Denise M. Monack

All Publications

• Salmonella enterica serovar Typhi uses two type 3 secretion systems to replicate in human macrophages and colonize humanized mice. mBio Hamblin, M., Schade, R., Narasimhan, R., Monack, D. M. 2023: e0113723

  Abstract

  Salmonella enterica serovar Typhi (S. Typhi) is a human-restricted pathogen that replicates in macrophages. In this study, we investigated the roles of the S. Typhi type 3 secretion systems (T3SSs) encoded on Salmonella pathogenicity islands (SPI)-1 (T3SS-1) and SPI-2 (T3SS-2) during human macrophage infection. We found that mutants of S. Typhi deficient for both T3SSs were defective for intramacrophage replication as measured by flow cytometry, viable bacterial counts, and live time-lapse microscopy. T3SS-secreted proteins PipB2 and SifA contributed to S. Typhi replication and were translocated into the cytosol of human macrophages through both T3SS-1 and T3SS-2, demonstrating functional redundancy for these secretion systems. Importantly, an S. Typhi mutant strain that is deficient for both T3SS-1 and T3SS-2 was severely attenuated in the ability to colonize systemic tissues in a humanized mouse model of typhoid fever. Overall, this study establishes a critical role for S. Typhi T3SSs during its replication within human macrophages and during systemic infection of humanized mice. IMPORTANCE Salmonella enterica serovar Typhi is a human-restricted pathogen that causes typhoid fever. Understanding the key virulence mechanisms that facilitate S. Typhi replication in human phagocytes will enable rational vaccine and antibiotic development to limit the spread of this pathogen. While S. Typhimurium replication in murine models has been studied extensively, there is limited information available about S. Typhi replication in human macrophages, some of which directly conflict with findings from S. Typhimurium murine models. This study establishes that both of S. Typhi's two type 3 secretion systems (T3SS-1 and T3SS-2) contribute to intramacrophage replication and virulence.

  View details for DOI 10.1128/mbio.01137-23

  View details for PubMedID 37341487

• Salmonella-liberated dietary L-arabinose promotes expansion in superspreaders. Cell host & microbe Ruddle, S. J., Massis, L. M., Cutter, A. C., Monack, D. M. 2023

  Abstract

  The molecular understanding of host-pathogen interactions in the gastrointestinal (GI) tract of superspreader hosts is incomplete. In a mouse model of chronic, asymptomatic Salmonella enterica serovar Typhimurium (S. Tm) infection, we performed untargeted metabolomics on the feces of mice and found that superspreader hosts possess distinct metabolic signatures compared with non-superspreaders, including differential levels of L-arabinose. RNA-seq on S. Tm from superspreader fecal samples showed increased expression of the L-arabinose catabolism pathway invivo. By combining bacterial genetics and diet manipulation, we demonstrate that diet-derived L-arabinose provides S. Tm a competitive advantage in the GI tract, and expansion of S. Tm in the GI tract requires an alpha-N-arabinofuranosidase that liberates L-arabinose from dietary polysaccharides. Ultimately, our work shows that pathogen-liberated L-arabinose from the diet provides a competitive advantage to S. Tm invivo. These findings propose L-arabinose as a critical driver of S. Tm expansion in the GI tracts of superspreader hosts.

  View details for DOI 10.1016/j.chom.2023.01.017

  View details for PubMedID 36812913

• Repairing the "Leaky Pipeline'' of Lost Talent CELL HOST & MICROBE Monack, D. M. 2020; 27 (3): 322

  View details for Web of Science ID 000519891100009

• Salmonella-Driven Polarization of Granuloma Macrophages Antagonizes TNF-Mediated Pathogen Restriction during Persistent Infection. Cell host & microbe Pham, T. H., Brewer, S. M., Thurston, T., Massis, L. M., Honeycutt, J., Lugo, K., Jacobson, A. R., Vilches-Moure, J. G., Hamblin, M., Helaine, S., Monack, D. M. 2019

