is thought to be more likely in persons infected with the human immunodeficiency virus (HIV). However, the relative importance of these two pathogenetic mechanisms in this population is unclear.Between December 1990 and April 1991, tuberculosis was diagnosed in 12 residents of a housing facility for HIV-infected persons. In the preceding six months, two patients being treated for tuberculosis had been admitted to the facility. We investigated this outbreak using standard procedures plus analysis of the cultured organisms with restriction-fragment-length polymorphisms (RFLPs).Organisms isolated from all 11 of the culture-positive residents had similar RFLP patterns, whereas the isolates from the 2 patients treated for tuberculosis in the previous six months were different strains. This implicated the first of the 12 patients with tuberculosis as the source of this outbreak. Among the 30 residents exposed to possible infection, active tuberculosis developed in 11 (37 percent), and 4 others (13 percent) had newly positive tuberculin skin tests. Of 28 staff members with possible exposure, at least 6 had positive tuberculin-test reactions, but none had tuberculosis.Newly acquired tuberculous infection in HIV-infected patients can spread readily and progress rapidly to active disease. There should be heightened surveillance for tuberculosis in facilities where HIV-infected persons live, and investigation of contacts must be undertaken promptly and be focused more broadly than is usual.

View details for Web of Science ID A1992HA43100004

View details for PubMedID 1345800
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
AN INDUCIBLE BUNDLE-FORMING PILUS OF ENTEROPATHOGENIC ESCHERICHIA-COLI SCIENCE Giron, J. A., HO, A. S., SCHOOLNIK, G. K. 1991; 254 (5032): 710-713
Abstract

Enteropathogenic Escherichia coli (EPEC), a cause of childhood diarrhea, grow on the surface of the small intestine and on cultured epithelial cells as colonies of adherent bacteria. When propagated on solid medium containing blood or attached to HEp-2 cells, EPEC express ropelike bundles of filaments, termed bundle-forming pili (BFP), that create a network of fibers that bind together the individual organisms. BFP were found to be expressed by five EPEC serogroups, each harboring a approximately 92-kilobase plasmid previously known to be important for virulence in humans. When two of these strains were cured of this plasmid, they neither expressed BFP nor grew as adherent colonies. An antiserum to BFP reduced the capacity of EPEC to infect cultured epithelial cells. BFP are composed of a repeating subunit of 19,500 daltons, the amino-terminal amino acid sequence of this subunit is homologous to that of the toxin-coregulated pilin of Vibrio cholerae.

View details for Web of Science ID A1991GN47400041

View details for PubMedID 1683004
LOCALIZED ADHERENCE BY ENTEROPATHOGENIC ESCHERICHIA-COLI IS AN INDUCIBLE PHENOTYPE ASSOCIATED WITH THE EXPRESSION OF NEW OUTER-MEMBRANE PROTEINS JOURNAL OF EXPERIMENTAL MEDICINE VUOPIOVARKILA, J., SCHOOLNIK, G. K. 1991; 174 (5): 1167-1177
Abstract

Enteropathogenic Escherichia coli grow as discrete colonies on the mucous membranes of the small intestine. A similar pattern can be demonstrated in vitro; termed localized adherence (LA), it is characterized by the presence of circumscribed clusters of bacteria attached to the surfaces of cultured epithelial cells. The LA phenotype was studied using B171, an O111:NM enteropathogenic E. coli (EPEC) strain, and HEp-2 cell monolayers. LA could be detected 30-60 min after exposure of HEp-2 cells to B171. However, bacteria transferred from infected HEp-2 cells to fresh monolayers exhibited LA within 15 min, indicating that LA is an inducible phenotype. Induction of the LA phenotype was found to be associated with de novo protein synthesis and changes in the outer membrane proteins, including the production of a new 18.5-kD polypeptide. A partial NH2-terminal amino acid sequence of this polypeptide was obtained and showed it to be identical through residue 12 to the recently described bundle-forming pilus subunit of EPEC. Expression of the 18.5-kD polypeptide required the 57-megadalton enteropathogenic E. coli adherence plasmid previously shown to be required for the LA phenotype in vitro and full virulence in vivo. This observation, the correspondence of the 18.5-kD polypeptide to an EPEC-specific pilus protein, and the temporal correlation of its expression with the development of the LA phenotype suggest that it may contribute to the EPEC colonial mode of growth.

View details for Web of Science ID A1991GM80300022

View details for PubMedID 1682410
THE ASSOCIATION BETWEEN HUMAN PAPILLOMAVIRUS DEOXYRIBONUCLEIC-ACID STATUS AND THE RESULTS OF CYTOLOGIC RESCREENING TESTS IN YOUNG, SEXUALLY ACTIVE WOMEN AMERICAN JOURNAL OF OBSTETRICS AND GYNECOLOGY Moscicki, A. B., Palefsky, J. M., Gonzales, J., SCHOOLNIK, G. K. 1991; 165 (1): 67-71
Abstract

We examined the utility of cytologic rescreening tests in women who had positive test results for human papillomavirus deoxyribonucleic acid but who were diagnosed as having benign conditions at cytologic testing. One hundred twenty-five Papanicolaou smears from women who were screened for human papillomavirus deoxyribonucleic acid were sent routinely to a private laboratory for diagnoses. These slides were then reviewed independently by two pathologists who were blinded to the human papillomavirus deoxyribonucleic acid results. The effects of cytologic rescreening in cases of both positive and negative human papillomavirus deoxyribonucleic acid were assessed by calculating z scores. Cervical intraepithelial neoplasia was diagnosed in 40% by pathologist A and in 20% by pathologist B of the human papillomavirus-positive subjects compared with none diagnosed by the private cytology laboratory (z = 3.09, p less than 0.005 and z = 1.98, p less than 0.05, respectively). No significant differences were found in the human papillomavirus-negative group. We conclude that cytologic rescreening in human papillomavirus deoxyribonucleic acid-positive women who were initially diagnosed as having benign cytologic results will yield a significant proportion of cases of cervical intraepithelial neoplasia.

View details for Web of Science ID A1991FY35800015

View details for PubMedID 1649551
LOCALIZATION OF THE RECEPTOR-BINDING REGION OF CLOSTRIDIUM-PERFRINGENS ENTEROTOXIN UTILIZING CLONED TOXIN FRAGMENTS AND SYNTHETIC PEPTIDES - THE 30 C-TERMINAL AMINO-ACIDS DEFINE A FUNCTIONAL BINDING REGION JOURNAL OF BIOLOGICAL CHEMISTRY Hanna, P. C., Mietzner, T. A., SCHOOLNIK, G. K., McClane, B. A. 1991; 266 (17): 11037-11043
Abstract

In this study a short sequence encoding the receptor-binding activity of the much larger 35-kDa enterotoxin elaborated by Clostridium perfringens was localized by recombinant DNA techniques. Defined fragments corresponding to portions of the enterotoxin gene were cloned into an Escherichia coli expression vector system, and these lysates were analyzed for their ability to compete for binding with native C. perfringens enterotoxin (CPE). The lysate containing CPE290-319 (CPE sequence encompassing residues 290-319) was shown to compete with 125I-CPE for specific binding sites on rabbit intestinal brush border membranes. To confirm this finding, a peptide corresponding to the CPE amino acid sequence 290-319 was synthesized and found to completely block CPE specific binding. To demonstrate directly that CPE290-319 can act as a competitive antagonist of CPE cytotoxicity for physiologic receptors, Vero cells were preincubated with either E. coli lysates containing CPE290-319 or the synthetic peptide corresponding to this sequence. Preincubation of Vero cells with either the lysate or the peptide completely protected these cells from CPE challenge. This information localizes the C-terminal 30 residues of CPE (CPE290-319) as a linear sequence sufficient for recognition and binding to the eukaryotic CPE receptor.

View details for Web of Science ID A1991FQ77400053

View details for PubMedID 1645721
CHARACTERIZATION OF INVIVO EXPRESSION OF THE HUMAN PAPILLOMAVIRUS TYPE-16 E4 PROTEIN IN CERVICAL BIOPSY TISSUES JOURNAL OF CLINICAL INVESTIGATION Palefsky, J. M., WINKLER, B., RABANUS, J. P., Clark, C., Chan, S., Nizet, V., SCHOOLNIK, G. K. 1991; 87 (6): 2132-2141
Abstract

The role of human papillomavirus (HPV) proteins in the pathogenesis of cervical intra-epithelial neoplasia (CIN) and invasive cervical cancer is poorly understood. To characterize E4 protein expression in 49 paraffin-embedded cervical biopsies representing different histopathologic grades of disease, antibodies were elicited to a synthetic peptide corresponding to amino acids 20-34 of a protein predicted to be encoded by the HPV 16 E4 open reading frame. The E4 protein was detected throughout the spectrum of CIN, from CIN1 to CIN3. Expression was localized to the cell nucleus, primarily in the superficial layers of the squamous cervical epithelium. Ultrastructural studies showed that the E4 protein was organized into compact, intranuclear arrays 25-35 nm in diameter. E4 protein expression was also demonstrated in some histologically normal tissues containing HPV 16 DNA, but not in any of five cervical cancers containing HPV 16 DNA. These results suggest that E4 protein expression may precede development of light microscopic tissue abnormalities, that it may continue through the spectrum of CIN, and that expression of this protein may be reduced or terminated in invasive cancer. The function of this protein remains unknown, but its nuclear localization may be consistent with a role in viral maturation.

View details for Web of Science ID A1991FP85100035

View details for PubMedID 1645754
Applications and transfer of technology to the nations of the South. Biotechnology and the control of childhood enteric infections. Infectious disease clinics of North America SCHOOLNIK, G. K. 1991; 5 (2): 265-275
Abstract

The optimal use of biotechnology to address the health care needs of developing countries entails the formation of interdisciplinary working groups linking basic biomedical scientists with public health workers, epidemiologists, physicians, and social scientists. Their mission should be congruent with and guided by the public health goals and primary care program of the country or region. Moreover, their research and development activities should lead to products that address a specific need and which are evaluable, cost-effective, and readily transferred to the public health sector. With respect to the enteric infections of childhood, the essential components of an integrated control effort are a field site where infections of this kind are common and readily studied; a local public health laboratory where the performance of new products can be tested and where technology transfer can occur; a basic science laboratory where molecular pathogenicity studies lead to new diagnostic tests, vaccines, and drugs; and a production laboratory where these products can be refined and prepared in sufficient amounts for field testing. This strategy is now being evaluated for the control of infantile diarrhea through the combined use of epidemiologic investigations, DNA probe and amplification techniques, and molecular fingerprinting. Together, these methods are yielding new information about microbial reservoirs and transmission systems; in turn, this information should lead to highly focused public health interventions.

View details for PubMedID 1869809
HUMAN PAPILLOMAVIRUS INFECTION IN SEXUALLY ACTIVE ADOLESCENT FEMALES - PREVALENCE AND RISK-FACTORS PEDIATRIC RESEARCH Moscicki, A. B., Palefsky, J., Gonzales, J., SCHOOLNIK, G. K. 1990; 28 (5): 507-513
Abstract

We examined the prevalence of human papillomavirus (HPV) infection, and associated risk factors for infection with HPV types 6, 11, 16, 18, 31, 33, and 35, in 661 sexually active adolescent females attending family planning clinics. Fifteen percent were positive for HPV DNA by RNA-DNA dot-blot hybridization. More than 60% of the HPV-positive subjects harbored at least one of the following cancer-related HPV types: 16, 18, 31, 33, or 35. Those with HPV had a mean range of four to 10 lifetime sexual partners compared with a mean range of one to three in those without HPV (p less than 0.001). After the analysis was adjusted for number of lifetime sexual partners, no other risk factor was associated with HPV infection. We conclude that oncogenic-related HPV types are common sexually transmitted organisms found in our population. The strong relationship with number of sexual partners suggests that acquisition of HPV infection is predominantly influenced by sexual behavior. However, in our population, confounders such as oral contraceptive use, past history of Chlamydia trachomatis infection, or substance abuse were not found to be significant independent risk factors.

View details for Web of Science ID A1990EE79800017

View details for PubMedID 2175024
HELA-CELL INVASION BY A STRAIN OF ENTEROPATHOGENIC ESCHERICHIA-COLI THAT LACKS THE O-ANTIGENIC POLYSACCHARIDE MOLECULAR MICROBIOLOGY Riley, L. W., JUNIO, L. N., SCHOOLNIK, G. K. 1990; 4 (10): 1661-1666
Abstract

The interaction with HeLa cells of an enteropathogenic Escherichia coli (EPEC) strain and its plasmid-cured derivative strain was examined. An O111:NM EPEC strain B171 harbours a 54 megadalton plasmid (pYR111) necessary for the expression of both localized adherence (LA) to HeLa cells and the O-repeating side chain of the lipopolysaccharide. Under light microscopy, the plasmid-cured derivative strain B171-4 was observed to interact with HeLa cells in a pattern distinct from LA. Transmission electron microscopy showed that the bacteria were internalized by HeLa cells. In contrast, strain B171 induced pedestal-like projections and invaginations of the plasma membrane, but was never completely internalized. A quantitative assay to determine the number of internalized bacteria revealed that strain B171-4 was internalized at levels 30-70-fold higher than those of avirulent E. coli strains. Cytochalasin B reduced the levels of internalization of both strain B171-4 and an enteroinvasive E. coli strain (E11), but did not affect LA by strain B171. These results suggest that EPEC strain B171 may carry a specific chromosomally determined surface factor needed to initiate internalization by HeLa cells. However, a plasmid-determined factor alters the nature of this interaction; the combined effects of the chromosomal and plasmid determinants lead to the characteristic attachment of the bacteria in clusters on the surface of the eukaryotic cell.

