Academic Appointments

Honors & Awards

  • Burroughs Wellcome Fund Recipient in Infectious Disease, The Burroughs Wellcome Fund (11/01/09-10/31/15)
  • G.J. Thorbecke Award, Society of Leukocyte Biology (2010)
  • Sidney Raffel Award for Outstanding Accomplishment in Graduate Study, Stanford University (2001)
  • Baxter Faculty Scholar Award, Baxter Foundation (May 2008)
  • Terman Fellowship, Terman Fellows Program (10/1/08-9/30/11)

Professional Education

  • Ph.D., Stanford University, Microbiology & Immunology (2002)

Research & Scholarship

Current Research and Scholarly Interests

The primary focus of my research is to understand the genetic and molecular mechanisms of intracellular bacterial pathogenesis. We use two model systems, Salmonella typhimurium and Francisella tularensis, to study the complex host-pathogen interactions.
Both of these organisms survive and multiply in macrophages, an important immune effector cell. Macrophages express Pattern Recognition Receptors on the surface as well as in the cytosol. My laboratory focuses on the cytosolic recognition of bacteria that leads to Type I Interferon signaling and Inflammasome activation. We take both a genetic and biochemical approach to understand the molecular mechanisms involved in host recognition pathways leading to inflammation and pathogen evasion mechanisms.
Salmonella typhi causes the systemic disease typhoid fever and Francisella tularensis causes the systemic disease tularemia (“rabbit fever”). Utilizing mouse models of systemic salmonellosis and tularemia, we would like to understand how Salmonella persists within certain hosts for years in the face of a robust immune response and how F. tularensis, a stealth invader, can cause a rapid, lethal infection.


2013-14 Courses


Journal Articles

  • The Systemic Immune State of Super-shedder Mice Is Characterized by a Unique Neutrophil-dependent Blunting of TH1 Responses. PLoS pathogens Gopinath, S., Hotson, A., Johns, J., Nolan, G., Monack, D. 2013; 9 (6)


    Host-to-host transmission of a pathogen ensures its successful propagation and maintenance within a host population. A striking feature of disease transmission is the heterogeneity in host infectiousness. It has been proposed that within a host population, 20% of the infected hosts, termed super-shedders, are responsible for 80% of disease transmission. However, very little is known about the immune state of these super-shedders. In this study, we used the model organism Salmonella enterica serovar Typhimurium, an important cause of disease in humans and animal hosts, to study the immune state of super-shedders. Compared to moderate shedders, super-shedder mice had an active inflammatory response in both the gastrointestinal tract and the spleen but a dampened TH1 response specific to the secondary lymphoid organs. Spleens from super-shedder mice had higher numbers of neutrophils, and a dampened T cell response, characterized by higher levels of regulatory T cells (Tregs), fewer T-bet(+) (TH1) T cells as well as blunted cytokine responsiveness. Administration of the cytokine granulocyte colony stimulating factor (G-CSF) and subsequent neutrophilia was sufficient to induce the super-shedder immune phenotype in moderate-shedder mice. Similar to super-shedders, these G-CSF-treated moderate-shedders had a dampened TH1 response with fewer T-bet(+) T cells and a loss of cytokine responsiveness. Additionally, G-CSF treatment inhibited IL-2-mediated TH1 expansion. Finally, depletion of neutrophils led to an increase in the number of T-bet(+) TH1 cells and restored their ability to respond to IL-2. Taken together, we demonstrate a novel role for neutrophils in blunting IL-2-mediated proliferation of the TH1 immune response in the spleens of mice that are colonized by high levels of S. Typhimurium in the gastrointestinal tract.

    View details for DOI 10.1371/journal.ppat.1003408

    View details for PubMedID 23754944

  • Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1 NATURE Broz, P., Ruby, T., Belhocine, K., Bouley, D. M., Kayagaki, N., Dixit, V. M., Monack, D. M. 2012; 490 (7419): 288-?


    Inflammasomes are cytosolic multiprotein complexes assembled by intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and they initiate innate immune responses to invading pathogens and danger signals by activating caspase-1 (ref. 1). Caspase-1 activation leads to the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1? and IL-18, as well as lytic inflammatory cell death known as pyroptosis. Recently, a new non-canonical inflammasome was described that activates caspase-11, a pro-inflammatory caspase required for lipopolysaccharide-induced lethality. This study also highlighted that previously generated caspase-1 knockout mice lack a functional allele of Casp11 (also known as Casp4), making them functionally Casp1?Casp11 double knockouts. Previous studies have shown that these mice are more susceptible to infections with microbial pathogens, including the bacterial pathogen Salmonella enterica serovar Typhimurium (S. typhimurium), but the individual contributions of caspase-1 and caspase-11 to this phenotype are not known. Here we show that non-canonical caspase-11 activation contributes to macrophage death during S. typhimurium infection. Toll-like receptor 4 (TLR4)-dependent and TIR-domain-containing adaptor-inducing interferon-? (TRIF)-dependent interferon-? production is crucial for caspase-11 activation in macrophages, but is only partially required for pro-caspase-11 expression, consistent with the existence of an interferon-inducible activator of caspase-11. Furthermore, Casp1(-/-) mice were significantly more susceptible to infection with S. typhimurium than mice lacking both pro-inflammatory caspases (Casp1(-/-)?Casp11(-/-)). This phenotype was accompanied by higher bacterial counts, the formation of extracellular bacterial microcolonies in the infected tissue and a defect in neutrophil-mediated clearance. These results indicate that caspase-11-dependent cell death is detrimental to the host in the absence of caspase-1-mediated innate immunity, resulting in extracellular replication of a facultative intracellular bacterial pathogen.

    View details for DOI 10.1038/nature11419

    View details for Web of Science ID 000309733300055

    View details for PubMedID 22895188

  • Differential Requirement for Caspase-1 Autoproteolysis in Pathogen-Induced Cell Death and Cytokine Processing CELL HOST & MICROBE Broz, P., von Moltke, J., Jones, J. W., Vance, R. E., Monack, D. M. 2010; 8 (6): 471-483


    Activation of the cysteine protease Caspase-1 is a key event in the innate immune response to infections. Synthesized as a proprotein, Caspase-1 undergoes autoproteolysis within multiprotein complexes called inflammasomes. Activated Caspase-1 is required for proteolytic processing and for release of the cytokines interleukin-1? and interleukin-18, and it can also cause rapid macrophage cell death. We show that macrophage cell death and cytokine maturation in response to infection with diverse bacterial pathogens can be separated genetically and that two distinct inflammasome complexes mediate these events. Inflammasomes containing the signaling adaptor Asc form a single large "focus" in which Caspase-1 undergoes autoproteolysis and processes IL-1?/IL-18. In contrast, Asc-independent inflammasomes activate Caspase-1 without autoproteolysis and do not form any large structures in the cytosol. Caspase-1 mutants unable to undergo autoproteolysis promoted rapid cell death, but processed IL-1?/18 inefficiently. Our results suggest the formation of spatially and functionally distinct inflammasomes complexes in response to bacterial pathogens.

    View details for DOI 10.1016/j.chom.2010.11.007

    View details for Web of Science ID 000287344400004

    View details for PubMedID 21147462

  • Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella JOURNAL OF EXPERIMENTAL MEDICINE Broz, P., Newton, K., Lamkanfi, M., Mariathasan, S., Dixit, V. M., Monack, D. M. 2010; 207 (8): 1745-1755


    Intracellular pathogens and endogenous danger signals in the cytosol engage NOD-like receptors (NLRs), which assemble inflammasome complexes to activate caspase-1 and promote the release of proinflammatory cytokines IL-1beta and IL-18. However, the NLRs that respond to microbial pathogens in vivo are poorly defined. We show that the NLRs NLRP3 and NLRC4 both activate caspase-1 in response to Salmonella typhimurium. Responding to distinct bacterial triggers, NLRP3 and NLRC4 recruited ASC and caspase-1 into a single cytoplasmic focus, which served as the site of pro-IL-1beta processing. Consistent with an important role for both NLRP3 and NLRC4 in innate immune defense against S. typhimurium, mice lacking both NLRs were markedly more susceptible to infection. These results reveal unexpected redundancy among NLRs in host defense against intracellular pathogens in vivo.