  Abstract

  Many intracellular bacteria can establish chronic infection and persist in tissues within granulomas composed of macrophages. Granuloma macrophages exhibit heterogeneous polarization states, or phenotypes, that may be functionally distinct. Here, we elucidate a host-pathogen interaction that controls granuloma macrophage polarization and long-term pathogen persistence during Salmonella Typhimurium (STm) infection. We show that STm persists within splenic granulomas that are densely populated by CD11b+CD11c+Ly6C+ macrophages. STm preferentially persists in granuloma macrophages reprogrammed to an M2 state, in part through the activity of the effector SteE, which contributes to the establishment of persistent infection. We demonstrate that tumor necrosis factor (TNF) signaling limits M2 granuloma macrophage polarization, thereby restricting STm persistence. TNF neutralization shifts granuloma macrophages toward an M2 state and increases bacterial persistence, and these effects are partially dependent on SteE activity. Thus, manipulating granuloma macrophage polarization represents a strategy for intracellular bacteria to overcome host restriction during persistent infection.

  View details for DOI 10.1016/j.chom.2019.11.011

  View details for PubMedID 31883922

• What career advice do you give your grad students or postdocs? TRENDS IN MICROBIOLOGY Jensen, G., Merrikh, H., Monack, D., Veening, J. 2019; 27 (6): 471–72

  View details for DOI 10.1016/j.tim.2019.03.009

  View details for Web of Science ID 000468064100001

  View details for PubMedID 31031091

• A Gut Commensal-Produced Metabolite Mediates Colonization Resistance to Salmonella Infection. Cell host & microbe Jacobson, A., Lam, L., Rajendram, M., Tamburini, F., Honeycutt, J., Pham, T., Van Treuren, W., Pruss, K., Stabler, S. R., Lugo, K., Bouley, D. M., Vilches-Moure, J. G., Smith, M., Sonnenburg, J. L., Bhatt, A. S., Huang, K. C., Monack, D. 2018

  Abstract

  The intestinal microbiota provides colonization resistance against pathogens, limiting pathogen expansion and transmission. These microbiota-mediated mechanisms were previously identified by observing loss of colonization resistance after antibiotic treatment or dietary changes, which severely disrupt microbiota communities. We identify a microbiota-mediated mechanism of colonization resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) by comparing high-complexity commensal communities with different levels of colonization resistance. Using inbred mouse strains with different infection dynamics and S. Typhimurium intestinal burdens, we demonstrate that Bacteroides species mediate colonization resistance against S. Typhimurium by producing the short-chain fatty acid propionate. Propionate directly inhibits pathogen growth invitro by disrupting intracellular pH homeostasis, and chemically increasing intestinal propionate levels protects mice from S.Typhimurium. In addition, administering susceptible mice Bacteroides, but not a propionate-production mutant, confers resistance to S. Typhimurium. This work provides mechanistic understanding into the role of individualized microbial communities in host-to-host variability of pathogen transmission.

  View details for PubMedID 30057174

• Intraspecies competition for niches in the distal gut dictate transmission during persistent Salmonella infection. PLoS pathogens Lam, L. H., Monack, D. M. 2014; 10 (12)

  Abstract

  In order to be transmitted, a pathogen must first successfully colonize and multiply within a host. Ecological principles can be applied to study host-pathogen interactions to predict transmission dynamics. Little is known about the population biology of Salmonella during persistent infection. To define Salmonella enterica serovar Typhimurium population structure in this context, 129SvJ mice were oral gavaged with a mixture of eight wild-type isogenic tagged Salmonella (WITS) strains. Distinct subpopulations arose within intestinal and systemic tissues after 35 days, and clonal expansion of the cecal and colonic subpopulation was responsible for increases in Salmonella fecal shedding. A co-infection system utilizing differentially marked isogenic strains was developed in which each mouse received one strain orally and the other systemically by intraperitoneal (IP) injection. Co-infections demonstrated that the intestinal subpopulation exerted intraspecies priority effects by excluding systemic S. Typhimurium from colonizing an extracellular niche within the cecum and colon. Importantly, the systemic strain was excluded from these distal gut sites and was not transmitted to naïve hosts. In addition, S. Typhimurium required hydrogenase, an enzyme that mediates acquisition of hydrogen from the gut microbiota, during the first week of infection to exert priority effects in the gut. Thus, early inhibitory priority effects are facilitated by the acquisition of nutrients, which allow S. Typhimurium to successfully compete for a nutritional niche in the distal gut. We also show that intraspecies colonization resistance is maintained by Salmonella Pathogenicity Islands SPI1 and SPI2 during persistent distal gut infection. Thus, important virulence effectors not only modulate interactions with host cells, but are crucial for Salmonella colonization of an extracellular intestinal niche and thereby also shape intraspecies dynamics. We conclude that priority effects and intraspecies competition for colonization niches in the distal gut control Salmonella population assembly and transmission.