View details for Web of Science ID A1990ED49600007

View details for PubMedID 1706454
THE PILI OF AEROMONAS-HYDROPHILA - IDENTIFICATION OF AN ENVIRONMENTALLY REGULATED MINI PILIN JOURNAL OF EXPERIMENTAL MEDICINE HO, A. S., Mietzner, T. A., SMITH, A. J., SCHOOLNIK, G. K. 1990; 172 (3): 795-806
Abstract

Ultrastructural studies of Aeromonas hydrophila strain AH26 revealed two distinctive pilus types: "straight" pili appear as brittle, rod-like filaments, whereas "flexible" pili are supple and curvilinear. Straight pili are produced constitutively under all tested conditions of growth. In contrast, the expression of flexible pili is regulated by physical and chemical variables, being produced at 22 vs. 37 degrees C, in a liquid vs. a solid medium, and when the availability of free-iron is reduced by the presence of deferoxamine mesylate. Both pilus proteins were purified and biochemically and functionally characterized. The major repeating subunit of the straight pilus is a 17,000-mol wt polypeptide with amino acid sequence homology with Escherichia coli type 1 and Pap pili. The flexible pilus filament is a homopolymer composed of a novel 46 amino acid polypeptide. Resistance of the flexible pilus filament to disaggregation using various chemical treatments was demonstrated; its stability as a polymer and its apparent mechanical strength seem to be conferred by a 20 amino acid hydrophobic, COOH-terminal domain. Purified straight pili lack hemagglutinating function. In contrast, purified flexible pili cause the agglutinin of human, guinea pig, ovine, bovine, and avian erythrocytes, although this property could only be demonstrated in the presence of divalent cations and was most evident at 4 vs. 22 degrees C. Taken together, these results suggest that the pathogenic and ecological roles of the flexible pilus are related to this species' existence as a free-living organism in aquatic environments and its ability to cause infections, both in cold-blooded vertebrates and the human intestine.

View details for Web of Science ID A1990DW49100014

View details for PubMedID 1974915
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
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
[Risk factors for acute infantile diarrhea in a rural community in Chiapas, Mexico. A strategy for intervention]. Boletín de la Oficina Sanitaria Panamericana. Pan American Sanitary Bureau Riley, L. W., Castro Muñoz, E., Zárate, R. J., SIBLEY, B., Keller, J., Zárate, L. G., Millán Velasco, F., SCHOOLNIK, G. K. 1990; 108 (2): 93-99
Abstract

A community-based, case-control study was conducted during the summer peak season for diarrhea in the highlands of Chiapas, Mexico, to identify risk and protective factors associated with acute diarrhea in children less than 6 years of age. To estimate the diarrheal morbidity rate, the community was divided into 13 sectors, each of about 20 households. A resident (volunteer mother) made daily visits to every household in her sector to identify new cases of diarrhea. During 3 weeks of surveillance, 63 children with diarrhea and 48 control children were identified. The diarrheal attack rate during this period for children less than 6 years of age was 30%. Analysis of 29 neighborhood-matched case-control pairs showed that children with diarrhea were more likely than their controls to have had a mother with diarrhea in the 2 weeks preceding the onset of the child's diarrhea (P less than 0.05; relative risk = 10). The association of childhood diarrhea with maternal diarrhea may serve as a focus for more detailed studies as well as an intervention that may be appropriate and effective for this community.

View details for PubMedID 2139786
Peptides: chemistry, biology, and pharmacology. Advances in pharmacology (San Diego, Calif.) Judd, A. K., SCHOOLNIK, G. K. 1990; 21: 221-285
View details for PubMedID 2265127
Risk factors for acute childhood diarrhea in the highlands of Chiapas, Mexico: a strategy for intervention. Bulletin of the Pan American Health Organization Riley, L. W., Castro Muñoz, E., Zárate, R. J., SIBLEY, B., Keller, J., Zárate, L. G., Millán Velasco, F., SCHOOLNIK, G. K. 1990; 24 (2): 210-216
Abstract

A case-control study was conducted in the highlands of Chiapas, Mexico, to identify factors associated with acute diarrhea in children less than six years old. The study found that the diarrhea attack rate among the children surveyed during three weeks in the month of August (the peak diarrhea season) was approximately 30%, and that children whose mothers had diarrhea were especially likely to contract the illness themselves. The methods employed could prove relevant to studies in other areas, and the results obtained could provide the basis for more detailed study of the area involved--and for preventive action.

View details for PubMedID 2379024
MULTIPLE T-CELL AND B-CELL EPITOPES IN THE S1 SUBUNIT (A-MONOMER) OF THE PERTUSSIS TOXIN MOLECULE JOURNAL OF IMMUNOLOGY Oksenberg, J. R., Ko, C., Judd, A. K., Lim, M., Kent, A., SCHOOLNIK, G. K., Steinman, L. 1989; 143 (12): 4227-4231
Abstract

The immunogenicity and reactogenicity of Bordetella pertussis vaccine are mediated in part by the S1 subunit of pertussis toxin (PT). To identify the immune epitopes in the S1 subunit of PT, synthetic peptides were prepared and tested for their capacity to induce antibodies in mice with different MHC genotypes. In BALB/c mice, peptides corresponding to sequences 1-17, 70-82 and 189-199 generate T cell proliferative responses, induce the production of antibodies capable of neutralization of the toxin in the Chinese hamster ovary-cell assay, and protect mice from a shock-like syndrome caused by alternate injections of BSA and PT. Protection and neutralization correlated with the ability of these peptides to elicit high anti-PT titers. Different B cell epitopes were detected in other inbred mouse strains. The antibody reactivity against synthetic peptides from two infants vaccinated with pertussis vaccine was tested. These infants had antibodies reactive to a variety of epitopes in the S1 subunit, including peptides 1-17, 70-82, 99-112, 135-145, and 189-199. Thus, it appears that there are multiple T and B cell epitopes in the S1 subunit of PT.

View details for Web of Science ID A1989CD70600054

View details for PubMedID 2480389
MULTI-GENE AMPLIFICATION - SIMULTANEOUS DETECTION OF 3 VIRULENCE GENES IN DIARRHEAL STOOL MOLECULAR MICROBIOLOGY Frankel, G., Giron, J. A., VALMASSOI, J., SCHOOLNIK, G. K. 1989; 3 (12): 1729-1734
Abstract

Enterotoxigenic Escherichia coli (ETEC) and Shigella account for a substantial proportion of acute diarrhoeal illnesses among Third-World children. Rapid detection of these infectious agents in faeces followed by the prompt implementation of public health measures could help reduce their spread during the early phase of epidemics. Towards this end, three pairs of synthetic oligonucleotide primers were prepared and shown to hybridize specifically to the genes encoding the heat-stable (ST) and the heat-labile (LT) enterotoxins of ETEC and to invasion-associated loci (ial) of the large Shigella virulence plasmid. When the three primer pairs were used together in the polymerase chain reaction (PCR), the three corresponding genetic loci could be simultaneously amplified using DNA extracted directly from stool; the amplified products were readily detected by ST-, LT- and ial-specific, alkaline phosphatase-labelled oligonucleotide probes (AP probes). The performance of this system was evaluated in a Mayan community in southeastern Mexico, where diarrhoeal illnesses are a common cause of childhood morbidity and mortality. Using only simple and inexpensive laboratory equipment, multigene amplification with these primers and probes led to the identification of ETEC and/or Shigella in the stools of 20 out of 71 children with diarrhoea; the procedure could be completed in seven hours and was more sensitive than conventional diagnostic tests or DNA probes used without amplification.

View details for Web of Science ID A1989CH04400006

View details for PubMedID 2695745
INTRAGENIC RECOMBINATION IN A FLAGELLIN GENE - CHARACTERIZATION OF THE H1-J GENE OF SALMONELLA-TYPHI EMBO JOURNAL Frankel, G., Newton, S. M., SCHOOLNIK, G. K., STOCKER, B. A. 1989; 8 (10): 3149-3152
Abstract

Salmonella typhi, the etiologic agent of typhoid fever, typically has only a phase-1 flagellar antigen, d, but some isolates, found only in Indonesia, have antigen j instead, and may have a second flagellar antigen, z66. It appears that intragenic recombination involving a directly repeated 11 bp sequence in the H1-d flagellin gene changed the flagellar antigen to j, by deleting 261 bp in its central, antigenically determinant, part. Sequencing of the hypervariable regions of genes H1-d and H1-j, and hybridization of such genes, after amplification by the polymerase chain reaction, with oligonucleotide probes specific for the deleted segment or for the sequence produced by the recombination confirmed that all the j alleles have the postulated deletion. By applying the polymerase chain reaction to study S. typhi isolates from Jakarta, not previously tested in respect to flagellar antigen, we showed that gene H1-j was nearly as common as H1-d in these isolates.

View details for Web of Science ID A1989AR14000046

View details for PubMedID 2583095
UNIQUE SEQUENCES IN REGION-VI OF THE FLAGELLIN GENE OF SALMONELLA-TYPHI MOLECULAR MICROBIOLOGY Frankel, G., Newton, S. M., SCHOOLNIK, G. K., STOCKER, B. A. 1989; 3 (10): 1379-1383
Abstract

The H1 (now renamed fliC; lino et al., 1988) alleles specifying antigenically different Salmonella flagellins are identical at their ends but differ greatly towards the middle, where there are two hypervariable segments (regions IV and VI). The flagellar antigen, d, of Salmonella typhi, is found also as phase-1 antigen in many other Salmonella species. We cloned the H1-d gene of a strain of S. typhi and determined the nucleotide sequence of its two hypervariable regions. Comparison with gene H1-d of Salmonella muenchen showed substantial differences in region VI: four scattered amino acid differences and ten adjacent amino acids in the inferred S. typhi sequence, all of which differ from the corresponding nine amino acids in the S. muenchen sequence. The results of polymerase chain reaction amplification indicated the presence of the S. typhi version in all of 18 additional S. typhi strains and the presence of the S. muenchen version in all four non-S. typhi species with flagellar antigen d. The difference in amino acid sequence in segment VI may be responsible for the minor serological differences between antigens d of S. typhi and antigen d of S. muenchen.

View details for Web of Science ID A1989AW76900008

View details for PubMedID 2615651
PROBING THE SURFACE OF NEISSERIA-GONORRHOEAE - IMMUNOELECTRON MICROSCOPIC STUDIES TO LOCALIZE CYANOGEN-BROMIDE FRAGMENT-2 IN GONOCOCCAL PILI MOLECULAR MICROBIOLOGY Robinson, E. N., Clemens, C. M., SCHOOLNIK, G. K., McGee, Z. A. 1989; 3 (1): 57-64
Abstract

Common epitopes accessible to antibody on purified macromolecules or structurally altered gonococci may not be accessible to antibody when those macromolecules are in their native state on the surface of intact organisms. To determine the immunologic accessibility of cyanogen bromide fragment 2 (CNBr2), a portion of the gonococcal pilin molecule that is common to all gonococcal strains on the surface of viable gonococci, probes composed of specific CNBr2 antibodies linked to gold spheres were manufactured. When whole piliated gonococci were exposed to these anti-CNBr2 immunological probes and examined using transmission electron microscopy, no significant marketing of native pili was evident. These probes, however, detected CNBr2 in purified form. The epitopes encompassed within the CNBr2 portion of pili appear to be inaccessible to anti-CNBr2 probes within native gonococcal pili.

View details for Web of Science ID A1989T347800008

View details for PubMedID 2469938
STRUCTURE-FUNCTION ANALYSIS OF PROTEIN ACTIVE-SITES WITH ANTIIDIOTYPIC ANTIBODY METHODS IN ENZYMOLOGY Ludwig, D. S., SCHOOLNIK, G. K. 1989; 178: 163-171
Abstract

Antigen and internal image-bearing anti-idiotypic antibody, owing to potential differences in size and chemical nature, need not necessarily demonstrate identical binding specificities. Such differences, termed "dissociability," may be exploited in structure-function analysis of receptor-ligand interaction to identify functionally important amino acid residues, define receptor class, or distinguish receptor conformation. In this sense, ligand and the anti-idiotypes they elicit constitute alternative and complementary probes of protein active sites.

View details for Web of Science ID A1989CX43700011

View details for PubMedID 2601623
MHC-RESTRICTED RECOGNITION OF IMMUNOGENIC T-CELL EPITOPES OF PERTUSSIS TOXIN REVEALS DETERMINANTS IN MAN DISTINCT FROM THE ADP-RIBOSYLASE ACTIVE-SITE JOURNAL OF EXPERIMENTAL MEDICINE Oksenberg, J. R., Judd, A. K., Ko, C., Lim, M., Fernandez, R., SCHOOLNIK, G. K., Steinman, L. 1988; 168 (5): 1855-1864
Abstract

The S1 subunit of Pertussis toxin (PT) is responsible for the reactogenicity and in part the immunogenicity of Bordetella pertussis vaccine. The critical residues associated with the immunomodulatory effects of PT were located around Glu140 in the S1 subunit. In man, T cell responses to PT are directed at S1 peptides distinct from Glu140. Two such epitopes, p64-75 and p151-161, are immunogenic in a panel of individuals covering a wide range of HLA genotypes. The response to PT peptides is HLA class II restricted. The response to p64-75 is blocked by an anti-HLA-DQ mAb, while that to p151-161 is blocked by an anti-HLA-DR mAb. These findings may allow for the development of a B. pertussis vaccine free from reactogenicity.