    View details for DOI 10.1084/jem.20100257

    View details for Web of Science ID 000280709900016

    View details for PubMedID 20603313

  • Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jones, J. W., Kayagaki, N., Broz, P., Henry, T., Newton, K., O'Rourke, K., Chan, S., Dong, J., Qu, Y., Roose-Girma, M., Dixit, V. M., Monack, D. M. 2010; 107 (21): 9771-9776


    Macrophages respond to cytosolic nucleic acids by activating cysteine protease caspase-1 within a complex called the inflammasome. Subsequent cleavage and secretion of proinflammatory cytokines IL-1beta and IL-18 are critical for innate immunity. Here, we show that macrophages from mice lacking absent in melanoma 2 (AIM2) cannot sense cytosolic double-stranded DNA and fail to trigger inflammasome assembly. Caspase-1 activation in response to intracellular pathogen Francisella tularensis also required AIM2. Immunofluorescence microscopy of macrophages infected with F. tularensis revealed striking colocalization of bacterial DNA with endogenous AIM2 and inflammasome adaptor ASC. By contrast, type I IFN (IFN-alpha and -beta) secretion in response to F. tularensis did not require AIM2. IFN-I did, however, boost AIM2-dependent caspase-1 activation by increasing AIM2 protein levels. Thus, inflammasome activation was reduced in infected macrophages lacking either the IFN-I receptor or stimulator of interferon genes (STING). Finally, AIM2-deficient mice displayed increased susceptibility to F. tularensis infection compared with wild-type mice. Their increased bacterial burden in vivo confirmed that AIM2 is essential for an effective innate immune response.

    View details for DOI 10.1073/pnas.1003738107

    View details for Web of Science ID 000278054700054

    View details for PubMedID 20457908

  • Type I IFN Signaling Constrains IL-17A/F Secretion by gamma delta T Cells during Bacterial Infections JOURNAL OF IMMUNOLOGY Henry, T., Kirimanjeswara, G. S., Ruby, T., Jones, J. W., Peng, K., Perret, M., Ho, L., Sauer, J., Iwakura, Y., Metzger, D. W., Monack, D. M. 2010; 184 (7): 3755-3767


    Recognition of intracellular bacteria by macrophages leads to secretion of type I IFNs. However, the role of type I IFN during bacterial infection is still poorly understood. Francisella tularensis, the causative agent of tularemia, is a pathogenic bacterium that replicates in the cytosol of macrophages leading to secretion of type I IFN. In this study, we investigated the role of type I IFNs in a mouse model of tularemia. Mice deficient for type I IFN receptor (IFNAR1(-/-)) are more resistant to intradermal infection with F. tularensis subspecies novicida (F. novicida). Increased resistance to infection was associated with a specific increase in IL-17A/F and a corresponding expansion of an IL-17A(+) gammadelta T cell population, indicating that type I IFNs negatively regulate the number of IL-17A(+) gammadelta T cells during infection. Furthermore, IL-17A-deficient mice contained fewer neutrophils compared with wild-type mice during infection, indicating that IL-17A contributes to neutrophil expansion during F. novicida infection. Accordingly, an increase in IL-17A in IFNAR1(-/-) mice correlated with an increase in splenic neutrophil numbers. Similar results were obtained in a mouse model of pneumonic tularemia using the highly virulent F. tularensis subspecies tularensis SchuS4 strain and in a mouse model of systemic Listeria monocytogenes infection. Our results indicate that the type I IFN-mediated negative regulation of IL-17A(+) gammadelta T cell expansion is conserved during bacterial infections. We propose that this newly described activity of type I IFN signaling might participate in the resistance of the IFNAR1(-/-) mice to infection with F. novicida and other intracellular bacteria.

    View details for DOI 10.4049/jimmunol.0902065

    View details for Web of Science ID 000275927600054

    View details for PubMedID 20176744

  • The Salmonella SPI2 Effector SseI Mediates Long-Term Systemic Infection by Modulating Host Cell Migration PLOS PATHOGENS McLaughlin, L. M., Govoni, G. R., Gerke, C., Gopinath, S., Peng, K., Laidlaw, G., Chien, Y., Jeong, H., Li, Z., Brown, M. D., Sacks, D. B., Monack, D. 2009; 5 (11)


    Host-adapted strains of Salmonella enterica cause systemic infections and have the ability to persist systemically for long periods of time despite the presence of a robust immune response. Chronically infected hosts are asymptomatic and transmit disease to naïve hosts via fecal shedding of bacteria, thereby serving as a critical reservoir for disease. We show that the bacterial effector protein SseI (also called SrfH), which is translocated into host cells by the Salmonella Pathogenicity Island 2 (SPI2) type III secretion system (T3SS), is required for Salmonella typhimurium to maintain a long-term chronic systemic infection in mice. SseI inhibits normal cell migration of primary macrophages and dendritic cells (DC) in vitro, and such inhibition requires the host factor IQ motif containing GTPase activating protein 1 (IQGAP1), an important regulator of cell migration. SseI binds directly to IQGAP1 and co-localizes with this factor at the cell periphery. The C-terminal domain of SseI is similar to PMT/ToxA, a bacterial toxin that contains a cysteine residue (C1165) that is critical for activity. Mutation of the corresponding residue in SseI (C178A) eliminates SseI function in vitro and in vivo, but not binding to IQGAP1. In addition, infection with wild-type (WT) S. typhimurium suppressed DC migration to the spleen in vivo in an SseI-dependent manner. Correspondingly, examination of spleens from mice infected with WT S. typhimurium revealed fewer DC and CD4(+) T lymphocytes compared to mice infected with Delta sseI S. typhimurium. Taken together, our results demonstrate that SseI inhibits normal host cell migration, which ultimately counteracts the ability of the host to clear systemic bacteria.

    View details for DOI 10.1371/journal.ppat.1000671

    View details for Web of Science ID 000274226600025

    View details for PubMedID 19956712

  • Host transmission of Salmonella enterica serovar Typhimurium is controlled by virulence factors and indigenous intestinal microbiota INFECTION AND IMMUNITY Lawley, T. D., Bouley, D. A., Hoy, Y. E., Gerke, C., Relman, D. A., Monack, D. M. 2008; 76 (1): 403-416


    Transmission is an essential stage of a pathogen's life cycle and remains poorly understood. We describe here a model in which persistently infected 129X1/SvJ mice provide a natural model of Salmonella enterica serovar Typhimurium transmission. In this model only a subset of the infected mice, termed supershedders, shed high levels (>10(8) CFU/g) of Salmonella serovar Typhimurium in their feces and, as a result, rapidly transmit infection. While most Salmonella serovar Typhimurium-infected mice show signs of intestinal inflammation, only supershedder mice develop colitis. Development of the supershedder phenotype depends on the virulence determinants Salmonella pathogenicity islands 1 and 2, and it is characterized by mucosal invasion and, importantly, high luminal abundance of Salmonella serovar Typhimurium within the colon. Immunosuppression of infected mice does not induce the supershedder phenotype, demonstrating that the immune response is not the main determinant of Salmonella serovar Typhimurium levels within the colon. In contrast, treatment of mice with antibiotics that alter the health-associated indigenous intestinal microbiota rapidly induces the supershedder phenotype in infected mice and predisposes uninfected mice to the supershedder phenotype for several days. These results demonstrate that the intestinal microbiota plays a critical role in controlling Salmonella serovar Typhimurium infection, disease, and transmissibility. This novel model should facilitate the study of host, pathogen, and intestinal microbiota factors that contribute to infectious disease transmission.