  View details for DOI 10.1371/journal.ppat.1004527

  View details for PubMedID 25474319

• Role of disease-associated tolerance in infectious superspreaders PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gopinath, S., Lichtman, J. S., Bouley, D. M., Elias, J. E., Monack, D. M. 2014; 111 (44): 15780-15785

  Abstract

  Natural populations show striking heterogeneity in their ability to transmit disease. For example, a minority of infected individuals known as superspreaders carries out the majority of pathogen transmission events. In a mouse model of Salmonella infection, a subset of infected hosts becomes superspreaders, shedding high levels of bacteria (>10(8) cfu per g of feces) but remain asymptomatic with a dampened systemic immune state. Here we show that superspreader hosts remain asymptomatic when they are treated with oral antibiotics. In contrast, nonsuperspreader Salmonella-infected hosts that are treated with oral antibiotics rapidly shed superspreader levels of the pathogen but display signs of morbidity. This morbidity is linked to an increase in inflammatory myeloid cells in the spleen followed by increased production of acute-phase proteins and proinflammatory cytokines. The degree of colonic inflammation is similar in antibiotic-treated superspreader and nonsuperspreader hosts, indicating that the superspreader hosts are tolerant of antibiotic-mediated perturbations in the intestinal tract. Importantly, neutralization of acute-phase proinflammatory cytokines in antibiotic-induced superspreaders suppresses the expansion of inflammatory myeloid cells and reduces morbidity. We describe a unique disease-associated tolerance to oral antibiotics in superspreaders that facilitates continued transmission of the pathogen.

  View details for DOI 10.1073/pnas.1409968111

  View details for Web of Science ID 000344088100047

  View details for PubMedCentralID PMC4226084

• Corrigendum to "Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathy" [Journal: Molecular of and Cellular Cardiology (2019) May 130;160-169]. Journal of molecular and cellular cardiology Haileselassie, B., Mukherjee, R., Joshi, A. U., Napier, B. A., Massis, L. M., Ostberg, N. P., Queliconi, B. B., Monack, D., Bernstein, D., Mochly-Rosen, D. 2023

  View details for DOI 10.1016/j.yjmcc.2023.11.004

  View details for PubMedID 38000978

• Turning foes into permissive hosts: manipulation of macrophage polarization by intracellular bacteria. Current opinion in immunology Pham, T. H., Monack, D. M. 2023; 84: 102367

  Abstract

  Macrophages function as tissue-immune sentinels and mediate key antimicrobial responses against bacterial pathogens. Yet, they can also act as a cellular niche for intracellular bacteria, such as Salmonella enterica, to persist in infected tissues. Macrophages exhibit heterogeneous activation or polarization, states that are linked to differential antibacterial responses and bacteria permissiveness. Remarkably, recent studies demonstrate that Salmonella and other intracellular bacteria inject virulence effectors into the cellular cytoplasm to skew the macrophage polarization state and reprogram these immune cells into a permissive niche. Here, we review mechanisms of macrophage reprogramming by Salmonella and highlight manipulation of macrophage polarization as a shared bacterial pathogenesis strategy. In addition, we discuss how the interplay of bacterial effector mechanisms, microenvironmental signals, and ontogeny may shape macrophage cell states and functions. Finally, we propose ideas of how further research will advance our understanding of macrophage functional diversity and immunobiology.