View details for Web of Science ID A1988Q904300025

View details for PubMedID 2460578
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
COMMON EPITOPES OF PILIN OF NEISSERIA-MENINGITIDIS JOURNAL OF INFECTIOUS DISEASES Stephens, D. S., Whitney, A. M., SCHOOLNIK, G. K., Zollinger, W. D. 1988; 158 (2): 332-342
Abstract

We assessed distribution, immunogenicity, and accessibility of common epitopes of meningococcal pilins. When used as probes in western immunoblots, antisera to gonococcal pili; gonococcal pilin fragment CNBR-2; peptide sequences 41-50, 48-60, and 69-84 of gonococcal pilin; and monoclonal antibody 2-1-Fc recognized meningococcal pilins of different isolates. Meningococcal pilins could be separated into distinct groups based on reactivity with these probes. Common epitopes were conserved on meningococcal pilins isolated during laboratory passage and during infection of human nasopharyngeal organ cultures. Antibodies to common epitopes of pilin were, however, rarely detected in convalescent sera from patients with meningococcal or gonococcal bacteremia. Except for an epitope involving peptide sequence 48-60, immunoelectron microscopy indicated that common epitopes of pilin were not readily accessible on pili assembled on meningococci. Our antisera also did not block attachment of piliated meningococci to human buccal epithelial cells.

View details for Web of Science ID A1988P697200008

View details for PubMedID 2457056
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
3-DIMENSIONAL STRUCTURE OF CHOLERA-TOXIN PENETRATING A LIPID-MEMBRANE SCIENCE RIBI, H. O., Ludwig, D. S., Mercer, K. L., SCHOOLNIK, G. K., Kornberg, R. D. 1988; 239 (4845): 1272-1276
Abstract

Two-dimensional crystals of cholera toxin bound to receptors in a lipid membrane give diffraction extending to 15 A resolution. Three-dimensional structure determination reveals a ring of five B subunits on the membrane surface, with one-third of the A subunit occupying the center of the ring. The remaining mass of the A subunit appears to penetrate the hydrophobic interior of the membrane. Cleavage of a disulfide bond in the A subunit, which activates the toxin, causes a major conformational change, with the A subunit mostly exiting from the B ring.

View details for Web of Science ID A1988M425400021

View details for PubMedID 3344432
HIGH-RESOLUTION MAPPING OF SEROVAR-SPECIFIC AND COMMON ANTIGENIC DETERMINANTS OF THE MAJOR OUTER-MEMBRANE PROTEIN OF CHLAMYDIA-TRACHOMATIS JOURNAL OF EXPERIMENTAL MEDICINE Stephens, R. S., Wagar, E. A., SCHOOLNIK, G. K. 1988; 167 (3): 817-831
Abstract

The principal surface protein antigen of Chlamydia trachomatis is the major outer membrane protein (MOMP). The MOMP is antigenically complex. Among the 15 serovars of C. trachomatis, mAbs define serovar-, subspecies-, and species-specific determinants on MOMP. The molecular basis of the antigenic diversity of these proteins is reflected in amino acid variable sequence domains. We have mapped the dominant topographic antigenic determinants of MOMP that are defined by mAbs. Using recombinant DNA approaches we have identified the linear distribution of two antigenic domains. One domain contains a serovar-specific determinant and the other contains subspecies- and species-specific determinants. These antigenic domains correspond to two amino acid sequence variable domains. Synthetic peptides were immunogenic and these resolved the serovar-specific determinant within a 14-amino acid peptide. The subspecies- and species-specific determinants were overlapping within a 16-amino acid peptide.

View details for Web of Science ID A1988M691400007

View details for PubMedID 2450954
SYNTHETIC PEPTIDES CORRESPONDING TO PROTECTIVE EPITOPES OF ESCHERICHIA-COLI DIGALACTOSIDE-BINDING PILIN PREVENT INFECTION IN A MURINE PYELONEPHRITIS MODEL PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schmidt, M. A., OHANLEY, P., LARK, D., SCHOOLNIK, G. K. 1988; 85 (4): 1247-1251
Abstract

Synthetic peptides corresponding to five segments of a globoside (Gal-Gal)-binding pilin sequence [residues 5-12 (R5-12), R65-75, R93-104, R103-116, and R131-143], cyanogen bromide fragment II (CNBr-II, R53-163), and purified, intact Gal-Gal pili were prepared as vaccines and tested for their efficacy in a BALB/c murine model of pyelonephritis. Intact Gal-Gal pili, CNBr-II, and synthetic peptides R5-12 and R65-75 engendered antibodies that bound the homologous pilin protein and prevented urine and renal colonization in most vaccine recipients. Protection correlated with serum anti-pilus IgG ELISA titers greater than or equal to 1:250. The efficacy afforded by synthetic peptides R5-12 and R65-75 in vaccinated mice indicates that linear "antigenic" determinants in separate cyanogen bromide fragments encode "protective" epitopes. Peptides R93-104, R103-116, and R131-143 lacked efficacy, indicating that not all regions of the sequence are serologically equivalent. The crossreactivity of the peptide antisera for different Gal-Gal pilins was also assessed and correlated with the sequence homology of the corresponding regions. Antiserum to peptide R65-75, which corresponds to a region of unconserved sequence in heterologous pilins, bound only the homologous pilin. Thus, it specifies a type-specific protective epitope. Antiserum to synthetic peptide R5-12, which corresponds to a region of conserved sequence, bound Gal-Gal pilins from seven of eight pyelonephritis strains, indicating that it specifies a crossreacting protective epitope.

View details for Web of Science ID A1988M196200063

View details for PubMedID 2448796
HLA-A2 PEPTIDES CAN REGULATE CYTOLYSIS BY HUMAN ALLOGENEIC LYMPHOCYTES-T NATURE CLAYBERGER, C., Parham, P., Rothbard, J., Ludwig, D. S., SCHOOLNIK, G. K., KRENSKY, A. M. 1987; 330 (6150): 763-765
Abstract

The class-I and class-II molecules encoded by the major histocompatibility complex (MHC) are homologous proteins which allow cytotoxic and helper T cells to recognize foreign antigens. Recent studies have shown that the form of the antigen recognized by T cells is generally not a native protein but rather a short peptide fragment and that class-II molecules specifically bind antigenic peptides. Furthermore, the three-dimensional structure of the human MHC class-I molecule, HLA-A2, is consistent with a peptide-binding function for MHC class-I molecules. An outstanding question concerns the molecular nature and involvement of MHC-bound peptides in antigens recognized by alloreactive T cells. In this study the effects of peptides derived from HLA-A2 on cytolysis of alloreactive cytotoxic T cells (TC) cells are presented. Peptides can inhibit lysis by binding to the T cell or sensitize to lysis by binding an HLA-A2-related class-I molecule (HLA-Aw69) on the target cell. Thus, allospecific TC cells can recognize HLA-derived peptides in the context of the MHC.

View details for Web of Science ID A1987L431600073

View details for PubMedID 3501071
IMPORTANCE OF DISULFIDE BRIDGES IN THE STRUCTURE AND ACTIVITY OF ESCHERICHIA-COLI ENTERO-TOXIN ST1B PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gariepy, J., Judd, A. K., SCHOOLNIK, G. K. 1987; 84 (24): 8907-8911
Abstract

A 13-amino acid sequence of the Escherichia coli heat-stable enterotoxin ST1b encodes its receptor-binding and diarrheal functions. This sequence includes six cysteines involved in three intramolecular disulfide bridges. To determine the importance of disulfide bridges to the biological activity of ST1b, we synthesized 15 analogues of the tridecapeptide representing all possible replacements of two of the six cysteines by alanines. Only 2 analogues--namely, A6,11ST1b-(6-18) and A10,18ST1b(6-18)--could inhibit the binding of a radiolabeled analogue of ST1b to rat intestinal cells. The purified peptides were, respectively, 4200 and 130 times less effective as inhibitors than ST1b(6-18), the sequence that includes all six cysteines. In addition, both peptides produce diarrhea when given orally to suckling mice. These analogues share in common only two cysteines (Cys-7 and Cys-15), suggesting that four cysteines, two of which are Cys-7 and Cys-15, are necessary for activity. A pattern of disulfide linkages is proposed where Cys-7 is paired to Cys-15, Cys-6 to Cys-11, and Cys-10 to Cys-18, the preceding disulfide bridges being ranked in descending order of importance in terms of their respective contribution to the activity of the enterotoxin. Using this disulfide bridge arrangement and constraints derived from NMR spectroscopy, we propose a folding pattern for the toxic domain of ST1b.

View details for Web of Science ID A1987L760100033

View details for PubMedID 2827159
PLASMID-ENCODED EXPRESSION OF LIPOPOLYSACCHARIDE O-ANTIGENIC POLYSACCHARIDE IN ENTEROPATHOGENIC ESCHERICHIA-COLI INFECTION AND IMMUNITY Riley, L. W., JUNIO, L. N., LIBAEK, L. B., SCHOOLNIK, G. K. 1987; 55 (9): 2052-2056
Abstract

The role of a plasmid in the virulence activity of an enteropathogenic Escherichia coli (EPEC) strain belonging to serotype 0111:NM was examined. EPEC strain B171, which is resistant to chloramphenicol, streptomycin, sulfathiazole, and tetracycline, harbors a 54-megadalton plasmid, pYR111, and exhibits localized adherence (LA) with HeLa cells. Curing the plasmid yielded strain B171-4, which had lost the ability to exhibit LA, resistance to the antibiotics, and the lipopolysaccharide (LPS) O-antigenic polysaccharide. To confirm that these phenotypic characteristics were specified by pYR111, the plasmid was transferred by conjugation into a nalidixic acid-resistant strain of E. coli HB101. LA and antimicrobial resistance were expressed in most of the transconjugants examined. The O-polysaccharide side chains, antigenically reactive with O111-specific antiserum, were also expressed by the transconjugants. Although EPEC plasmids coding for both drug resistance and LA have been described, an EPEC plasmid encoding the expression of an LPS O antigen has not been previously reported. Similar findings described for some Shigella and Salmonella strains suggest that plasmid-encoded modification of the LPS in some enteric bacterial species may be more common than previously recognized and may contribute to the characteristic virulence activity of the organism.

View details for Web of Science ID A1987J684900017

View details for PubMedID 3305360
ANTIRECEPTOR ANTIBODIES DESIGNED TO ELICIT INTERNAL IMAGE-BEARING ANTIIDIOTYPES - A POSSIBLE AIDS VACCINE MEDICAL HYPOTHESES Ludwig, D. S., SCHOOLNIK, G. K. 1987; 23 (3): 303-307
Abstract

Two obstacles hinder the development of an AIDS vaccine: (1) the AIDS virus exhibits extensive amino acid heterogeneity between isolates and (2) antibodies elicited by virus during the course of natural infection are often non-neutralizing. A vaccine designed to induce anti-idiotypic antibodies against the virus' receptor on T-cells, T4, should, in principle, overcome these obstacles. Such antibody could contain an "internal image" of T4 and bind the receptor binding domain of the virus. Since this domain is both critical to function and, therefore, poorly susceptible to antigenic variation, anti-receptor anti-idiotypic antibodies may demonstrate broad, strain-independent crossreactivity and block viral adherence.

View details for Web of Science ID A1987J123000010

View details for PubMedID 3039322
ANTIIDIOTYPIC ANTIBODIES AS PROBES OF PROTEIN ACTIVE-SITES - APPLICATION TO CHOLERA-TOXIN SUBUNIT-B PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ludwig, D. S., Finkelstein, R. A., Karu, A. E., Dallas, W. S., Ashby, E. R., SCHOOLNIK, G. K. 1987; 84 (11): 3673-3677
Abstract

Since Jerne proposed a "network" theory of immune regulation, the properties of anti-idiotypic antibodies (anti-IdAb) have been investigated widely. Anti-IdAb raised against antibodies to a variety of ligands have been shown to bind the ligands' receptors. Thus, the combining site of an anti-IdAb may contain information regarding the three-dimensional structure of an antigen. However, this remarkable property of "internal imagery" has not been exploited for structural investigation at the molecular level. In the present report, a monoclonal "auto"-anti-IdAb was raised against ganglioside GM1 (a cell-surface glycolipid that binds cholera toxin) and was shown to crossreact with the B subunit of cholera toxin. This antibody was presumed to recognize amino acid residues located within the GM1 binding domain. To identify these residues, the antibody was screened against homologous toxins purified from enterotoxigenic strains of Escherichia coli and chimeric peptides produced by recombinant methods. Amino acid variation at position 4 from the N terminus of these proteins was found to disrupt antibody binding. Since the toxins and chimera are all closely related in structure and function, the residue at position 4 (an asparagine in cholera toxin B subunit) appears to be in the epitope of the antibody and, by implication, in the GM1 binding site. Of particular significance, this structural detail could not be deduced with GM1 alone. It would seem that ligand and anti-ligand anti-IdAb encode similar stereochemical information but do so with different "chemical alphabets," giving rise to distinct binding specificities.