    View details for DOI 10.1128/IAI.01189-07

    View details for Web of Science ID 000252126000043

    View details for PubMedID 17967858

  • Type I interferon signaling is required for activation of the inflammasome during Francisella infection JOURNAL OF EXPERIMENTAL MEDICINE Henry, T., Brotcke, A., Weiss, D. S., Thompson, L. J., Monack, D. M. 2007; 204 (5): 987-994


    Francisella tularensis is a pathogenic bacterium whose virulence is linked to its ability to replicate within the host cell cytosol. Entry into the macrophage cytosol activates a host-protective multimolecular complex called the inflammasome to release the proinflammatory cytokines interleukin (IL)-1beta and -18 and trigger caspase-1-dependent cell death. In this study, we show that cytosolic F. tularensis subspecies novicida (F. novicida) induces a type I interferon (IFN) response that is essential for caspase-1 activation, inflammasome-mediated cell death, and release of IL-1beta and -18. Extensive type I IFN-dependent cell death resulting in macrophage depletion occurs in vivo during F. novicida infection. Type I IFN is also necessary for inflammasome activation in response to cytosolic Listeria monocytogenes but not vacuole-localized Salmonella enterica serovar Typhimurium or extracellular adenosine triphosphate. These results show the specific connection between type I IFN signaling and inflammasome activation, which are two sequential events triggered by the recognition of cytosolic bacteria. To our knowledge, this is the first example of the positive regulation of inflammasome activation. This connection underscores the importance of the cytosolic recognition of pathogens and highlights how multiple innate immunity pathways interact before commitment to critical host responses.

    View details for DOI 10.1084/jem.20062665

    View details for Web of Science ID 000246467600004

    View details for PubMedID 17452523

  • In vivo negative selection screen identifies genes required for Francisella virulence PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Weiss, D. S., Brotcke, A., Henry, T., Margolis, J. J., Chan, K., Monack, D. M. 2007; 104 (14): 6037-6042


    Francisella tularensis subverts the immune system to rapidly grow within mammalian hosts, often causing tularemia, a fatal disease. This pathogen targets the cytosol of macrophages where it replicates by using the genes encoded in the Francisella pathogenicity island. However, the bacteria are recognized in the cytosol by the host's ASC/caspase-1 pathway, which is essential for host defense, and leads to macrophage cell death and proinflammatory cytokine production. We used a microarray-based negative selection screen to identify Francisella genes that contribute to growth and/or survival in mice. The screen identified many known virulence factors including all of the Francisella pathogenicity island genes, LPS O-antigen synthetic genes, and capsule synthetic genes. We also identified 44 previously unidentified genes that were required for Francisella virulence in vivo, indicating that this pathogen may use uncharacterized mechanisms to cause disease. Among these, we discovered a class of Francisella virulence genes that are essential for growth and survival in vivo but do not play a role in intracellular replication within macrophages. Instead, these genes modulate the host ASC/caspase-1 pathway, a previously unidentified mechanism of Francisella pathogenesis. This finding indicates that the elucidation of the molecular mechanisms used by other uncharacterized genes identified in our screen will increase our understanding of the ways in which bacterial pathogens subvert the immune system.

    View details for DOI 10.1073/pnas.0609675104

    View details for Web of Science ID 000245657600060

    View details for PubMedID 17389372

  • Genome-wide screen for Salmonella genes required for long-term systemic infection of the mouse PLOS PATHOGENS Lawley, T. D., Chan, K., Thompson, L. J., Kim, C. C., Govoni, G. R., Monack, D. M. 2006; 2 (2): 87-100


    A microarray-based negative selection screen was performed to identify Salmonella enterica serovar Typhimurium (serovar Typhimurium) genes that contribute to long-term systemic infection in 129X1/SvJ (Nramp1(r)) mice. A high-complexity transposon-mutagenized library was used to infect mice intraperitoneally, and the selective disappearance of mutants was monitored after 7, 14, 21, and 28 d postinfection. One hundred and eighteen genes were identified to contribute to serovar Typhimurium infection of the spleens of mice by 28 d postinfection. The negatively selected mutants represent many known aspects of Salmonella physiology and pathogenesis, although the majority of the identified genes are of putative or unknown function. Approximately 30% of the negatively selected genes correspond to horizontally acquired regions such as those within Salmonella pathogenicity islands (SPI 1-5), prophages (Gifsy-1 and -2 and remnant), and the pSLT virulence plasmid. In addition, mutations in genes responsible for outer membrane structure and remodeling, such as LPS- and PhoP-regulated and fimbrial genes, were also selected against. Competitive index experiments demonstrated that the secreted SPI2 effectors SseK2 and SseJ as well as the SPI4 locus are attenuated relative to wild-type bacteria during systemic infection. Interestingly, several SPI1-encoded type III secretion system effectors/translocases are required by serovar Typhimurium to establish and, unexpectedly, to persist systemically, challenging the present description of Salmonella pathogenesis. Moreover, we observed a progressive selection against serovar Typhimurium mutants based upon the duration of the infection, suggesting that different classes of genes may be required at distinct stages of infection. Overall, these data indicate that Salmonella long-term systemic infection in the mouse requires a diverse repertoire of virulence factors. This diversity of genes presumably reflects the fact that bacteria sequentially encounter a variety of host environments and that Salmonella has evolved to respond to these selective forces in a way that permits both the bacteria and the host to survive.

    View details for DOI 10.1371/journal.ppat.0020011

    View details for Web of Science ID 000202894200004

    View details for PubMedID 16518469

  • Revisiting caspase-11 function in host defense. Cell host & microbe Ng, T. M., Monack, D. M. 2013; 14 (1): 9-14


    Proinflammatory caspases play important roles in innate immunity. Much attention has focused on caspase-1, which acts to eliminate pathogens by obliterating their replicative niches as well as alerting the host to their presence. Now, emerging data have shed light on the lesser-studied proinflammatory caspase-11 in the combat between host and pathogens. Using the new tools available, researchers are further elucidating the mechanisms by which caspase-11 contributes to host defense. Here, we review the emerging understanding of caspase-11 functions and the mechanisms of activation and discuss the implications for human disease.

    View details for DOI 10.1016/j.chom.2013.06.009

    View details for PubMedID 23870309

  • The NeST Long ncRNA Controls Microbial Susceptibility and Epigenetic Activation of the Interferon-gamma Locus CELL Gomez, J. A., Wapinski, O. L., Yang, Y. W., Bureau, J., Gopinath, S., Monack, D. M., Chang, H. Y., Brahic, M., Kirkegaard, K. 2013; 152 (4): 743-754


    Long noncoding RNAs (lncRNAs) are increasingly appreciated as regulators of cell-specific gene expression. Here, an enhancer-like lncRNA termed NeST (nettoie Salmonella pas Theiler's [cleanup Salmonella not Theiler's]) is shown to be causal for all phenotypes conferred by murine viral susceptibility locus Tmevp3. This locus was defined by crosses between SJL/J and B10.S mice and contains several candidate genes, including NeST. The SJL/J-derived locus confers higher lncRNA expression, increased interferon-? (IFN-?) abundance in activated CD8(+) T cells, increased Theiler's virus persistence, and decreased Salmonella enterica pathogenesis. Transgenic expression of NeST lncRNA alone was sufficient to confer all phenotypes of the SJL/J locus. NeST RNA was found to bind WDR5, a component of the histone H3 lysine 4 methyltransferase complex, and to alter histone 3 methylation at the IFN-? locus. Thus, this lncRNA regulates epigenetic marking of IFN-?-encoding chromatin, expression of IFN-?, and susceptibility to a viral and a bacterial pathogen.