  View details for DOI 10.1016/j.coi.2023.102367

  View details for PubMedID 37437470

• One species, different diseases: the unique molecular mechanisms that underlie the pathogenesis of typhoidal Salmonella infections. Current opinion in microbiology Wang, B. X., Butler, D. S., Hamblin, M., Monack, D. M. 2023; 72: 102262

  Abstract

  Salmonella enterica is one of the most widespread bacterial pathogens found worldwide, resulting in approximately 100 million infections and over 200 000 deaths per year. Salmonella isolates, termed 'serovars', can largely be classified as either nontyphoidal or typhoidal Salmonella, which differ in regard to disease manifestation and host tropism. Nontyphoidal Salmonella causes gastroenteritis in many hosts, while typhoidal Salmonella is human-restricted and causes typhoid fever, a systemic disease with a mortality rate of up to 30% without treatment. There has been considerable interest in understanding how different Salmonella serovars cause different diseases, but the molecular details that underlie these infections have not yet been fully characterized, especially in the case of typhoidal Salmonella. In this review, we highlight the current state of research into understanding the pathogenesis of both nontyphoidal and typhoidal Salmonella, with a specific interest in serovar-specific traits that allow human-adapted strains of Salmonella to cause enteric fever. Overall, a more detailed molecular understanding of how different Salmonella isolates infect humans will provide critical insights into how we can eradicate these dangerous enteric pathogens.

  View details for DOI 10.1016/j.mib.2022.102262

  View details for PubMedID 36640585

• Single-cell profiling identifies ACE+ granuloma macrophages as a nonpermissive niche for intracellular bacteria during persistent Salmonella infection. Science advances Pham, T. H., Xue, Y., Brewer, S. M., Bernstein, K. E., Quake, S. R., Monack, D. M. 2023; 9 (1): eadd4333

  Abstract

  Macrophages mediate key antimicrobial responses against intracellular bacterial pathogens, such as Salmonella enterica. Yet, they can also act as a permissive niche for these pathogens to persist in infected tissues within granulomas, which are immunological structures composed of macrophages and other immune cells. We apply single-cell transcriptomics to investigate macrophage functional diversity during persistent S. enterica serovar Typhimurium (STm) infection in mice. We identify determinants of macrophage heterogeneity in infected spleens and describe populations of distinct phenotypes, functional programming, and spatial localization. Using an STm mutant with impaired ability to polarize macrophage phenotypes, we find that angiotensin-converting enzyme (ACE) defines a granuloma macrophage population that is nonpermissive for intracellular bacteria, and their abundance anticorrelates with tissue bacterial burden. Disruption of pathogen control by neutralizing TNF is linked to preferential depletion of ACE+ macrophages in infected tissues. Thus, ACE+ macrophages have limited capacity to serve as cellular niche for intracellular bacteria to establish persistent infection.

  View details for DOI 10.1126/sciadv.add4333

  View details for PubMedID 36608122

• Drp1/Fis1-Dependent Pathologic Fission and Associated Damaged Extracellular Mitochondria Contribute to Macrophage Dysfunction in Endotoxin Tolerance. Critical care medicine Mukherjee, R., Tompkins, C. A., Ostberg, N. P., Joshi, A. U., Massis, L. M., Vijayan, V., Gera, K., Monack, D., Cornell, T. T., Hall, M. W., Mochly-Rosen, D., Haileselassie, B. 1800