View details for Web of Science ID A1987H603000029

View details for PubMedID 3473474
PURIFICATION AND CHARACTERIZATION OF THE MAJOR IRON-REGULATED PROTEIN EXPRESSED BY PATHOGENIC NEISSERIAE JOURNAL OF EXPERIMENTAL MEDICINE Mietzner, T. A., Bolan, G., SCHOOLNIK, G. K., Morse, S. A. 1987; 165 (4): 1041-1057
Abstract

This report describes a method to purify the major iron-regulated protein (MIRP) expressed by N. gonorrhoeae and N. meningitidis. This purification procedure involves maximal expression of the MIRP by growing the organisms on iron-limited media; cellular disruption by sonication followed by centrifugal fractionation; selective solubilization of the MIRP with the cationic detergent hexadecyltrimethylammonium bromide; cation-exchange chromatography in the presence of this detergent; and gel filtration chromatography. The MIRP purified by this technique migrates as a single band when analyzed by SDS-PAGE. The purified MIRP displayed an unusually basic isoelectric point, this value being greater than 9.35. Further biochemical analysis revealed the highly conserved nature of this protein isolated from the two pathogenic species of the genus Neisseria. For example, the amino acid composition of the meningococcal and gonococcal MIRPs were nearly identical and amino terminal sequence analysis showed that both shared the identical primary sequence through residue 48. Surprisingly, the first five NH2-terminal residues of the MIRPs exhibited homology with the first five residues of the gonococcal porin, protein I. Purified preparations of the MIRP exhibited a characteristic pink color reminiscent of the basic iron-binding protein lactoferrin. This observation coupled with the property of iron-regulation prompted us to analyze purified MIRP for iron-content. Approximately 0.5 mol iron per 1 mol of MIRP was detected. This study is the first to show that iron is associated with the MIRP, a property that may implicate this protein as playing a direct role in neisserial iron assimilation. While the precise function of the MIRP is not known, the availability of this protein in pure and biologically relevant quantities will allow further studies to elucidate its pathobiologic function.

View details for Web of Science ID A1987G732700009

View details for PubMedID 3559476
INHIBITION OF ALLOREACTIVE CYTOTOXIC LYMPHOCYTES-T BY PEPTIDES FROM THE ALPHA-2 DOMAIN OF HLA-A2 NATURE Parham, P., CLAYBERGER, C., ZORN, S. L., Ludwig, D. S., SCHOOLNIK, G. K., KRENSKY, A. M. 1987; 325 (6105): 625-628
Abstract

Class I major histocompatibility complex (MHC) molecules function in the recognition of antigens by cytotoxic T lymphocytes (CTL). Although this biological role is firmly established and much has been learnt about their structure and polymorphic variation, little is known of the regions of class I molecules that are involved in functional interactions with components of the T-cell surface. Here we show that peptides derived from residues 98-113 of the alpha 2 domain of HLA-A2 specifically inhibit the recognition of target cells by many HLA-A2-specific CTL. In addition to identifying a region that is probably involved in binding the T-cell receptor these results raise the possibility that alloreactive CTL may recognize degraded fragments of class I histocompatibility antigens.

View details for Web of Science ID A1987F973500058

View details for PubMedID 2433598
CHARACTERIZATION OF THE MAJOR IRON-REGULATED PROTEIN OF NEISSERIA-GONORRHOEAE AND NEISSERIA-MENINGITIS ANTONIE VAN LEEUWENHOEK JOURNAL OF MICROBIOLOGY Morse, S. A., Mietzner, T. A., BOLEN, G., Lefaou, A., SCHOOLNIK, G. 1987; 53 (6): 465-469
Abstract

The major iron-regulated protein (MIRP) was purified, from both Neisseria gonorrhoeae and N. meningitidis by selective extraction with cetyltrimethylammonium bromide followed by ion-exchange and moleculair-seive chromatography. Solutions of the purified proteins had a characteristic pink color. The overall amino acid composition of these proteins was similar, although differences were noted in the number of serine, threonine, and lysine residues. Nevertheless, the N-terminal amino acid sequence was identical through 47 residues for both the meningococcal and gonococcal MIRP. Plasma emission spectrophotometry revealed that the meningococcal 37K protein contained ca. 1 mole Fe/mole protein.

View details for Web of Science ID A1987M330400016

View details for PubMedID 3130784
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
STRUCTURE OF THE TOXIC DOMAIN OF THE ESCHERICHIA-COLI HEAT-STABLE ENTEROTOXIN ST-I BIOCHEMISTRY Gariepy, J., Lane, A., FRAYMAN, F., Wilbur, D., Robien, W., SCHOOLNIK, G. K., Jardetzky, O. 1986; 25 (24): 7854-7866
Abstract

Active fragments of the heat-stable enterotoxin ST I of Escherichia coli were chemically synthesized with the sequence Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-(Tyr) and studied by proton (1H NMR) and carbon-13 (13C NMR) nuclear magnetic resonance spectroscopy as a function of pH and temperature. All of the nonexchangeable protons in the 1H NMR spectrum were assigned. Although all amide protons were present at temperatures below 25 degrees C and and pH values below 6, some of the resonances are broad and could not be assigned. The temperature dependence of these broad resonances indicates a change in conformation that is localized in the N-terminus. Other amide protons disappear at higher temperatures owing to chemical exchange with the solvent. Sufficient resonance assignments can be made at high and low temperatures to permit structural conclusions to be made. The chemical shifts of the alpha-carbon protons indicate the presence of substantial structure, which was further defined with the observed pattern of nuclear Overhauser enhancements (NOEs), coupling constants, and exchange rates. The NMR data identify a turn from Ala-14 to Cys-18. A second likely turn is centered around the proline residue. An interresidue NOE between the alpha-carbon protons of Asn-12 and Gly-17 indicates that the molecule folds back on itself. The NMR information is sufficient to define the structure of the C-terminal region of ST I. Manual model building then indicated that one arrangement of the three disulfides is particularly compatible with the NMR data and van der Waals constraints. A model incorporating the disulfide arrangement proposed by Houghten and his co-workers [Houghten, R.A., Ostresh, J.M., & Klipstein, F.A. (1984) Eur. J. Biochem. 145, 157-162] and the NMR constraints was derived with the programs PROTO [Frayman, F. (1985) Ph.D. Thesis, Northwestern University] and NOEMOT [Lane, A.N., Lefévre, J.-F., & Jardetsky, O. (1986) Biochim. Biophys. Acta 867, 45-56].

View details for Web of Science ID A1986F129500011

View details for PubMedID 3801445
PEPTIDE ANTISERA AS SEQUENCE-SPECIFIC PROBES OF PROTEIN CONFORMATIONAL TRANSITIONS - CALMODULIN EXHIBITS CALCIUM-DEPENDENT CHANGES IN ANTIGENICITY PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gariepy, J., Mietzner, T. A., SCHOOLNIK, G. K. 1986; 83 (23): 8888-8892
Abstract

Local changes in conformation between the calcium-saturated and calcium-free forms of calmodulin were monitored using antisera to four peptides corresponding to three helical regions of the calcium-saturated protein. The N-terminal helix was monitored using antiserum to residues 9-19, calmodulin-(9-19); the C-terminal helix using antiserum to residues 141-148, calmodulin-(141-148); and the long central helix with antisera to residues 68-79 and 80-92, calmodulin-(68-79) and -(80-92). Crossreactivities of peptide antisera with calmodulin (either in the presence or absence of calcium) were determined using solution-phase and solid-phase immunoassays. When examined by the fluid-phase assay, all four peptides elicited antibody that precipitated radiolabeled apocalmodulin but not the calcium-saturated form of the protein. Similarly, when calmodulin was immobilized on a solid-support, only the calcium-free form readily bound the antibodies to calmodulin-(80-92) and -(141-148). In addition, the crossreactivity of antiserum to calmodulin-(68-79) with calcium-saturated calmodulin in solid phase was reduced by approximately equal to 40% relative to reactivity with apocalmodulin. According to the x-ray crystal structure of Ca2+-saturated calmodulin and the antigenic reactivity of calmodulin for the peptide antisera in the absence of calcium, the regions of the protein monitored by these antisera are exposed to the surface in both conformational states and probably accessible to specific antibodies. The apparent preference of peptide antibodies for one conformation of the molecule suggests that changes in the conformation of calmodulin occur in cognate sequences that are transformed by calcium from antigenic, flexible structures to less antigenic, relatively helical structures. Peptide antibodies may be employed as sequence-specific reporter molecules to monitor local conformational changes providing the cognate sequence is sterically accessible to antibody in both states but antigenic in only one.

View details for Web of Science ID A1986F147100015

View details for PubMedID 2431410
TWO-DIMENSIONAL CRYSTALS OF CHOLERA-TOXIN B-SUBUNIT RECEPTOR COMPLEXES - PROJECTED STRUCTURE AT 17-A RESOLUTION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ludwig, D. S., RIBI, H. O., SCHOOLNIK, G. K., Kornberg, R. D. 1986; 83 (22): 8585-8588
Abstract

The B subunit of cholera toxin forms two-dimensional crystals when bound to its membrane receptor, ganglioside GM1, in phospholipid layers. A rectangular crystal lattice gives diffraction extending to 15-A resolution in negative stain, and image-processing of electron micrographs reveals a ring of five protein densities. The diameter of the central hole and the outer diameter of the ring are about 20 and 60 A, respectively. These data are consistent with a pentameric, doughnut-shaped structure of the B subunit that lies flat on a membrane surface. A hexagonal crystal lattice is obtained as well, and results of image processing and chemical crosslinking allow two interpretations: the B subunit may exist in both pentameric and hexameric forms or, more likely, the hexagonal lattice may represent a disordered or liquid crystalline form, in which a pentamer undergoes rotational averaging about its 5-fold axis.

View details for Web of Science ID A1986E906700032

View details for PubMedID 3464971
SYNTHETIC PEPTIDES AS NUCLEAR-LOCALIZATION SIGNALS NATURE Goldfarb, D. S., Gariepy, J., SCHOOLNIK, G., Kornberg, R. D. 1986; 322 (6080): 641-644
Abstract

The nuclear envelope defines a compartment boundary which is penetrated by pores that mediate a remarkable transport process. Precursor RNAs are retained in the nucleus, while processed messenger RNA, transfer RNA and ribosomal subunits are transported to the cytoplasm. Proteins destined for the nucleus become localized soon after synthesis and again following mitosis, while cytoplasmic proteins are excluded. The process is highly specific: a single base change in vertebrate initiator tRNAMet (tRNAiMet) reduces the rate of export 20-fold; a point mutation within the simian virus 40 (SV40) large-T antigen, converting Lys 128 to Thr or Asn, prevents import. Lys 128 lies within a short 'signal' sequence which, when fused to large non-nuclear proteins, causes their accumulation in nuclei. Regions of other eukaryotic proteins also seem to contain nuclear localization signals, although a single consensus sequence has not emerged. We report here that a synthetic peptide containing 10 residues of large-T antigen sequence serves as a nuclear localization signal when cross-linked to bovine serum albumin (BSA) or immunoglobulin G (IgG) and microinjected in Xenopus oocytes. Substitution of Thr at the position of Lys 128 in this peptide renders it six- to sevenfold less effective. The uptake of peptide-linked BSA is saturable, and the rate is diminished by co-injection of free peptide. These findings are indicative of a receptor-mediated uptake process. With the use of anti-peptide antibodies, a family of proteins is revealed in nuclear but not cytoplasmic extracts of human lymphocytes which contain large-T antigen-like sequences.

View details for Web of Science ID A1986D628900057

View details for PubMedID 3638500
CHARACTERIZATION OF THE RECEPTOR FOR HEAT-STABLE ENTEROTOXIN FROM ESCHERICHIA-COLI IN RAT INTESTINE JOURNAL OF BIOLOGICAL CHEMISTRY Kuno, T., Kamisaki, Y., Waldman, S. A., Gariepy, J., SCHOOLNIK, G., Murad, F. 1986; 261 (3): 1470-1476
Abstract

The receptor for the heat-stable enterotoxin (ST) from Escherichia coli was solubilized with Lubrol-PX from rat intestinal brush-border membranes and characterized. The binding kinetics and analog specificity of the solubilized receptor were virtually identical to those obtained with the membrane-bound receptor. Furthermore, the regulation of the receptor's affinity by cations was also maintained after solubilization, indicating a conservation of the toxin-binding site after removal of the receptor from its membrane environment. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 5.5 nm and a sedimentation coefficient of 7.0 S for the solubilized receptor. The isoelectric point of the receptor was determined as 5.5 using Sephadex isoelectric focusing electrophoresis. In all of these separation techniques, the ST receptor showed a single peak of activity that was clearly separated from that of guanylate cyclase. When 125I-ST was cross-linked to brush-border membranes with disuccinimidyl suberate, the affinity-labeled receptor solubilized with 0.1% Lubrol-PX eluted at a similar position as the native receptor on gel filtration chromatography. Analysis of the affinity-labeled receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent and by autoradiography revealed the presence of three specifically labeled polypeptides with apparent molecular weights of 80,000, 68,000, and 60,000. These results suggest that the ST receptor is solubilized by Lubrol-PX in an active form with preservation of its regulation by cations. Also, the ST receptor is separable from particulate guanylate cyclase indicating that the receptor is coupled to the activation of guanylate cyclase by an as yet undefined mechanism. Three subunit peptides may constitute a binding region of the receptor.