    View details for DOI 10.1016/j.cell.2013.01.015

    View details for Web of Science ID 000314945600010

    View details for PubMedID 23415224

  • Policing the cytosol-bacterial-sensing inflammasome receptors and pathways CURRENT OPINION IN IMMUNOLOGY Ng, T. M., Kortmann, J., Monack, D. M. 2013; 25 (1): 34-39


    Pattern recognition receptors recognize signals originating from pathogens and comprise a large part of the arsenal in innate immune responses. The NOD-like receptors (NLRs) are one particular class of these receptors that survey the cytoplasm for signs of pathogen invasion. Upon detection, they trigger the formation of a macromolecular complex called the inflammasome that is required for elimination of the pathogen, as well as amplifying a pro-inflammatory response. Although the core machinery has been defined, recent data emphasize the complexity of how NLR inflammasomes function. Here, we highlight new discoveries that reveal how precisely fine-tuned NLR inflammasome functions are, and how that may be modulated by antagonistic effects of concomitant inflammasome activation as well as novel regulatory factors.

    View details for DOI 10.1016/j.coi.2012.11.009

    View details for Web of Science ID 000316777300005

    View details for PubMedID 23261344

  • The complex interactions of bacterial pathogens and host defenses. Current opinion in microbiology Monack, D. M., Hultgren, S. J. 2013; 16 (1): 1-3

    View details for DOI 10.1016/j.mib.2013.03.001

    View details for PubMedID 23518336

  • Measuring inflammasome activation in response to bacterial infection. Methods in molecular biology (Clifton, N.J.) Broz, P., Monack, D. M. 2013; 1040: 65-84


    Inflammasomes are multi-protein signaling platforms assembled in response to viral and bacterial pathogens as well as endogenous danger signals. Inflammasomes serve as activation platforms for the mammalian cysteine protease caspase-1, a central mediator of innate immunity. The hallmarks of inflammasome activation are the processing of caspase-1, the maturation and release of interleukin-1β (IL-1β) and the induction of pyroptosis, a lytic inflammatory cell death. This protocol describes methods for studying inflammasome activation in response to bacterial pathogens in bone-marrow derived murine macrophages (BMDMs). In particular, we outline the protocols to measure cytokine maturation by ELISA and pyroptosis by the release of Lactate Dehydrogenase (LDH). In addition, we describe methods to visualize endogenous ASC specks or foci in infected cells and to study the release of processed caspase-1, caspase-11 and mature cytokines into the cell supernatant by Western blotting. General considerations are discussed to design and optimize the infection protocol for the study of inflammasome activation by other bacterial pathogens.

    View details for DOI 10.1007/978-1-62703-523-1_6

    View details for PubMedID 23852597

  • Caspase-1 activity is required to bypass macrophage apoptosis upon Salmonella infection NATURE CHEMICAL BIOLOGY Puri, A. W., Broz, P., Shen, A., Monack, D. M., Bogyo, M. 2012; 8 (9): 745-747


    Here we report AWP28, an activity-based probe that can be used to biochemically monitor caspase-1 activation in response to proinflammatory stimuli. Using AWP28, we show that apoptosis is triggered upon Salmonella enterica var. Typhimurium infection in primary mouse bone marrow macrophages lacking caspase-1. Furthermore, we report that upon Salmonella infection, inflammasome-mediated caspase-1 activity is required to bypass apoptosis in favor of proinflammatory pyroptotic cell death.

    View details for DOI 10.1038/NCHEMBIO.1023

    View details for Web of Science ID 000308077600004

    View details for PubMedID 22797665

  • Shedding light on Salmonella carriers TRENDS IN MICROBIOLOGY Gopinath, S., Carden, S., Monack, D. 2012; 20 (7): 320-327


    Host-to-host transmission in most Salmonella serovars occurs primarily via the fecal-oral route. Salmonella enterica serovar Typhi is a human host-adapted pathogen and some S. Typhi patients become asymptomatic carriers. These individuals excrete large numbers of the bacteria in their feces and transmit the pathogen by contaminating water or food sources. The carrier state has also been described in livestock animals and is responsible for food-borne epidemics. Identification and treatment of carriers are crucial for the control of disease outbreaks. In this review, we describe recent advances in molecular profiling of human carriers and the use of animal models to identify potential host and bacterial genes involved in the establishment of the carrier state.

    View details for DOI 10.1016/j.tim.2012.04.004

    View details for Web of Science ID 000306868100003

    View details for PubMedID 22591832

  • Salmonella's long-term relationship with its host FEMS MICROBIOLOGY REVIEWS Ruby, T., McLaughlin, L., Gopinath, S., Monack, D. 2012; 36 (3): 600-615


    Host-adapted strains of Salmonella enterica cause systemic infections and have the ability to persist systemically for long periods of time and pose significant public-health problems. Multidrug-resistant S. enterica serovar Typhi (S. Typhi) and nontyphoidal Salmonella (NTS) are on the increase and are often associated with HIV infection. Chronically infected hosts are often asymptomatic and transmit disease to naïve hosts via fecal shedding of bacteria, thereby serving as a critical reservoir for disease. Salmonella utilizes multiple ways to evade and modulate host innate and adaptive immune responses in order to persist in the presence of a robust immune response. Survival in macrophages and modulation of immune cells migration allow Salmonella to evade various immune responses. The ability of Salmonella to persist depends on a balance between immune responses that lead to the clearance of the pathogen and avoidance of damage to host tissues.

    View details for DOI 10.1111/j.1574-6976.2012.00332.x

    View details for Web of Science ID 000302240500005

    View details for PubMedID 22335190

  • Delayed activation of host innate immune pathways in streptozotocin-induced diabetic hosts leads to more severe disease during infection with Burkholderia pseudomallei IMMUNOLOGY Chin, C., Monack, D. M., Nathan, S. 2012; 135 (4): 312-332


    Diabetes mellitus is a predisposing factor of melioidosis, contributing to higher mortality rates in diabetics infected with Burkholderia pseudomallei. To investigate how diabetes alters the inflammatory response, we established a streptozotocin (STZ) -induced diabetic murine acute-phase melioidosis model. Viable B. pseudomallei cells were consistently detected in the blood, liver and spleen during the 42-hr course of infection but the hyperglycaemic environment did not increase the bacterial burden. However, after 24 hr, granulocyte counts increased in response to infection, whereas blood glucose concentrations decreased over the course of infection. A genome-wide expression analysis of the STZ-diabetic murine acute melioidosis liver identified ~1000 genes whose expression was altered in the STZ-diabetic mice. The STZ-diabetic host transcriptional response was compared with the normoglycaemic host transcriptional response recently reported by our group. The microarray data suggest that the presence of elevated glucose levels impairs the host innate immune system by delaying the identification and recognition of B. pseudomallei surface structures. Consequently, the host is unable to activate the appropriate innate immune response over time, which may explain the increased susceptibility to melioidosis in the STZ-diabetic host. Nevertheless, a general 'alarm signal' of infection as well as defence programmes are still triggered by the STZ-diabetic host, although only 24 hr after infection. In summary, this study demonstrates that in the face of a B. pseudomallei acute infection, poor glycaemic control impaired innate responses during the early stages of B. pseudomallei infection, contributing to the increased susceptibility of STZ-induced diabetics to this fatal disease.

    View details for DOI 10.1111/j.1365-2567.2011.03544.x

    View details for Web of Science ID 000300982500007

    View details for PubMedID 22136109

  • Innate immune response to Salmonella typhimurium, a model enteric pathogen. Gut microbes Broz, P., Ohlson, M. B., Monack, D. M. 2012; 3 (2): 62-70


    The innate immune system provides the first line of defense against invading microorganisms by inducing a variety of inflammatory and antimicrobial responses. These responses are particularly important in the gastrointestinal tract, where the needs for efficient nutrient uptake and host defense collide. Many pathogens have evolved to specifically colonize the intestine, causing millions of cases of enteric infections a year. A paradigm of an enteric pathogen is Salmonella enterica, a gram-negative bacterium that causes a wide range of gastrointestinal and systemic diseases. Infections with Salmonella enterica serovar Typhimurium (S. typhimurium) lead to an acute intestinal inflammation in human and animal hosts, as a result of the bacterium invading the mucosa. A distinctive feature of Salmonella is that it has not only adapted to survive in a strong inflammatory environment, but it also uses this adaptation as a strategy to gain a growth advantage over the intestinal microbiota. We will use the model organism S. typhimurium to discuss the innate immune mechanisms employed by the mammalian gastrointestinal system and how the pathogen responds and subverts these mechanisms. In particular, we focus on the recognition of extra- and intra-cellular Salmonellae by germline-encoded pattern recognition receptors of the TLR and NLR families, and how Salmonella might profit from the activation of these receptors.