  Abstract

  OBJECTIVES: Recent publications have shown that mitochondrial dynamics can govern the quality and quantity of extracellular mitochondria subsequently impacting immune phenotypes. This study aims to determine if pathologic mitochondrial fission mediated by Drp1/Fis1 interaction impacts extracellular mitochondrial content and macrophage function in sepsis-induced immunoparalysis.DESIGN: Laboratory investigation.SETTING: University laboratory.SUBJECTS: C57BL/6 and BALB/C mice.INTERVENTIONS: Using in vitro and murine models of endotoxin tolerance (ET), we evaluated changes in Drp1/Fis1-dependent pathologic fission and simultaneously measured the quantity and quality of extracellular mitochondria. Next, by priming mouse macrophages with isolated healthy mitochondria (MC) and damaged mitochondria, we determined if damaged extracellular mitochondria are capable of inducing tolerance to subsequent endotoxin challenge. Finally, we determined if inhibition of Drp1/Fis1-mediated pathologic fission abrogates release of damaged extracellular mitochondria and improves macrophage response to subsequent endotoxin challenge.MEASUREMENTS AND MAIN RESULTS: When compared with naive macrophages (NMs), endotoxin-tolerant macrophages (ETM) demonstrated Drp1/Fis1-dependent mitochondrial dysfunction and higher levels of damaged extracellular mitochondria (Mitotracker-Green + events/50 muL: ETM = 2.42 * 106 ± 4,391 vs NM = 5.69 * 105 ± 2,478; p < 0.001). Exposure of NMs to damaged extracellular mitochondria (MH) induced cross-tolerance to subsequent endotoxin challenge, whereas MC had minimal effect (tumor necrosis factor [TNF]-alpha [pg/mL]: NM = 668 ± 3, NM + MH = 221 ± 15, and NM + Mc = 881 ± 15; p < 0.0001). Inhibiting Drp1/Fis1-dependent mitochondrial fission using heptapeptide (P110), a selective inhibitor of Drp1/Fis1 interaction, improved extracellular mitochondrial function (extracellular mitochondrial membrane potential, JC-1 [R/G] ETM = 7 ± 0.5 vs ETM + P110 = 19 ± 2.0; p < 0.001) and subsequently improved immune response in ETMs (TNF-alpha [pg/mL]; ETM = 149 ± 1 vs ETM + P110 = 1,150 ± 4; p < 0.0001). Similarly, P110-treated endotoxin tolerant mice had lower amounts of damaged extracellular mitochondria in plasma (represented by higher extracellular mitochondrial membrane potential, TMRM/MT-G: endotoxin tolerant [ET] = 0.04 ± 0.02 vs ET + P110 = 0.21 ± 0.02; p = 0.03) and improved immune response to subsequent endotoxin treatment as well as cecal ligation and puncture.CONCLUSIONS: Inhibition of Drp1/Fis1-dependent mitochondrial fragmentation improved macrophage function and immune response in both in vitro and in vivo models of ET. This benefit is mediated, at least in part, by decreasing the release of damaged extracellular mitochondria, which contributes to endotoxin cross-tolerance. Altogether, these data suggest that alterations in mitochondrial dynamics may play an important role in sepsis-induced immunoparalysis.

  View details for DOI 10.1097/CCM.0000000000005437

  View details for PubMedID 35067534

• Controlling the polarity of human gastrointestinal organoids to investigate epithelial biology and infectious diseases. Nature protocols Co, J. Y., Margalef-Catala, M., Monack, D. M., Amieva, M. R. 2021

  Abstract

  Human epithelial organoids-3D spheroids derived from adult tissue stem cells-enable investigation of epithelial physiology and disease and host interactions with microorganisms, viruses and bioactive molecules. One challenge in using organoids is the difficulty in accessing the apical, or luminal, surface of the epithelium, which is enclosed within the organoid interior. This protocol describes a method we previously developed to control human and mouse organoid polarity in suspension culture such that the apical surface faces outward to the medium (apical-out organoids). Our protocol establishes apical-out polarity rapidly (24-48 h), preserves epithelial integrity, maintains secretory and absorptive functions and allows regulation of differentiation. Here, we provide a detailed description of the organoid polarity reversal method, compatible characterization assays and an example of an application of the technology-specifically the impact of host-microbe interactions on epithelial function. Control of organoid polarity expands the possibilities of organoid use in gastrointestinal and respiratory health and disease research.