View details for Web of Science ID A1986AYH9300076

View details for PubMedID 3944095
Synthetic peptides protect against infection in a model of experimental pyelonephritis in mice. Annali Sclavo. Collana monografica Schmidt, M. A., O'Hanley, P., SCHOOLNIK, G. K. 1986; 3 (1-2): 389-398
View details for PubMedID 3322342
DESIGN OF A PHOTOREACTIVE ANALOG OF THE ESCHERICHIA-COLI HEAT-STABLE ENTEROTOXIN STIB - USE IN IDENTIFYING ITS RECEPTOR ON RAT BRUSH-BORDER MEMBRANES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gariepy, J., SCHOOLNIK, G. K. 1986; 83 (2): 483-487
Abstract

The Escherichia coli heat-stable enterotoxin, STIb was prepared by solid-phase peptide synthesis and purified to homogeneity by high-pressure liquid chromatography. This analogue was iodinated and shown to bind specifically to rat intestinal membranes. The radiolabeled peptide was derivatized at the amino terminus with the photoreactive heterobifunctional crosslinking agent N-hydroxysuccinimidyl p-benzoylbenzoate. This photoreactive probe also exhibited binding specificity. It was mixed with rat intestinal brush border membranes and photolyzed in the presence or absence of excess unlabeled STIb. Polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate and 2-mercaptoethanol indicated that the peptide probe was crosslinked specifically to two molecular species of 57 and 75 kDa. One or both of these molecules appear to constitute the enterotoxin receptor or to be in close proximity to it.

View details for Web of Science ID A1986AYP2200062

View details for PubMedID 3510436
INTESTINAL RECEPTOR FOR HEAT-STABLE ENTEROTOXIN OF ESCHERICHIA-COLI IS TIGHTLY COUPLED TO A NOVEL FORM OF PARTICULATE GUANYLATE-CYCLASE INFECTION AND IMMUNITY Waldman, S. A., Kuno, T., Kamisaki, Y., Chang, L. Y., Gariepy, J., OHANLEY, P., SCHOOLNIK, G., Murad, F. 1986; 51 (1): 320-326
Abstract

A novel form of particulate guanylate cyclase tightly coupled by cytoskeletal components to receptors for heat-stable enterotoxin (ST) produced by Escherichia coli can be found in membranes from rat intestinal mucosa. Intestinal particulate guanylate cyclase was resistant to solubilization with detergent alone, with only 30% of the total enzyme activity being extracted with Lubrol-PX. Under similar conditions, 70% of this enzyme was solubilized from rat lung membranes. The addition of high concentrations of sodium chloride to the extraction buffer resulted in greater solubilization of particulate guanylate cyclase from intestinal membranes. Although extraction of intestinal membranes with detergent and salt resulted in greater solubilization of guanylate cyclase, a small fraction of the enzyme activity remained associated with the particulate fraction. This activity was completely resistant to solubilization with a variety of detergents and chaotropes. Particulate guanylate cyclase and the ST receptor solubilized by detergent retained their abilities to produce cyclic GMP and bind ST, respectively. However, ST failed to activate particulate guanylate cyclase in detergent extracts. In contrast, guanylate cyclase resistant to solubilization remained functional and coupled to the ST receptor since enzyme activation by ST was unaffected by various extraction procedures. The possibility that the ST receptor and particulate guanylate cyclase were the same molecule was explored. ST binding and cyclic GMP production were separated by affinity chromatography on GTP-agarose. Similarly, guanylate cyclase migrated as a 300,000-dalton protein, while the ST receptor migrated as a 240,000-dalton protein on gel filtration chromatography. Also, thiol-reactive agents such as cystamine and N-ethylmaleimide inhibited guanylate cyclase activation by ST, with no effect on receptor binding of ST. These data suggest that guanylate cyclase and the ST receptor are independent proteins coupled by cytoskeletal components in membranes of intestinal mucosa.

View details for Web of Science ID A1986AXB5200050

View details for PubMedID 2867046
Chemical and immunochemical studies on the receptor binding domain of cholera toxin B subunit. journal of biological chemistry Ludwig, D. S., Holmes, R. K., SCHOOLNIK, G. K. 1985; 260 (23): 12528-12534
Abstract

The contributions of various amino acids to the structure and function of cholera toxin B subunit were assessed with quantifiable, chemically conservative, reversible derivatizations, and sensitive assays of activity. A panel of monoclonal antibodies was employed to monitor the conformational integrity of modified protein and help distinguish the direct from indirect effects of chemical derivatization. We describe a novel monoclonal antibody, which competes with the receptor GM1 for binding to cholera toxin B subunit, and use this reagent to help identify critically located residues. Our data support the hypothesis that tryptophan participates directly in binding GM1. In addition, we propose a dual role for lysine: first, these basic residues maintain an electrostatic attraction vital to receptor recognition; second, at least 1 lysine resides near the receptor binding domain and may interact with GM1. The influence of arginyl and tyrosyl residues upon activity is re-examined. Finally, we present data which suggest, in variance with previous studies, that the intramolecular disulfide bond is vital to the structure and function of cholera toxin B subunit.

View details for PubMedID 2413025
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
THE PRIMARY SEQUENCE AND ANTIGENIC STRUCTURE OF GONOCOCCAL PILIN - APPROACHES TOWARDS A GONOCOCCAL VACCINE ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Rothbard, J. B., SCHOOLNIK, G. K. 1985; 185: 247-273
View details for Web of Science ID A1985AUS7800017

View details for PubMedID 2416202
AFA-I, A CLONED AFIMBRIAL X-TYPE ADHESIN FROM A HUMAN PYELONEPHRITIC ESCHERICHIA-COLI STRAIN - PURIFICATION AND CHEMICAL, FUNCTIONAL AND SEROLOGIC CHARACTERIZATION EUROPEAN JOURNAL OF BIOCHEMISTRY Walz, W., Schmidt, M. A., LABIGNEROUSSEL, A. F., FALKOW, S., SCHOOLNIK, G. 1985; 152 (2): 315-321
Abstract

AFA-I, a mannose-resistant, P-independent, X-binding afimbrial Escherichia coli adhesin was purified from a recombinant strain and chemically, functionally and serologically characterized. AFA-I exists on the bacterial surface and free as a macromolecular aggregate in the supernatant of spent culture medium. It is composed of a single, repeating 16-kDa polypeptide subunit. The AFA-I protein amino acid composition is remarkable for the presence of 22% non-polar hydrophobic residues and 2.5-3.0 cysteines per subunit. Since AFA-I travels as a monomer in sodium dodecyl sulfate/polyacrylamide gel electrophoresis under non-reducing conditions, no disulfide bonds exist between subunits and at least one free sulfhydryl per subunit is available. The AFA-I N-terminal amino acid sequence residues 1-24 was unrelated to E. coli fimbrial sequences; however, the N-terminus of AFA-I and GV-12, another E. coli afimbrial protein, was asparagine. HB101 (pIL 14), the AFA-I recombinant strain, agglutinated only human and gorilla erythrocytes, indicating a preference for receptor molecules on the red cells of man and the anthropoid apes. AFA-I did not bind glycophorin A or sialyl glycosides and is therefore distinct from the E. coli X-binding adhesins with M and S specificity. The AFA-I receptor was found to be abundant and diffusely distributed on HeLa tissue culture monolayer cell surfaces by indirect fluorescent microscopy. Anti-AFA-I sera bound AFA-I in Western blots of 4 out of 16 X-binding E. coli urine isolates. They did not bind MS or P pili. AFA-I may be exemplary of an adhesin class significant for the pathogenesis of human urinary tract infections.

View details for Web of Science ID A1985ASZ0300012

View details for PubMedID 2865133
GAL-GAL BINDING AND HEMOLYSIN PHENOTYPES AND GENOTYPES ASSOCIATED WITH UROPATHOGENIC ESCHERICHIA-COLI NEW ENGLAND JOURNAL OF MEDICINE OHANLEY, P., Low, D., Romero, I., LARK, D., Vosti, K., FALKOW, S., SCHOOLNIK, G. 1985; 313 (7): 414-420
Abstract

To determine whether uropathogenic strains of Escherichia coli exhibit a distinctive constellation of phenotypes, we examined 44 urinary isolates from women with radiologically normal urinary tracts and pyelonephritis, cystitis, or asymptomatic bacteriuria and 73 fecal isolates from healthy control subjects. The strains were characterized by their O serogroup, by their binding specificity (as determined by adhesins), and by their production of hemolysin and colicin V. In addition, the strains were assessed for homologous gene sequences by means of DNA-hybridization probes prepared from cistrons that encode hemolysin and the Gal-Gal binding adhesin--two determinants of virulence, which cause tissue injury and promote bacterial colonization of uroepithelia, respectively. In contrast to most isolates from normal feces and from the urine of patients with asymptomatic bacteriuria, pyelonephritis strains belong to a small number of O serogroups; all express the Gal--Gal binding adhesin and 75 per cent are hemolytic. A gene probe for the Gal--Gal binding adhesin, derived from the chromosome of one strain from a patient with pyelonephritis, hybridized with the DNA of all other pyelonephritis strains. The probe for the hemolysin gene hybridized with DNA from all other hemolytic strains. These data indicate that most cases of pyelonephritis are due to a small number of pathogenic clones that express critical determinants of virulence, and that the nucleotide sequences for hemolysin and the Gal--Gal binding adhesin in heterologous strains share homology. We are tempted to speculate that the gene products of these shared regions of the genome might form the basis for a vaccine against pyelonephritis.

View details for Web of Science ID A1985ANV8600004

View details for PubMedID 2862582
CLONING AND SEQUENCING OF A MORAXELLA-BOVIS PILIN GENE JOURNAL OF BACTERIOLOGY Marrs, C. F., SCHOOLNIK, G., Koomey, J. M., Hardy, J., Rothbard, J., FALKOW, S. 1985; 163 (1): 132-139
Abstract

Moraxella bovis pili have been shown to play a major role in both infectivity and protective immunity of bovine infectious keratoconjunctivitis. Sonicated M. bovis DNA from the piliated strain EPP63 was inserted into the vector lambda gt11 with EcoRI linkers. Recombinant phage were screened with an oligonucleotide probe based on the amino-terminal portion of the DNA sequence of a Neisseria gonorrhoeae pilin gene. Two candidate phages produced a protein that comigrated with EPP63 beta pilin in sodium dodecyl sulfate-polyacrylamide gels and bound anti-pilus antisera. The 1.9-kilobase insert from one of these, lambda gt11M182, was subcloned in both orientations into pBR322, forming the plasmids pMxB7 and pMxB9, both of which produced beta pilin, as did pMxB12, a HindIII deletion derivative of pMxB7. In HB101(pMxB12), the M. bovis pilin protein was shown to be primarily localized in the inner membrane. The entire 939-base-pair insert of pMxB12 was sequenced, revealing a ribosome binding site just upstream of the coding region and an AT-rich region further upstream containing some potential RNA polymerase recognition sites. The translation of the sequence predicts a six-amino-acid leader sequence preceding the phenylalanine that begins the mature protein. Codon usage analysis of the M. bovis beta pilin gene revealed greater use of the CUA codon for leucine than usual for a well-expressed Escherichia coli gene. Comparisons of the M. bovis EPP63 beta pilin protein sequence with other pilin gene sequences are presented.

View details for Web of Science ID A1985ALK8400019

View details for PubMedID 2861194
PILI OF NEISSERIA-MENINGITIDIS - ANALYSIS OF STRUCTURE AND INVESTIGATION OF STRUCTURAL AND ANTIGENIC RELATIONSHIPS TO GONOCOCCAL PILI JOURNAL OF EXPERIMENTAL MEDICINE Stephens, D. S., Whitney, A. M., Rothbard, J., SCHOOLNIK, G. K. 1985; 161 (6): 1539-1553
Abstract

To provide information useful for the design of a pilus vaccine effective for the prevention of both meningococcal and gonococcal disease, the electron microscopic morphology of meningococcal pili and the structural and antigenic relationships of meningococcal pili to gonococcal pili were investigated. Meningococcal pili were 4-6 nm in width, extended 500-6,000 nm from the organism surface, and occurred singly or in bundles composed of 8-10 pili per bundle. Meningococcal pilin varied between 17,250 and 20,600 daltons. Pilin was present in outer membrane preparations of some meningococcal isolates that were nonpiliated by electron microscopic examination. Antibodies to gonococcal pili, cyanogen bromide cleavage fragments of gonococcal pilin, or synthetic peptide analogues corresponding to regions of the gonococcal pilin sequence, were used to detect common meningococcal and gonococcal antigenic determinants that might indicate the existence of a conserved sequence beyond residue 29. Antibody to intact gonococcal pili or to the variable CNBR-3 region of gonococcal pilin detected little shared antigenicity with meningococcal pilin. However, pilin from all tested meningococcal isolates reacted with antibody to the CNBR-2 fragment of gonococcal pilin, a region highly conserved among gonococcal strains. Meningococcal pilins were also broadly crossreactive with antibody to a synthetic peptide corresponding to residues 69-84 of the gonococcal sequence, a part of the CNBR-2 region that appears to be critical for gonococcal receptor-binding function. If a sequence similar to 69-84 is also important for receptor-binding function in meningococcal pili, a peptide corresponding to this region may elicit antibodies that block the adherence function of pili elaborated by both Neisseria gonorrhoeae and N. meningitidis.