    View details for DOI 10.4161/gmic.19141

    View details for PubMedID 22198618

  • Salmonella persistence and transmission strategies CURRENT OPINION IN MICROBIOLOGY Monack, D. M. 2012; 15 (1): 100-107


    Host-adapted strains of Salmonella enterica cause systemic infections and have the ability to persist systemically for long periods of time and pose significant public-health problems. Multidrug-resistant Salmonella enteric serovar Typhi (S. Typhi) and non-Typhoidal Salmonella (NTS) are on the increase, and are often associated with HIV infection. Chronically infected hosts are often asymptomatic and transmit disease to naïve hosts via fecal shedding of bacteria, thereby serving as a critical reservoir for disease. Salmonella utilizes multiple strategies to evade and modulate host innate and adaptive immune responses in order to persist in the presence of a robust immune response. In addition, the intestinal microbiota plays a critical role in controlling Salmonella infection, disease, and transmissibility.

    View details for DOI 10.1016/j.mib.2011.10.013

    View details for Web of Science ID 000301314200016

    View details for PubMedID 22137596

  • Francisella infection triggers activation of the AIM2 inflammasome in murine dendritic cells CELLULAR MICROBIOLOGY Belhocine, K., Monack, D. M. 2012; 14 (1): 71-80


    The intracellular bacterium Francisella tularensis is the causative agent of tularemia, a potentially fatal disease. In macrophages, Francisella escapes the initial phagosome and replicates in the cytosol, where it is detected by the cytosolic DNA sensor AIM2 leading to activation of the AIM2 inflammasome. However, during aerosol infection, Francisella is also taken up by dendritic cells. In this study, we show that Francisella novicida escapes into the cytosol of bone marrow-derived dendritic cells (BMDC) where it undergoes rapid replication. We show that F. novicida activates the AIM2 inflammasome in BMDC, causing release of large amounts of IL-1? and rapid host cell death. The Francisella Pathogenicity Island is required for bacterial escape and replication and for inflammasome activation in dendritic cells. In addition, we show that bacterial DNA is bound by AIM2, which leads to inflammasome assembly in infected dendritic cells. IFN-? is upregulated in BMDC following Francisella infection, and the IFN-? signalling pathway is partially required for inflammasome activation in this cell type. Taken together, our results demonstrate that F. novicida induces inflammasome activation in dendritic cells. The resulting inflammatory cell death may be beneficial to remove the bacterial replicative niche and protect the host.

    View details for DOI 10.1111/j.1462-5822.2011.01700.x

    View details for Web of Science ID 000298061800007

    View details for PubMedID 21902795

  • Elevated AIM2-mediated pyroptosis triggered by hypercytotoxic Francisella mutant strains is attributed to increased intracellular bacteriolysis CELLULAR MICROBIOLOGY Peng, K., Broz, P., Jones, J., Joubert, L., Monack, D. 2011; 13 (10): 1586-1600


    Intracellular bacterial pathogens Francisella novicida and the Live Vaccine Strain (LVS) are recognized in the macrophage cytosol by the AIM2 inflammasome, which leads to the activation of caspase-1 and the processing and secretion of active IL-1?, IL-18 and pyroptosis. Previous studies have reported that F. novicida and LVS mutants in specific genes (e.g. FTT0584, mviN and ripA) induce elevated inflammasome activation and hypercytotoxicity in host cells, leading to the proposal that F. novicida and LVS may have proteins that actively modulate inflammasome activation. However, there has been no direct evidence of such inflammasome evasion mechanisms. Here, we demonstrate for the first time that the above mutants, along with a wide range of F. novicida hypercytotoxic mutants that are deficient for membrane-associated proteins (?FTT0584, ?mviN, ?ripA, ?fopA and ?FTN1217) or deficient for genes involved in O-antigen or LPS biosynthesis (?wbtA and ?lpxH) lyse more intracellularly, thus activating increased levels of AIM2-dependent pyroptosis and other innate immune signalling pathways. This suggests that an inflammasome-specific evasion mechanism may not be present in F. novicida and LVS. Furthermore, future studies may need to consider increased bacterial lysis as a possible cause of elevated stimulation of multiple innate immune pathways when the protein composition or surface carbohydrates of the bacterial membrane is altered.

    View details for DOI 10.1111/j.1462-5822.2011.01643.x

    View details for Web of Science ID 000294924500012

    View details for PubMedID 21883803

  • The two-component sensor kinase KdpD is required for Salmonella typhimurium colonization of Caenorhabditis elegans and survival in macrophages CELLULAR MICROBIOLOGY Alegado, R. A., Chin, C., Monack, D. M., Tan, M. 2011; 13 (10): 1618-1637


    The ability of enteric pathogens to perceive and adapt to distinct environments within the metazoan intestinal tract is critical for pathogenesis; however, the preponderance of interactions between microbe- and host-derived factors remain to be fully understood. Salmonella enterica serovar Typhimurium is a medically important enteric bacterium that colonizes, proliferates and persists in the intestinal lumen of the nematode Caenorhabditis elegans. Several Salmonella virulence factors important in murine and tissue culture models also contribute to worm mortality and intestinal persistence. For example, PhoP and the virulence plasmid pSLT are virulence factors required for resistance to the C. elegans antimicrobial peptide SPP-1. To uncover additional determinants required for Salmonella typhimurium pathogenesis in vivo, we devised a genetic screen to identify bacterial mutants defective in establishing a persistent infection in the intestine of C. elegans. Here we report on identification of 14 loci required for persistence in the C. elegans intestine and characterization of KdpD, a sensor kinase of a two-component system in S. typhimurium pathogenesis. We show that kdpD mutants are profoundly attenuated in intestinal persistence in the nematode and in macrophage survival. These findings may be attributed to the essential role KdpD plays in promoting resistance to osmotic, oxidative and antimicrobial stresses.

    View details for DOI 10.1111/j.1462-5822.2011.01645.x

    View details for Web of Science ID 000294924500014

    View details for PubMedID 21790938

  • IMMUNOLOGY Recognition of a unique partner NATURE Monack, D. M. 2011; 477 (7366): 543-544

    View details for Web of Science ID 000295320900027

    View details for PubMedID 21956324

  • Molecular mechanisms of inflammasome activation during microbial infections IMMUNOLOGICAL REVIEWS Broz, P., Monack, D. M. 2011; 243: 174-190


    The innate immune system plays a crucial role in the rapid recognition and elimination of invading microbes. Detection of microbes relies on germ-line encoded pattern recognition receptors (PRRs) that recognize essential bacterial molecules, so-called pathogen-associated molecular patterns (PAMPs). A subset of PRRs, belonging to the NOD-like receptor (NLR) and the PYHIN protein families, detects viral and bacterial pathogens in the cytosol of host cells and induces the assembly of a multi-protein signaling platform called the inflammasome. The inflammasome serves as an activation platform for the mammalian cysteine protease caspase-1, a central mediator of innate immunity. Active caspase-1 promotes the maturation and release of interleukin-1? (IL-1?) and IL-18 as well as protein involved in cytoprotection and tissue repair. In addition, caspase-1 initiates a novel form of cell death called pyroptosis. Here, we discuss latest advances and our insights on inflammasome stimulation by two model intracellular pathogens, Francisella tularensis and Salmonella typhimurium. Recent studies on these pathogens have significantly shaped our understanding of the molecular mechanisms of inflammasome activation and how microbes can evade or manipulate inflammasome activity. In addition, we review the role of the inflammasome adapter ASC in caspase-1 autoproteolysis and new insights into the structure of the inflammasome complex.