View details for Web of Science ID A1985AKB7800020

View details for PubMedID 2409203
ANTIBODY-RESPONSE IN HUMAN NOCARDIOSIS - IDENTIFICATION OF 2 IMMUNODOMINANT CULTURE-FILTRATE ANTIGENS DERIVED FROM NOCARDIA-ASTEROIDES JOURNAL OF INFECTIOUS DISEASES Sugar, A. M., SCHOOLNIK, G. K., Stevens, D. A. 1985; 151 (5): 895-901
Abstract

An ammonium sulfate precipitate derived from a culture filtrate of Nocardia asteroides contained 20-30 protein subunits, as identified by gel electrophoresis. Western blot analysis of these proteins (obtained from serum samples from patients with systemic nocardiosis) revealed that all of the 17 samples reacted with a subunit of a protein with an apparent molecular weight of 55,000 and that 11 of the 17 samples reacted with a subunit with a molecular weight of 31,000. In contrast, only two of 25 serum samples obtained from healthy controls or patients hospitalized without nocardial or mycobacterial disease reacted with these subunits. Sera from 21 patients infected with Mycobacterium tuberculosis, a historically troublesome cross-reactive group, failed to react with either the 55,000 or the 31,000 molecular weight subunit. The presence of only two protein subunits that reacted with sera from humans with nocardiosis suggests that these molecules may have use as diagnostic reagents or probes of the immunologic reaction to Nocardia.

View details for Web of Science ID A1985AHU9700021

View details for PubMedID 2580915
EXPRESSION OF THE L11 NEUROPEPTIDE GENE IN THE APLYSIA CENTRAL NERVOUS-SYSTEM JOURNAL OF COMPARATIVE NEUROLOGY Taussig, R., KALDANY, R. R., Rothbard, J. B., SCHOOLNIK, G., Scheller, R. H. 1985; 238 (1): 53-64
Abstract

Neuron L11 in the abdominal ganglion of Aplysia californica is thought to be both cholinergic and peptidergic. In previous studies, we isolated a cDNA clone encoding the precursor for an L11 secreted protein(s) by differentially screening an abdominal ganglion cDNA library. We now report the isolation of genomic clones encoding the L11 cDNA sequences. Analysis of these clones reveals that the gene is present in a single copy per haploid genome. RNA blotting and cDNA cloning demonstrate that the L11 gene is expressed not only in the abdominal ganglion but in the head ganglia as well. To define the positions of cells expressing this gene and to follow their processes, we raised antibodies to synthetic peptides defined by the cDNA sequence. Histochemistry revealed about 100 neurons containing immunoreactive material. These cells arborize in the neuropil and are distributed throughout the central nervous system, representing about 0.5% of the Aplysia central neurons. In addition, cells in the abdominal ganglion send processes to the mantle floor at the base of the gill via the genital and branchial nerves. Our data suggest that this network of cells expresses the single L11 peptide gene.

View details for Web of Science ID A1985AMM9800004

View details for PubMedID 3900151
ANTIBODIES TO PEPTIDES CORRESPONDING TO A CONSERVED SEQUENCE OF GONOCOCCAL PILINS BLOCK BACTERIAL ADHESION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rothbard, J. B., Fernandez, R., Wang, L., Teng, N. N., SCHOOLNIK, G. K. 1985; 82 (3): 915-919
Abstract

Antisera generated against each of seven synthetic peptides corresponding to constant and variable sequences of the pilin from gonococcal strain MS11 were assayed for their ability to crossreact with intact pili from both homologous and heterologous strains. The peptides elicited roughly equal antipeptide responses but varied substantially in their ability to elicit antisera that crossreacted with intact pili. Of the antisera to peptides corresponding to regions of conserved sequence, antisera directed against residues 69-84 were the most efficient in binding pili from all strains tested in both solid-phase assays and immunoblots. Anti-69-84 also efficiently precipitated a tryptic fragment of pilin known to bind human endocervical cells. Sera against the two peptides (121-134 and 135-151) previously shown to contain strain-specific epitopes crossreacted with MS11 pili equally well, but differed in their ability to bind pili from heterologous strains. Anti-121-134 was strain-specific whereas anti-135-151 bound all pilin tested. Each of the sera was examined for its ability to inhibit bacterial adhesion to a human endometrial carcinoma cell line. Sera generated against residues 41-50 and 69-84 successfully inhibited a heterologous gonococcal strain from binding. These peptides could be important components of an effective vaccine for the prevention of gonorrhea.

View details for Web of Science ID A1985ADL2400060

View details for PubMedID 3919385
PURIFICATION AND CHARACTERIZATION OF HEMOPHILUS-INFLUENZAE PILI, AND THEIR STRUCTURAL AND SEROLOGICAL RELATEDNESS TO ESCHERICHIA-COLI P AND MANNOSE-SENSITIVE PILI JOURNAL OF EXPERIMENTAL MEDICINE Guerina, N. G., Langermann, S., SCHOOLNIK, G. K., KESSLER, T. W., GOLDMANN, D. A. 1985; 161 (1): 145-159
Abstract

Haemophilus influenzae pili were purified, and their physical and serological properties were examined. The solution properties of the pili were determined, and then a purification scheme involving repeated cycles of precipitation and solubilization was developed. The purified pili from one type b isolate (A02) were found to consist of multiple copies of a 25,000 mol wt subunit. Amino-terminal sequence analysis of A02 pili was carried out to 40 amino acid residues, and a remarkable degree of sequence homology was found with E. coli P and mannose-sensitive (MS) pili (27.5 and 25% homology, respectively). Purified A02 pili were found to be highly immunogenic, and serological analysis by enzyme-linked immunosorbent assay and whole piliated cell agglutination revealed significant cross-reactivity between A02 pilus antiserum and the pili of seven other H. influenzae strains tested (heterologous titers = 2-100% of the homologous titer). Cross-reactivity was also observed between the H. influenzae pili (five of eight strains tested) and the P pili from E. coli strains HU849 and 3669; no cross-reactivity was detected with MS pili from E. coli strain H10407 and C94. The structural similarities between H. influenzae and E. coli P and MS pili suggest a common gene ancestry.

View details for Web of Science ID A1985AAV9200011

View details for PubMedID 2857190
GAL-GAL PYELONEPHRITIS ESCHERICHIA-COLI PILI LINEAR IMMUNOGENIC AND ANTIGENIC EPITOPES JOURNAL OF EXPERIMENTAL MEDICINE Schmidt, M. A., OHANLEY, P., SCHOOLNIK, G. K. 1985; 161 (4): 705-717
Abstract

The linear immunogenic and antigenic structure of E. coli Gal-Gal pili from the recombinant strain HU 849 was investigated with nine synthetic peptides corresponding to regions of the pilus sequence predicted to contain hydrophilic beta-turns. Five peptides, as bovine serum albumin conjugates, were found by anti-HU 849 pilus serum and were thus designated "immunogenic epitopes." Peptides corresponding to R 25-38, R 38-50, and R 48-61 (which jointly comprise the single intramolecular disulfide loop), and R 103-116, were bound in low titer. A prominent immunogenic epitope was specified by a peptide corresponding to R 65-75. Four peptides, as thyroglobulin conjugates, elicited antisera in rabbits that bound intact HU 849 pili. These were designated "antigenic epitopes." Two prominent antigenic epitopes were localized to peptides corresponding to R 5-12 and R 93-104, whereas peptides corresponding to R 65-75 and R 119-131 represented two minor antigenic epitopes. None of the peptide antisera bound Gal-Gal pili from heterologous strains except anti-R 93-104 and anti-R 5-12. In 8 of the 10 Gal-Gal-binding pyelonephritis isolates tested, anti-R 5-12 detected a protein with an apparent molecular weight of 18,000 co-migrating with several Gal-Gal pili. Anti-R 93-104 detected a corresponding protein in 4 of 8 fecal and 7 of 12 pyelonephritis Gal-Gal-binding isolates; however, it also bound apparently unrelated proteins of higher molecular weight.

View details for Web of Science ID A1985AFC1800005

View details for PubMedID 2580037
ANTIBODY-ANTIGEN SPECIFICITY IN THE IMMUNE-RESPONSE TO INFECTION WITH NEISSERIA-GONORRHOEAE JOURNAL OF INFECTIOUS DISEASES Lammel, C. J., Sweet, R. L., Rice, P. A., Knapp, J. S., SCHOOLNIK, G. K., Heilbron, D. C., Brooks, G. F. 1985; 152 (5): 990-1001
Abstract

This study was done to define antigens important in the immune response to infection with Neisseria gonorrhoeae. Sera were obtained from men and women with uncomplicated gonorrhea (UGC), women with disseminated gonococcal infection, and women with gonococcal pelvic inflammatory disease (PID); sera were also obtained from uninfected controls. Vaginal fluids were taken from 15 patients with UCG or PID. The sera and vaginal fluids were tested against gonococcal isolates from the same patients to examine homologous antibody-antigen interactions by use of the western blot technique. Antibodies in the serum reacted with more gonococcal antigens compared with antibodies in the vaginal fluid. IgG in serum and vaginal fluid reacted with more antigens than did IgA in the same specimens. The predominant antigens reactive with IgG in serum were pili, protein II, a broad 23-33-kDa band of antigen, and presumptive lipopolysaccharide; and for IgA, protein II and a 46-48-kDa protein. The control sera also reacted with the 46-48-kDa protein. The predominant antigens reactive with IgG in vaginal fluid were protein I, protein II, pili, and the 46-48-kDa protein; and for IgA, protein I, protein II, and pili. Immunoglobulin in vaginal fluid reacted comparatively more with protein I than did immunoglobulin in serum.

View details for Web of Science ID A1985AUD8600018

View details for PubMedID 2413148
T-CELL ACTIVATION BY PEPTIDE ANTIGEN - EFFECT OF PEPTIDE SEQUENCE AND METHOD OF ANTIGEN PRESENTATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Watts, T. H., Gariepy, J., SCHOOLNIK, G. K., McConnell, H. M. 1985; 82 (16): 5480-5484
Abstract

A series of synthetic peptide analogues of a determinant recognized by the ovalbumin-specific, I-Ad-restricted, T-cell hybridoma 3DO-54.8 were synthesized. The resulting peptides were tested for activation of 3DO-54.8 cells by using glutaraldehyde-fixed cells as well as reconstituted membranes as antigen-presenting surfaces. The results show that the minimum epitope for activation of this T cell is between 7 and 11 amino acids in length. This region includes two important histidine residues. The order of preference of the various peptide analogues was the same regardless of the method of antigen presentation. However, the amount of peptide required for T-cell activation was considerably higher when reconstituted membranes, rather than fixed cells, were used as antigen-presenting surfaces.

View details for Web of Science ID A1985APP1500052

View details for PubMedID 2410926
GENE CLUSTERS GOVERNING THE PRODUCTION OF HEMOLYSIN AND MANNOSE-RESISTANT HEMAGGLUTINATION ARE CLOSELY LINKED IN ESCHERICHIA-COLI SEROTYPE-O4 AND O6-ISOLATES FROM URINARY-TRACT INFECTIONS INFECTION AND IMMUNITY Low, D., David, V., LARK, D., SCHOOLNIK, G., FALKOW, S. 1984; 43 (1): 353-358
Abstract

The genes encoding alpha-hemolysin and mannose-resistant hemagglutination were shown to be closely linked in cloned DNA from two Escherichia coli urinary tract isolates of serotypes O4 (J96) and O6 (C1212). DNA hybridization experiments demonstrated that the hly and mrh gene clusters of other E. coli O6 serotypes were also linked. Colony hybridizations showed that most normal fecal E. coli do not contain hly and mrh DNA but much of the intervening DNA between these two gene clusters is common among all E. coli. We have further demonstrated that there is a small (about 1 kilobase) region of homology located on both sides of the hly sequence and present elsewhere in the C1212 strain. We suggest that linkage of hly and mrh occurred through a transposition event, and we discuss the potential significance of this linkage in the acquisition of virulence determinants by these bacteria.

View details for Web of Science ID A1984RW80600058

View details for PubMedID 6317570
ALL 8 UNASSIGNED READING FRAMES OF MOUSE MITOCHONDRIAL-DNA ARE EXPRESSED EMBO JOURNAL Michael, N. L., Rothbard, J. B., Shiurba, R. A., LINKE, H. K., SCHOOLNIK, G. K., Clayton, D. A. 1984; 3 (13): 3165-3175
Abstract

Animal mitochondrial DNA contains genes for 13 potential polypeptides of significant size. Five of these genes have been assigned to distinct proteins and eight remained unassigned reading frames (URFs). Short peptides corresponding to URF protein sequences were synthesized chemically. Antibodies raised to these synthetic peptides were used to establish the existence of all eight URF proteins in mouse tissues and cells by the complementary techniques of immunoperoxidase staining, protein blotting and immunoprecipitation. Immunoperoxidase staining of thin-sectioned, freeze-substituted tissue may prove generally useful for the identification of gene products for which no formal genetic data exist. Furthermore, the ability to determine the cellular and tissue distribution of such proteins may provide the first insight into their function.