    View details for DOI 10.1111/j.1600-065X.2011.01041.x

    View details for Web of Science ID 000295016800014

    View details for PubMedID 21884176

  • Francisella tularensis Schu S4 O-Antigen and Capsule Biosynthesis Gene Mutants Induce Early Cell Death in Human Macrophages INFECTION AND IMMUNITY Lindemann, S. R., Peng, K., Long, M. E., Hunt, J. R., Apicella, M. A., Monack, D. M., Allen, L. H., Jones, B. D. 2011; 79 (2): 581-594


    Francisella tularensis is capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains of Francisella, relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238. Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest that FTT1236, FTT1237, and FTT1238 are important for polysaccharide biosynthesis and that the Francisella O antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.

    View details for DOI 10.1128/IAI.00863-10

    View details for Web of Science ID 000286462000004

    View details for PubMedID 21078861

  • At home with hostility: How do pathogenic bacteria evade mammalian immune surveillance to establish persistent infection? F1000 biology reports Ruby, T., Monack, D. M. 2011; 3: 1-?


    Bacterial persistence is of major concern as persistent bacterial infections involving bacteria such as Helicobacter pylori, Salmonella enterica serotype Typhi, and Mycobacterium tuberculosis pose significant public health problems worldwide. This report discusses the recent advances in understanding the strategies used by bacteria during persistent infection that allow them to colonize specific sites in the host and evade immune surveillance.

    View details for DOI 10.3410/B3-1

    View details for PubMedID 21399762

  • Innate immune recognition of francisella tularensis: activation of type-I interferons and the inflammasome. Frontiers in microbiology Jones, J. W., Broz, P., Monack, D. M. 2011; 2: 16-?


    Francisella tularensis is an intracellular pathogen that can cause severe disease in a wide range of mammalian hosts. Primarily residing in host macrophages, F. tularensis escapes phagosomal degradation, and replicates in the macrophage cytosol. The macrophage uses a series of pattern recognition receptors to detect conserved microbial molecules from invading pathogens, and initiates an appropriate host response. In the cytosol, F. tularensis is recognized by the inflammasome, a multiprotein complex responsible for the activation of the cysteine protease caspase-1. Caspase-1 activation leads to processing and release of proinflammatory cytokines and host cell death. Here we review recent work on the molecular mechanisms of inflammasome activation by F. tularensis, and its consequences both in vitro and in vivo. Finally, we discuss the coordination between the inflammasome and other cytosolic host responses, and the evidence for F. tularensis virulence factors that suppress inflammasome activation.

    View details for DOI 10.3389/fmicb.2011.00016

    View details for PubMedID 21687410

  • Reciprocal Analysis of Francisella novicida Infections of a Drosophila melanogaster Model Reveal Host-Pathogen Conflicts Mediated by Reactive Oxygen and imd-Regulated Innate Immune Response PLOS PATHOGENS Moule, M. G., Monack, D. M., Schneider, D. S. 2010; 6 (8)


    The survival of a bacterial pathogen within a host depends upon its ability to outmaneuver the host immune response. Thus, mutant pathogens provide a useful tool for dissecting host-pathogen relationships, as the strategies the microbe has evolved to counteract immunity reveal a host's immune mechanisms. In this study, we examined the pathogen Francisella novicida and identified new bacterial virulence factors that interact with different parts of the Drosophila melanogaster innate immune system. We performed a genome-wide screen to identify F. novicida genes required for growth and survival within the fly and identified a set of 149 negatively selected mutants. Among these, we identified a class of genes including the transcription factor oxyR, and the DNA repair proteins uvrB, recB, and ruvC that help F. novicida resist oxidative stress. We determined that these bacterial genes are virulence factors that allow F. novicida to counteract the fly melanization immune response. We then performed a second in vivo screen to identify an additional subset of bacterial genes that interact specifically with the imd signaling pathway. Most of these mutants have decreased resistance to the antimicrobial peptide polymyxin B. Characterization of a mutation in the putative transglutaminase FTN_0869 produced a curious result that could not easily be explained using known Drosophila immune responses. By using an unbiased genetic screen, these studies provide a new view of the Drosophila immune response from the perspective of a pathogen. We show that two branches of the fly's immunity are important for fighting F. novicida infections in a model host: melanization and an imd-regulated immune response, and identify bacterial genes that specifically counteract these host responses. Our work suggests that there may be more to learn about the fly immune system, as not all of the phenotypes we observe can be readily explained by its interactions with known immune responses.

    View details for DOI 10.1371/journal.ppat.1001065

    View details for Web of Science ID 000281399900037

    View details for PubMedID 20865166

  • Indoleamine 2,3-Dioxygenase 1 Is a Lung-Specific Innate Immune Defense Mechanism That Inhibits Growth of Francisella tularensis Tryptophan Auxotrophs INFECTION AND IMMUNITY Peng, K., Monack, D. M. 2010; 78 (6): 2723-2733


    Upon microbial challenge, organs at various anatomic sites of the body employ different innate immune mechanisms to defend against potential infections. Accordingly, microbial pathogens evolved to subvert these organ-specific host immune mechanisms to survive and grow in infected organs. Francisella tularensis is a bacterium capable of infecting multiple organs and thus encounters a myriad of organ-specific defense mechanisms. This suggests that F. tularensis may possess specific factors that aid in evasion of these innate immune defenses. We carried out a microarray-based, negative-selection screen in an intranasal model of Francisella novicida infection to identify Francisella genes that contribute to bacterial growth specifically in the lungs of mice. Genes in the bacterial tryptophan biosynthetic pathway were identified as being important for F. novicida growth specifically in the lungs. In addition, a host tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase 1 (IDO1), is induced specifically in the lungs of mice infected with F. novicida or Streptococcus pneumoniae. Furthermore, the attenuation of F. novicida tryptophan mutant bacteria was rescued in the lungs of IDO1(-/-) mice. IDO1 is a lung-specific innate immune mechanism that controls pulmonary Francisella infections.

    View details for DOI 10.1128/IAI.00008-10

    View details for Web of Science ID 000277841300035

    View details for PubMedID 20385761

  • Two physically, functionally, and developmentally distinct peritoneal macrophage subsets PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bou Ghosn, E. E., Cassado, A. A., Govoni, G. R., Fukuhara, T., Yang, Y., Monack, D. M., Bortoluci, K. R., Almeida, S. R., Herzenberg, L. A., Herzenberg, L. A. 2010; 107 (6): 2568-2573


    The peritoneal cavity (PerC) is a unique compartment within which a variety of immune cells reside, and from which macrophages (MØ) are commonly drawn for functional studies. Here we define two MØ subsets that coexist in PerC in adult mice. One, provisionally called the large peritoneal MØ (LPM), contains approximately 90% of the PerC MØ in unstimulated animals but disappears rapidly from PerC following lipopolysaccharide (LPS) or thioglycolate stimulation. These cells express high levels of the canonical MØ surface markers, CD11b and F4/80. The second subset, referred to as small peritoneal MØ (SPM), expresses substantially lower levels of CD11b and F4/80 but expresses high levels of MHC-II, which is not expressed on LPM. SPM, which predominates in PerC after LPS or thioglycolate stimulation, does not derive from LPM. Instead, it derives from blood monocytes that rapidly enter the PerC after stimulation and differentiate to mature SPM within 2 to 4 d. Both subsets show clear phagocytic activity and both produce nitric oxide (NO) in response to LPS stimulation in vivo. However, their responses to LPS show key differences: in vitro, LPS stimulates LPM, but not SPM, to produce NO; in vivo, LPS stimulates both subsets to produce NO, albeit with different response patterns. These findings extend current models of MØ heterogeneity and shed new light on PerC MØ diversity, development, and function. Thus, they introduce a new context for interpreting (and reinterpreting) data from ex vivo studies with PerC MØ.

    View details for DOI 10.1073/pnas.0915000107

    View details for Web of Science ID 000274408100039

    View details for PubMedID 20133793

  • Contributions of Francisella tularensis subsp novicida Chitinases and Sec Secretion System to Biofilm Formation on Chitin APPLIED AND ENVIRONMENTAL MICROBIOLOGY Margolis, J. J., El-Etr, S., Joubert, L., Moore, E., Robison, R., Rasley, A., Spormann, A. M., Monack, D. M. 2010; 76 (2): 596-608


    Francisella tularensis, the zoonotic cause of tularemia, can infect numerous mammals and other eukaryotes. Although studying F. tularensis pathogenesis is essential to comprehending disease, mammalian infection is just one step in the ecology of Francisella species. F. tularensis has been isolated from aquatic environments and arthropod vectors, environments in which chitin could serve as a potential carbon source and as a surface for attachment and growth. We show that F. tularensis subsp. novicida forms biofilms during the colonization of chitin surfaces. The ability of F. tularensis to persist using chitin as a sole carbon source is dependent on chitinases, since mutants lacking chiA or chiB are attenuated for chitin colonization and biofilm formation in the absence of exogenous sugar. A genetic screen for biofilm mutants identified the Sec translocon export pathway and 14 secreted proteins. We show that these genes are important for initial attachment during biofilm formation. We generated defined deletion mutants by targeting two chaperone genes (secB1 and secB2) involved in Sec-dependent secretion and four genes that encode putative secreted proteins. All of the mutants were deficient in attachment to polystyrene and chitin surfaces and for biofilm formation compared to wild-type F. novicida. In contrast, mutations in the Sec translocon and secreted factors did not affect virulence. Our data suggest that biofilm formation by F. tularensis promotes persistence on chitin surfaces. Further study of the interaction of F. tularensis with the chitin microenvironment may provide insight into the environmental survival and transmission mechanisms of this pathogen.

    View details for DOI 10.1128/AEM.02037-09

    View details for Web of Science ID 000273354200027

    View details for PubMedID 19948864

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


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

    View details for DOI 10.1073/pnas.0912386106

    View details for Web of Science ID 000273178700071

    View details for PubMedID 20018727

  • Contribution of Flagellin Pattern Recognition to Intestinal Inflammation during Salmonella enterica Serotype Typhimurium Infection INFECTION AND IMMUNITY Winter, S. E., Thiennimitr, P., Nuccio, S., Haneda, T., Winter, M. G., Wilson, R. P., Russell, J. M., Henry, T., Tran, Q. T., Lawhon, S. D., Gomez, G., Bevins, C. L., Ruessmann, H., Monack, D. M., Adams, L. G., Baeumler, A. J. 2009; 77 (5): 1904-1916


    Salmonella enterica serotype Typhimurium causes acute inflammatory diarrhea in humans. Flagella contribute to intestinal inflammation, but the mechanism remains unclear since most mutations abrogating pattern recognition of flagellin also prevent motility and reduce bacterial invasion. To determine the contribution of flagellin pattern recognition to the generation of innate immune responses, we compared in two animal models a nonmotile, but flagellin-expressing and -secreting serotype Typhimurium strain (flgK mutant) to a nonmotile, non-flagellin-expressing strain (flgK fliC fljB mutant). In vitro, caspase-1 can be activated by cytosolic delivery of flagellin, resulting in release of the interferon gamma inducing factor interleukin-18 (IL-18). Experiments with streptomycin-pretreated caspase-1-deficient mice suggested that induction of gamma interferon expression in the murine cecum early (12 h) after serotype Typhimurium infection was caspase-1 dependent but independent of flagellin pattern recognition. In addition, mRNA levels of the CXC chemokines macrophage inflammatory protein 2 and keratinocyte-derived chemokine were markedly increased early after serotype Typhimurium infection of streptomycin-pretreated wild-type mice regardless of flagellin expression. In contrast, in bovine ligated ileal loops, flagellin pattern recognition contributed to increased mRNA levels of macrophage inflammatory protein 3alpha and more fluid accumulation at 2 h after infection. Collectively, our data suggest that pattern recognition of flagellin contributes to early innate host responses in the bovine ileal mucosa but not in the murine cecal mucosa.

    View details for DOI 10.1128/IAI.01341-08

    View details for Web of Science ID 000265279900019

    View details for PubMedID 19237529

  • Identification of fevR, a novel regulator of virulence gene expression in Francisella novicida INFECTION AND IMMUNITY Broteke, A., Monack, D. M. 2008; 76 (8): 3473-3480


    Francisella tularensis infects wild animals and humans to cause tularemia. This pathogen targets the cytosol of macrophages, where it replicates using the genes in the Francisella pathogenicity island (FPI). Virulence gene regulation in Francisella is complex, but transcriptional regulators MglA and SspA have been shown to regulate the expression of approximately 100 genes, including the entire FPI. We utilized a Francisella novicida transposon mutant library to identify additional regulatory factors and identified five additional genes that are essential for virulence gene expression. One regulatory gene, FTN_0480 (fevR, Francisella effector of virulence regulation), present in all Francisella species, is required for expression of the FPI genes and other genes in the MglA/SspA regulon. The expression of fevR is positively regulated by MglA. However, constitutive expression of fevR in an mglA mutant strain did not restore expression of the MglA/SspA regulon, demonstrating that mglA and fevR act in parallel to positively regulate virulence gene expression. Virulence studies revealed that fevR is essential for bacterial replication in macrophages and in mice, where we additionally show that fevR is required for the expression of genes in the MglA/SspA regulon in vivo. Thus, fevR is a crucial virulence gene in Francisella, required for the expression of virulence factors known to be essential for this pathogen's subversion of host defenses and pathogenesis in vivo.

    View details for DOI 10.1128/IAI.00430-08

    View details for Web of Science ID 000258480900011

    View details for PubMedID 18559431

  • The inflammasome: a key player in the inflammation triggered in response to bacterial pathogens. Journal of pediatric gastroenterology and nutrition Monack, D. M. 2008; 46: E14-?

    View details for DOI 10.1097/01.mpg.0000313827.16713.5e

    View details for PubMedID 18354318

  • Activation of the inflammasome upon Francisella tularensis infection: interplay of innate immune pathways and virulence factors CELLULAR MICROBIOLOGY Henry, T., Monack, D. M. 2007; 9 (11): 2543-2551


    Tularaemia is a zoonotic disease caused by the facultative intracellular bacterium Francisella tularensis. The virulence of this pathogen depends on its ability to escape into the cytosol of host cells. Pathogens are detected by the innate immune system's pattern recognition receptors which are activated in response to conserved microbial molecules (pathogen-associated molecular patterns). Cytosolic bacteria are sensed intracellularly, often leading to activation of the cysteine protease caspase-1 within a multimolecular complex called the inflammasome. Caspase-1 activation leads to both host cell death and release of pro-inflammatory cytokines in a process called pyroptosis. Here we review the pathway leading to, and the consequences of, inflammasome activation upon F. tularensis infection both in vitro and in vivo. Finally, we discuss recent data on how other innate immune pathways and F. tularensis virulence factors control the activation of the inflammasome during infection.

    View details for DOI 10.1111/j.1462-5822.2007.01022.x

    View details for Web of Science ID 000249824300001

    View details for PubMedID 17662071

  • Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation NATURE REVIEWS IMMUNOLOGY Mariathasan, S., Monack, D. M. 2007; 7 (1): 31-40


    The NOD-like receptors have important roles in innate immunity as intracellular sensors of microbial components and cell injury. It has been proposed that these cytosolic proteins regulate the cysteine protease caspase-1 within a multiprotein complex known as the 'inflammasome'. Activation of caspase-1 leads to the cleavage and activation of pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-18, as well as host-cell death. The analysis of mice that are deficient in various inflammasome components has revealed that the inflammasome is a dynamic entity that is assembled from different adaptors in a stimulus-dependent manner. Here we review recent work on the activation of the inflammasome in response to various bacterial pathogens and tissue damage.