View details for Web of Science ID A1984TX37100020

View details for PubMedID 6241149
CLONING AND EXPRESSION OF AN AFIMBRIAL ADHESIN (AFA-I) RESPONSIBLE FOR P-BLOOD GROUP-INDEPENDENT, MANNOSE-RESISTANT HEMAGGLUTINATION FROM A PYELONEPHRITIC ESCHERICHIA-COLI STRAIN INFECTION AND IMMUNITY LABIGNEROUSSEL, A. F., LARK, D., SCHOOLNIK, G., FALKOW, S. 1984; 46 (1): 251-259
Abstract

The uropathogenic Escherichia coli KS52 strain expresses a mannose-resistant hemagglutinin involving an erythrocyte recognition site distinct from the alpha-digalactoside glycosphingolipid receptor identified for the uropathogenic E. coli strains specifying a P adhesin. The KS52 strain showed three major properties. (i) It agglutinated human erythrocytes of all tested blood groups. (ii) Hemagglutinin activity was found both in the supernatant fluid L-broth cultures and in cells grown on L-agar plates. (iii) No fimbriae in organisms grown on L-agar plates were detected by electron microscopy. Whole-cell DNA from the KS52 strain was size fractionated and cloned into the pHC79 cosmid vector. Three recombinant cosmids expressing a mannose-resistant hemagglutination (MRHA) phenotype were characterized and used to subclone the smallest DNA fragment able to confer the same MRHA properties as the parent strain. A 6.7-kilobase chromosomal DNA fragment cloned in pBR322 (pIL14) was shown to be necessary for host-cell MRHA expression and uroepithelial cell adherence. The insert encoded the production of a 16,000-dalton hemagglutinin. This polypeptide could be detected in culture supernatant fluids, in E. coli minicells harboring the pIL14 plasmid, and, by immunoblotting, in the KS52 strain and E. coli whole cells harboring the pIL14 plasmid. No homology was detected by Southern hybridization between the cloned insert and the DNA of the operon responsible for MRHA in the P-specifying, fimbriate strains (pap operon).

View details for Web of Science ID A1984TL29100039

View details for PubMedID 6148308
NUCLEOTIDE-SEQUENCE OF THE PAPA-GENE ENCODING THE PAP PILUS SUBUNIT OF HUMAN UROPATHOGENIC ESCHERICHIA-COLI JOURNAL OF BACTERIOLOGY BAGA, M., Normark, S., Hardy, J., OHANLEY, P., LARK, D., Olsson, O., SCHOOLNIK, G., FALKOW, S. 1984; 157 (1): 330-333
Abstract

The papA gene of the uropathogenic strain Escherichia coli J96, coding for the Pap pili subunit, was subjected to DNA sequencing, and found to code for an 185-amino acid-long polypeptide with a 22-amino acid-long signal peptide. Here we present the primary sequence, the hydrophilicity profile, and the predicted polypeptide secondary structure of the Pap pili subunit.

View details for Web of Science ID A1984RY22100055

View details for PubMedID 6140260
ANTIBODIES TO SYNTHETIC PEPTIDES DEFINED BY CDNA CLONING REVEAL A NETWORK OF PEPTIDERGIC NEURONS IN APLYSIA JOURNAL OF NEUROSCIENCE Kreiner, T., Rothbard, J. B., SCHOOLNIK, G. K., Scheller, R. H. 1984; 4 (10): 2581-2589
Abstract

We previously isolated and characterized a cDNA clone specifically expressed in neurons R3 to R8 and R14 of the Aplysia abdominal ganglion (Nambu, J.R., R. Taussig, A.C. Mahon, and R.H. Scheller (1983) Cell 35: 47-56). The cDNA nucleotide sequence and the inferred protein amino acid sequence suggest that this gene encodes the precursor for neuroactive peptides used by these cells. Peptides corresponding to three regions of the precursor were synthesized, coupled to a protein carrier, and used to generate antibodies. These antibodies stain a set of cell bodies, R3 to R14, and their processes in the abdominal ganglion; no other cells in the nervous system or the periphery are immunoreactive. R3 to R14 send numerous fine immunoreactive processes into the vascularized sheath that surrounds the ganglion. Each of these cells also has a large axon which exits the ganglion via the branchial nerve and terminates on the heart. In addition, R14 is anatomically distinct from R3 to R13 in that it sends additional immunoreactive processes to the vasculature near the ganglion. Immunoreactive processes and varicosities were observed on the efferent vein of the gill, the abdominal ganglion artery, and the anterior aorta. These data are consistent with previous studies suggesting that one or more neuropeptides released from R3 to R14 function as modulators of cardiovascular physiology.

View details for Web of Science ID A1984TN29200017

View details for PubMedID 6491724
STRAIN-SPECIFIC AND COMMON EPITOPES OF GONOCOCCAL PILI JOURNAL OF EXPERIMENTAL MEDICINE Rothbard, J. B., Fernandez, R., SCHOOLNIK, G. K. 1984; 160 (1): 208-221
Abstract

The antigenic structure of gonococcal pilin, strain MS11 (Tr), was investigated by assaying the binding of antisera engendered by intact pili from strains MS11 and R10 and their two major cyanogen bromide-generated fragments, CNBr-2 (residues 9-92) and CNBr-2 (residues 93-159), to synthetic peptides corresponding to the amino acid sequence of MS11 pilin. Four peptides were synthesized corresponding to regions of sequence variation between MS11 and R10 gonococcal pilin. Antisera against the homologous pilus filament and against its CNBr-3 fragment bind peptides equivalent to residues 121-134 and 135-151, which comprise the 30 amino acid disulfide loop near the carboxyl terminus of the protein. Heterologous pili antisera did not bind these peptides. Absorption studies proved that each peptide contained an independent, strain-specific epitope. Synthetic peptides corresponding to regions of identical sequence between MS11 and R10 pilin were used in similar binding experiments to localize a weakly immunogenic, common determinant between residues 48 and 60. less than 15% of the antibodies raised against intact pili were directed at this site. Antisera raised against MS11 or R10 CNBr-2 bind a separate peptide, residues 69-80. This region is immunogenic only as a fragment, not in the intact pilus filament.

View details for Web of Science ID A1984SZ32600016

View details for PubMedID 6203999
MUTATIONS IN ESCHERICHIA-COLI CISTRONS AFFECTING ADHESION TO HUMAN-CELLS TO NOT ABOLISH PAP PILI FIBER FORMATION EMBO JOURNAL Norgren, M., Normark, S., LARK, D., OHANLEY, P., SCHOOLNIK, G., FALKOW, S., SVANBORGEDEN, C., BAGA, M., Uhlin, B. E. 1984; 3 (5): 1159-1165
Abstract

A chromosomal DNA fragment which mediates Pap (pili associated with pyelonephritis) pili formation, mannose-resistant hemagglutination ( MRHA ) and binding to uroepithelial cells has been isolated from the uropathogenic Escherichia coli clinical isolate J96 , and genetically studied. Analysis of polypeptides expressed by the Pap DNA led to detection of a number of polypeptides ranging in mol. wt. from 13 000 to 81 000 daltons. The gene order and transcriptional orientation for four of the corresponding cistrons was: 13 000 ( papB ) 19 500 ( papA , structural gene for the Pap pilus subunit), 81 000 ( papC ) and 28 500 ( papD ). Analyses of a lacZ- papA gene fusion located a promoter upstream from papA within the cloned DNA. Transposon Tn5 insertions in any of these four cistrons decreased or eliminated Pap pili formation. A number of transposon Tn5 mutations were identified in a region distal to papD that expressed normal levels of the papA protein on the cell surface in the form of recognizable pili structures but did not agglutinate human erythrocytes or adhere to uroepithelial cells. This region expressed polypeptides of 15 000, 24 000, 26 000 and 35 000 daltons. This finding shows that Pap pili formation and binding properties can be genetically dissociated.

View details for Web of Science ID A1984SR31600033

View details for PubMedID 6145589
GONOCOCCAL PILI - PRIMARY STRUCTURE AND RECEPTOR-BINDING DOMAIN JOURNAL OF EXPERIMENTAL MEDICINE SCHOOLNIK, G. K., Fernandez, R., Tai, J. Y., Rothbard, J., GOTSCHLICH, E. C. 1984; 159 (5): 1351-1370
Abstract

The complete amino acid sequence of pilin from gonococcal strain MS11 and the sequence of constant and variable regions from strain R10 pilin have been determined in order to elucidate the structural basis for adherence function, antigenic diversity, and polymeric structure. The MS11 pilin sequence consists of 159 amino acids in a single polypeptide chain with two cysteines in disulfide linkage and serine-bonded phosphate residues. TC-2 (31-111), a soluble monomeric pilus peptide prepared by arginine-specific digestion, bound human endocervical, but not buccal or HeLa cells and therefore is postulated to encompass the receptor binding domain. Variable regions of CNBr-3 appear to confer antigenic diversity and comprise segments in which changes in the position of charged residues occur in hydrophilic, beta-turns. Residues 2-21 and 202-221 of gonococcal pilins and lower eucaryotic actins, respectively, exhibit 50% homology. When these residues are arranged at intervals of 100 degrees of arc on "helical wheels," the identical amino acids comprise a hydrophobic face on one side of the helix. This observation, the hydrophobic character of this region and the tendency for TC-1 (residues 1-30) to aggregate in water, suggest that this stretch interacts with other subunits to stabilize polymeric structure.

View details for Web of Science ID A1984SQ91000005

View details for PubMedID 6143785
A COMMON ANTIGENIC DETERMINANT FOUND IN 2 FUNCTIONALLY UNRELATED TOXINS JOURNAL OF EXPERIMENTAL MEDICINE Gariepy, J., OHANLEY, P., Waldman, S. A., Murad, F., SCHOOLNIK, G. K. 1984; 160 (4): 1253-1258
Abstract

The heat-stable enterotoxin ST Ib produced by enterotoxigenic E. coli strains shares a sequence homology with the sea snail neurotoxin, conotoxin GI. Rabbit antisera were raised against synthetic analogs of these toxins and to a six-residue peptide representing the region common to both toxins. Results from enzyme-linked immunosorbent assays indicate that the homologous region of both toxins represents part of their antigenic site. The lack of cross-reactivity exhibited by the six-residue common domain with serum directed against either toxin suggests that this region probably retains a similar conformation in the intact toxins but not in the isolated fragment.

View details for Web of Science ID A1984TM66100023

View details for PubMedID 6207263
A SIMPLE, SENSITIVE, AND SPECIFIC ASSAY FOR THE HEAT-STABLE ENTERO-TOXIN OF ESCHERICHIA-COLI JOURNAL OF INFECTIOUS DISEASES Waldman, S. A., OHANLEY, P., FALKOW, S., SCHOOLNIK, G., Murad, F. 1984; 149 (1): 83-89
Abstract

A simple assay for the heat-stable enterotoxin (ST) of Escherichia coli was developed on the basis of ST activation of guanylate cyclase in membranes from the intestinal mucosa of mice. ST activated guanylate cyclase in mucosal membranes in a linear fashion over a 50-fold range of toxin concentrations with Mg++-guanosine 5'-triphosphate as substrate. Activation of guanylate cyclase was detectable at concentrations of ST that were five- to 10-fold lower than those resulting in increases in the ratio of gut weight to carcass weight of mice. This assay was used to quantify ST in crude and purified samples from culture filtrates of wild-type strains and recombinant strains of E coli containing the gene for ST. Activation of guanylate cyclase was specific for ST; purified cholera toxin and E coli heat-labile enterotoxin did not activate guanylate cyclase. Thus, this assay for ST is sensitive, specific, and will facilitate rapid analysis of samples for quantification of ST.

View details for Web of Science ID A1984SB62600012

View details for PubMedID 6141207
ULTRASTRUCTURAL-LOCALIZATION OF SPECIFIC GONOCOCCAL MACROMOLECULES WITH ANTIBODY-GOLD SPHERE IMMUNOLOGICAL PROBES INFECTION AND IMMUNITY Robinson, E. N., McGee, Z. A., Kaplan, J., Hammond, M. E., Larson, J. K., Buchanan, T. M., SCHOOLNIK, G. K. 1984; 46 (2): 361-366
Abstract

In an effort to determine the ultrastructural location of specific macromolecules on the surface of intact microorganisms and in experimentally infected tissues, a new method of rapidly conjugating antibodies to gold spheres via a staphylococcal protein A intermediary has been developed. This new technique provides the excellent density of marking and versatility of sphere size provided by existing gold methods, but decreases preparation time, decreases the chance of bacterial contamination of antibody reagents, and increases specificity of marking. Staphylococcal protein A-coated gold spheres were conjugated with antibodies from rabbits immunized with purified gonococcal pili. The resulting gold-antibody conjugates allowed demonstration of antibody binding to pilus structures of the same gonococcal strain whose pili were used to raise the antibody and demonstration of the lack of antibody recognition of pilus structures on two other gonococcal strains. The failure of gold spheres to attach to isogenic nonpiliated clones of the homologous gonococcus indicated the absence of pilus antigens on the surface of these organisms. The use of a double label--small gold spheres conjugated to anti-pilus antibody and larger gold spheres conjugated to anti-protein I antibody--allowed the simultaneous localization of two gonococcal antigens.

View details for Web of Science ID A1984TR41000012

View details for PubMedID 6150005
ANTIGENIC ANALYSIS OF GONOCOCCAL PILI USING MONOCLONAL-ANTIBODIES JOURNAL OF EXPERIMENTAL MEDICINE Edwards, M., MCDADE, R. L., SCHOOLNIK, G., Rothbard, J. B., GOTSCHLICH, E. C. 1984; 160 (6): 1782-1791
Abstract

A bank of mouse monoclonal antibodies has been produced with reactivity to gonococcal pili to investigate epitopes of the pilus structural protein, pilin. Pili of Neisseria gonorrhoeae strains R10 and MS11 were used as immunogens to elicit 19 monoclonal antibodies reactive with the homologous pili type in ELISA. Of the 19 antibodies, 16 demonstrated type-specific reactivity and 3 were cross-reactive with heterologous pili. Reactivity of the antibodies with the carboxyterminal, cyanogen bromide fragment (CB-3) of R10 pilin allowed their classification into three groups. The first group (10 antibodies) were R10 specific and equally reactive with the R10 CB-3 fragment. The second group (6) were also type specific but demonstrated poor reactivity with the CB-3 fragment. This suggested that the epitopes of the first group are linear, and those of the second group, nonlinear. The third group (3), consisting of the cross-reactive antibodies, were not reactive with the CB-3 fragment. Two of the antibodies in group 3 were examined in detail to localize their epitopes. The epitope of one, 9B9/H5, was shown to be a linear determinant. This antibody was reactive with a fragment of MS11 pilin (residues 31-111) and to a synthetic peptide representing residues 69-84 in MS11 pilin. The epitope was more finely mapped, with shorter synthetic peptides conjugated to bovine serum albumin, to an eight amino acid segment (residues 69-76). The epitope of 1E8/G8, a strongly reactive antibody, proved elusive to this type of analysis and probably results from conformational restraints. The significance of species-specific epitopes in the pilin protein is discussed.

View details for Web of Science ID A1984TX70100012

View details for PubMedID 6210338
GENETICS OF DIGALACTOSIDE-BINDING ADHESIN FROM A UROPATHOGENIC ESCHERICHIA-COLI STRAIN INFECTION AND IMMUNITY Normark, S., LARK, D., Hull, R., Norgren, M., BAGA, M., OHANLEY, P., SCHOOLNIK, G., FALKOW, S. 1983; 41 (3): 942-949
Abstract

The uropathogenic strain Escherichia coli J96 mediates mannose-resistant hemagglutination owing to production of a digalactoside-binding adhesin. A cosmid clone from this strain has been isolated that, when harbored in E. coli K-12, expressed Pap pili and this adhesin (R. Hull et al., Infect. Immun. 33:933-938, 1981). By transposon mutagenesis and by the construction of a number of hybrid plasmid derivatives, we have demonstrated that about 8.5 kilobases of DNA is required to generate a mannose-resistant hemagglutination-positive phenotype in E. coli K-12 strain P678-54. The structural gene for the Pap pili monomer, papA, has been identified and mapped close to the promotor-proximal end of the Pap operon. Although strain P678-54 that harbored a Tn5 insertion within papA showed a mannose-resistant hemagglutination-positive phenotype, it was negative in a competitive enzyme-linked immunosorbent assay with anti-Pap pilus serum. This could mean that a Pap adhesin is encoded by a region on the Pap operon that is distinct from papA.

View details for Web of Science ID A1983RE61100010

View details for PubMedID 6136465
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
ASPECTS ON STRUCTURE AND FUNCTION OF PILI ON UROPATHOGENIC ESCHERICHIA-COLI PROGRESS IN ALLERGY EDEN, C. S., GOTSCHLICH, E. C., KORHONEN, T. K., Leffler, H., SCHOOLNIK, G. 1983; 33: 189-202
View details for Web of Science ID A1983PW91800011

View details for PubMedID 6131425
A PILUS PEPTIDE VACCINE FOR THE PREVENTION OF GONORRHEA PROGRESS IN ALLERGY SCHOOLNIK, G. K., Tai, J. Y., GOTSCHLICH, E. C. 1983; 33: 314-331
View details for Web of Science ID A1983PW91800020

View details for PubMedID 6187017
INHIBITION OF EXPERIMENTAL ASCENDING URINARY-TRACT INFECTION BY AN EPITHELIAL CELL-SURFACE RECEPTOR ANALOG NATURE EDEN, C. S., Freter, R., HAGBERG, L., Hull, R., Hull, S., Leffler, H., SCHOOLNIK, G. 1982; 298 (5874): 560-562
Abstract

It has been shown that the establishment of urinary tract infection by Escherichia coli is dependent on attachment of the bacteria to epithelial cells. The attachment involves specific epithelial cell receptors, which have been characterized as glycolipids. Reversible binding to cell-surface mannosides may also be important. This suggests an approach to the treatment of infections--that of blocking bacterial attachment with cell membrane receptor analogues. Using E. coli mutants lacking one or other of the two binding specificities (glycolipid and mannose), we show here that glycolipid analogues can block in vitro adhesion and in vivo urinary tract infection.

View details for Web of Science ID A1982PA67200047

View details for PubMedID 7048106
CHEMICAL AND CLINICAL-STUDIES ON THE INTERACTION OF ESCHERICHIA-COLI WITH HOST GLYCOLIPID RECEPTORS IN URINARY-TRACT INFECTION SCANDINAVIAN JOURNAL OF INFECTIOUS DISEASES Leffler, H., LOMBERG, H., Gotschlich, E., HAGBERG, L., Jodal, U., Korhonen, T., Samuelsson, B. E., SCHOOLNIK, G., SVANBORGEDEN, C. 1982: 46-51
Abstract

The role of glycosphingolipids as host receptors, and fimbriae as bacterial ligands, for the adhesive and hemagglutinating reactions of uropathogenic E. coli was assessed. Glycolipids including globotetraosylceramide and globotriaosylceramide, which contain the disaccharide Gal alpha 1 leads to 4Gal were bound by many strains isolated from patients with pyelonephritis and cystitis. The fimbriae of one strain were shown to serve as ligands for these receptors. Although most pyelonephritic E. coli recognized globotetraosylceramide, as measured by the agglutination of glycolipid coated erythrocytes, some also recognized D-mannose residues (i.e. mannose-sensitive hemagglutination). Several strains were exclusively mannose-sensitive or bound neither globotetraosylceramide nor mannose. A genetic basis for susceptibility to infection was indicated. The erythrocytes of blood group P2 have lower amounts of Gal alpha 1 leads to 4Gal containing glycolipids than individuals of blood group P1. Blood group P2 was significantly less frequent (1/28) among patients with recurrent urinary tract infection compared to the normal population (10/40, p less than 0.02). Therefore, globoseries glycolipids may be determinants of susceptibility to urinary tract infection.

View details for Web of Science ID A1982PC20700009

View details for PubMedID 6127802
IMMUNOGLOBULIN CLASS RESPONSIBLE FOR GONOCOCCAL BACTERICIDAL ACTIVITY OF NORMAL HUMAN-SERA JOURNAL OF IMMUNOLOGY SCHOOLNIK, G. K., Ochs, H. D., Buchanan, T. M. 1979; 122 (5): 1771-1779
View details for Web of Science ID A1979GU71100024

View details for PubMedID 448108
PHENOTYPIC AND EPIDEMIOLOGIC CORRELATES OF AUXOTYPE IN NEISSERIA-GONORRHOEAE JOURNAL OF INFECTIOUS DISEASES Knapp, J. S., Thornsberry, C., SCHOOLNIK, G. A., WIESNER, P. J., HOLMES, K. K. 1978; 138 (2): 160-165
Abstract

Previous studies from Seattle, Wash., suggested that strains of Neisseria gonorrhoeae which require arginine, hypoxanthine, and uracil (Arg-Hyx-Ura- auxotype) are unifomly highly susceptible to penicillin G, are relatively resistant to complement-dependent killing by heated, pooled human serum, and are associated with disseminated gonococcal infection. For further study of the epidemiology of these strains and for analysis of the susceptibility to penicillin, serum sensitivity, and the nutritional requirements of gonococcal isolates from other cities, a survey was made of urethral and cervical strains isolated in 1972--1974 from 50 randomly selected pateit-s with uncomplicated gonorrhea from each of nine cities. Arg-Hyx-Ura-strains represented greater than 50% of isolates from Seattle and Des Moines, Iowa, 22% of isolates from Denver, Colo., and Dayton, Ohio, and less than or equal to 12% of the isolates from Boston, Mass., Newark, N.J., Norfork, Va., Miami, Fla., and Oakland, Calif. Arg-Hyx-Ura- strains were recovered from 42% of white and 9% of black patients (P less than 0.001), and clincis with the highest incidences of these strains had the highest proportion of white patients among those with gonorrhea. Arg-Hyx-Ura- strains were all susceptible to less than or equal to 0.125 microgram of penicillin G/ml and were more resistant than strains with other auxotypes to killing by heat-inactivated human serum plus complement (P less than 0.001).

View details for Web of Science ID A1978FM05400005

View details for PubMedID 98600
GENETIC STUDIES ON NEISSERIA-GONORRHOEAE FROM DISSEMINATED GONOCOCCAL INFECTIONS INFECTION AND IMMUNITY MAYER, L. W., SCHOOLNIK, G. K., FALKOW, S. 1977; 18 (1): 165-172
Abstract

Isolates from uncomplicated and disseminated gonococcal infections were analyzed by using deoxyribonucleic acid-mediated transformation. Most pairs of auxotrophs could recombine, producing independent transformants. When the constellation of arginine (Arg), hypoxanthine (Hyx), and uracil (Ura) requirements was present in donor and recipient, no recombination for these traits could be detected. Except for Arg to Hyx, no linkage between Arg, Hyx, Ura, penicillin G sensitivity, and serum resistance could be demonstrated. Some distant linkage of Ura to nalidixic acid and rifampin resistances was found. The data show that the traits associated with disseminated gonococcal infection strains are not closely linked but are identical in all strains, indicating a common origin.

View details for Web of Science ID A1977DX72000026

View details for PubMedID 409684
PROTECTION AGAINST INFECTION WITH NEISSERIA-GONORRHOEAE BY IMMUNIZATION WITH OUTER MEMBRANE-PROTEIN COMPLEX AND PURIFIED PILI JOURNAL OF INFECTIOUS DISEASES Buchanan, T. M., Pearce, W. A., SCHOOLNIK, G. K., ARKO, R. J. 1977; 136: S132-S137
Abstract

Some of the antigens that are capable of producing strain-related immunity to gonococcal infection in the guinea pig are located on the outer membrane of Neisseria gonorrhoeae. This finding has been demonstrated by immunization of guinea pigs with isolated outer membranes from two different strains of N. gonorrhoeae prior to challenged. Isolated principal outer membrane protein complex proved a better protective immunogen than purified pili from the same strain of N. gonorrhoeae. Principal outer membrane protein appears to react in antibody-complement-mediated killing of gonococci, whereas antibodies to pili are only weakly bactericidal. Pili-mediated attachment of N. gonorrhoeae to human cells is inhibited by antibodies to pili, and maximal inhibition occurs when antibodies are directed at pili antigenically identical to the pili mediating the attachment.

View details for Web of Science ID A1977DS68400021

View details for PubMedID 408428
GONOCOCCI CAUSING DISSEMINATED GONOCOCCAL INFECTION ARE RESISTANT TO BACTERICIDAL ACTION OF NORMAL HUMAN SERA JOURNAL OF CLINICAL INVESTIGATION SCHOOLNIK, G. K., Buchanan, T. M., HOLMES, K. K. 1976; 58 (5): 1163-1173
Abstract

The susceptibility of strains of Neisseria gonorrhoeae to the bactericidal action of normal human sera was determined for isolates from patients with disseminated gonococcal infection and uncomplicated gonorrhea. Serum susceptibility was correlated with penicillin susceptibility and auxotype. 38 of 39 strains (97%) of N. gonorrhoeae from Seattle patients with disseminated gonococcal infection were resistant to the complement-dependent bactericidal action of normal human sera. 36 of these were inhibited by less than or equal to mug/ml of penicillin G and required arginine, hypoxanthine, and uracil for growth on chemically defined medium (Arg-Hyx-Ura- auxotype). 12 of 43 isolates from patients with uncomplicated gonorrhea were also of the Arg-Hyx-Ura-auxotype, inhibited by less than or equal to 0.030 mug/ml of penicillin G, and serum resistant. Of the 31 remaining strains of other auxotypes isolated from patients with uncomplicated gonorrhea, 18 (58.1%) were sensitive to normal human sera in titers ranging from 2 to 2,048. The bactericidal action of normal human sera may prevent the dissemination of serum-sensitive gonococci. However, since only a small proportion of individuals infected by serum-resistant strains develop disseminated gonococcal infection, serum resistance appears to be a necessary but not a sufficient virulence factor for dissemination. Host factors such as menstruation and pharyngeal gonococcal infection may favor the dissemination of serum-resistant strains. Since serum-resistant Arg-Hyx-Ura strains are far more frequently isolated from patients with disseminated gonococcal infection than serum-resistant strains of other auxotypes, Arg-Hyx-Ura-strains may possess other virulence factors in addition to serum resistance.

View details for Web of Science ID A1976CJ24600015

View details for PubMedID 825532

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