    View details for DOI 10.1038/nri1997

    View details for Web of Science ID 000243036700013

    View details for PubMedID 17186029

  • Francisella tularensis: Activation of the inflammasome FRANCISELLA TULARENSIS: BIOLOGY, PATHOGENICITY, EPIDEMIOLOGY, AND BIODEFENSE Weiss, D. S., Henry, T., Monack, D. M. 2007; 1105: 219-237


    Francisella tularensis (F. tularensis) is a facultative intracellular pathogen that causes the systemic disease tularemia. This pathogen can replicate in the cytosol of macrophages, an ability that is linked with its virulence. We discuss recent data demonstrating that in macrophages, cytosolic Francisella induce the activation of the cysteine protease caspase-1 within a multiprotein complex called the inflammasome. NOD-like receptors (NLRs), which may have important roles in innate immunity as intracellular sensors of microbial components and cell injury, and the adaptor molecule ASC are thought to regulate caspase-1 within the inflammasome. Both ASC and caspase-1 play a critical role in host defense against Francisella infection in vivo. Activation of caspase-1 leads to the cleavage and activation of proinflammatory cytokines, such as interleukin-1beta (IL-1beta) and IL-18, as well as the induction of host cell death, which are required for innate immune defense against Francisella and other intracellular pathogens. The cytokine IFN-beta is secreted from infected cells in response to cytosolic Francisella and its signaling through the type I interferon receptor is required for activation of the inflammasome. Despite the effort of the host to induce inflammasome activation, Francisella modulates this host defense pathway, limiting its efficacy. These results highlight the role that the inflammasome plays in the tug-of-war between Francisella and the immune system.

    View details for DOI 10.1196/annals.1409.005

    View details for Web of Science ID 000248298500010

    View details for PubMedID 17395724

  • Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis INFECTION AND IMMUNITY Brotcke, A., Weiss, D. S., Kim, C. C., Chain, P., Malfatti, S., Garcia, E., Monack, D. M. 2006; 74 (12): 6642-6655


    The facultative intracellular bacterium Francisella tularensis causes the zoonotic disease tularemia. F. tularensis resides within host macrophages in vivo, and this ability is essential for pathogenesis. The transcription factor MglA is required for the expression of several Francisella genes that are necessary for replication in macrophages and for virulence in mice. We hypothesized that the identification of MglA-regulated genes in the Francisella genome by transcriptional profiling of wild-type and mglA mutant bacteria would lead to the discovery of new virulence factors utilized by F. tularensis. A total of 102 MglA-regulated genes were identified, the majority of which were positively regulated, including all of the Francisella pathogenicity island (FPI) genes. We mutated novel MglA-regulated genes and tested the mutants for their ability to replicate and induce cytotoxicity in macrophages and to grow in mice. Mutations in MglA-regulated genes within the FPI (pdpB and cds2) as well as outside the FPI (FTT0989, oppB, and FTT1209c) were either attenuated or hypervirulent in macrophages compared to the wild-type strain. All of these mutants exhibited decreased fitness in vivo in competition experiments with wild-type bacteria. We have identified five new Francisella virulence genes, and our results suggest that characterizations of additional MglA-regulated genes will yield further insights into the pathogenesis of this bacterium.

    View details for DOI 10.1128/IAI.01250-06

    View details for Web of Science ID 000242308100014

    View details for PubMedID 17000729

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


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

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

    View details for Web of Science ID 000226457800024

    View details for PubMedID 15661016

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


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

    View details for DOI 10.1038/nrmicro955

    View details for Web of Science ID 000223627200019

    View details for PubMedID 15372085

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


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

    View details for DOI 10.1084/jem.20031319

    View details for Web of Science ID 000188369700009

    View details for PubMedID 14734525

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


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

    View details for DOI 10.1128/IAI.71.6.3196-3205.2003

    View details for Web of Science ID 000183116300027

    View details for PubMedID 12761099

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


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

    View details for Web of Science ID 000182042800009

    View details for PubMedID 12675799

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


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

    View details for Web of Science ID 000172601200005

    View details for PubMedID 11736994

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


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

    View details for Web of Science ID 000173168500004

    View details for PubMedID 11755408

  • Actin-based motility is sufficient for bacterial membrane protrusion formation and host cell uptake CELLULAR MICROBIOLOGY Monack, D. M., Theriot, J. A. 2001; 3 (9): 633-647


    Shigella flexneri replicates in the cytoplasm of host cells, where it nucleates host cell actin filaments at one pole of the bacterial cell to form a 'comet tail' that propels the bacterium through the host's cytoplasm. To determine whether the ability to move by actin-based motility is sufficient for subsequent formation of membrane-bound protrusions and intercellular spread, we conferred the ability to nucleate actin on a heterologous bacterium, Escherichia coli. Previous work has shown that IcsA (VirG), the molecule that is necessary and sufficient for actin nucleation and actin-based motility, is distributed in a unipolar fashion on the surface of S. flexneri. Maintenance of the unipolar distribution of IcsA depends on both the S. flexneri outer membrane protease IcsP (SopA) and the structure of the lipopolysaccharide (LPS) in the outer membrane. We co-expressed IcsA and IcsP in two strains of E. coli that differed in their LPS structures. The E. coli were engineered to invade host cells by expression of invasin from Yersinia pseudotuberculosis and to escape the phagosome by incubation in purified listeriolysin O (LLO) from Listeria monocytogenes. All E. coli strains expressing IcsA replicated in host cell cytoplasm and moved by actin-based motility. Actin-based motility alone was sufficient for the formation of membrane protrusions and uptake by recipient host cells. The presence of IcsP and an elaborate LPS structure combined to enhance the ability of E. coli to form protrusions at the same frequency as S. flexneri, quantitatively reconstituting this step in pathogen intercellular spread in a heterologous organism. The frequency of membrane protrusion formation across all strains tested correlates with the efficiency of unidirectional actin-based movement, but not with bacterial speed.

    View details for Web of Science ID 000171021200006

    View details for PubMedID 11553015

  • The making of a gradient: IcsA (VirG) polarity in Shigella flexneri MOLECULAR MICROBIOLOGY Robbins, J. R., Monack, D., McCallum, S. J., Vegas, A., Pham, E., Goldberg, M. B., Theriot, J. A. 2001; 41 (4): 861-872


    The generation and maintenance of subcellular organization in bacteria is critical for many cell processes and properties, including growth, structural integrity and, in pathogens, virulence. Here, we investigate the mechanisms by which the virulence protein IcsA (VirG) is distributed on the bacterial surface to promote efficient transmission of the bacterium Shigella flexneri from one host cell to another. The outer membrane protein IcsA recruits host factors that result in actin filament nucleation and, when concentrated at one bacterial pole, promote unidirectional actin-based motility of the pathogen. We show here that the focused polar gradient of IcsA is generated by its delivery exclusively to one pole followed by lateral diffusion through the outer membrane. The resulting gradient can be modified by altering the composition of the outer membrane either genetically or pharmacologically. The gradient can be reshaped further by the action of the protease IcsP (SopA), whose activity we show to be near uniform on the bacterial surface. Further, we report polar delivery of IcsA in Escherichia coli and Yersinia pseudotuberculosis, suggesting that the mechanism for polar delivery of some outer membrane proteins is conserved across species and that the virulence function of IcsA capitalizes on a more global mechanism for subcellular organization.

    View details for Web of Science ID 000170904400008

    View details for PubMedID 11532149

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


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

    View details for Web of Science ID 000088261100011

    View details for PubMedID 10899911

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


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

    View details for Web of Science ID 000086862900002

    View details for PubMedID 11043977

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


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

    View details for Web of Science ID 000078956600106

    View details for PubMedID 10051653

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


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

    View details for Web of Science ID 000077484700016

    View details for PubMedID 9841926

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


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

    View details for Web of Science ID 000076538500015

    View details for PubMedID 9786194

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


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

    View details for Web of Science ID A1997XX39900070

    View details for PubMedID 9294220

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


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

    View details for Web of Science ID A1996VF61400093

    View details for PubMedID 8790417

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