  View details for DOI 10.1038/s41596-021-00607-0

  View details for PubMedID 34663962

• A Salmonella Typhi RNA thermosensor regulates virulence factors and innate immune evasion in response to host temperature. PLoS pathogens Brewer, S. M., Twittenhoff, C., Kortmann, J., Brubaker, S. W., Honeycutt, J., Massis, L. M., Pham, T. H., Narberhaus, F., Monack, D. M. 2021; 17 (3): e1009345

  Abstract

  Sensing and responding to environmental signals is critical for bacterial pathogens to successfully infect and persist within hosts. Many bacterial pathogens sense temperature as an indication they have entered a new host and must alter their virulence factor expression to evade immune detection. Using secondary structure prediction, we identified an RNA thermosensor (RNAT) in the 5' untranslated region (UTR) of tviA encoded by the typhoid fever-causing bacterium Salmonella enterica serovar Typhi (S. Typhi). Importantly, tviA is a transcriptional regulator of the critical virulence factors Vi capsule, flagellin, and type III secretion system-1 expression. By introducing point mutations to alter the mRNA secondary structure, we demonstrate that the 5' UTR of tviA contains a functional RNAT using in vitro expression, structure probing, and ribosome binding methods. Mutational inhibition of the RNAT in S. Typhi causes aberrant virulence factor expression, leading to enhanced innate immune responses during infection. In conclusion, we show that S. Typhi regulates virulence factor expression through an RNAT in the 5' UTR of tviA. Our findings demonstrate that limiting inflammation through RNAT-dependent regulation in response to host body temperature is important for S. Typhi's "stealthy" pathogenesis.

  View details for DOI 10.1371/journal.ppat.1009345

  View details for PubMedID 33651854

• A Rapid Caspase-11 Response Induced by IFNgamma Priming Is Independent of Guanylate Binding Proteins. iScience Brubaker, S. W., Brewer, S. M., Massis, L. M., Napier, B. A., Monack, D. M. 2020; 23 (10): 101612

  Abstract

  In mammalian cells, inflammatory caspases detect Gram-negative bacterial invasion by binding lipopolysaccharides (LPS). Murine caspase-11 binds cytosolic LPS, stimulates pyroptotic cell death, and drives sepsis pathogenesis. Extracellular priming factors enhance caspase-11-dependent pyroptosis. Herein we compare priming agents and demonstrate that IFNgamma priming elicits the most rapid and amplified macrophage response to cytosolic LPS. Previous studies indicate that IFN-induced expression of caspase-11 and guanylate binding proteins (GBPs) are causal events explaining the effects of priming on cytosolic LPS sensing. We demonstrate that these events cannot fully account for the increased response triggered by IFNgamma treatment. Indeed, IFNgamma priming elicits higher pyroptosis levels in response to cytosolic LPS when macrophages stably express caspase-11. In macrophages lacking GBPs encoded on chromosome 3, IFNgamma priming enhanced pyroptosis in response to cytosolic LPS as compared with other priming agents. These results suggest an unknown regulator of caspase-11-dependent pyroptosis exists, whose activity is upregulated by IFNgamma.

  View details for DOI 10.1016/j.isci.2020.101612

  View details for PubMedID 33089101

• Spraying Small Water Droplets Acts as a Bacteriocide. QRB discovery Dulay, M. T., Lee, J. K., Mody, A. C., Narasimhan, R., Monack, D. M., Zare, R. N. 2020; 1: e3

  Abstract

  Disinfectants are important for arresting the spread of pathogens in the environment. Frequently used disinfectants are often incompatible with certain surfaces, expensive and can produce hazardous by-products. We report that micron-sized water droplets can act as an effective disinfectant, which were formed by spraying pure bulk water with coaxial nebulizing airflow. Spraying for 20 min onto Escherichia coli and Salmonella typhimurium on stainless-steel discs caused inactivation of over 98% of the bacteria. Control experiments resulted in less than 10% inactivation (water stream only and gas only) and 55% inactivation with 3% hydrogen peroxide. Experiments have shown that cell death results from cell wall destruction. We suggest that the combined action of reactive oxygen species present in water droplets (but not in bulk water) along with the droplet surface charge is responsible for the observed bactericidal activity.

  View details for DOI 10.1017/qrd.2020.2

  View details for PubMedID 37528962

  View details for PubMedCentralID PMC10392691

• Genetic variation in the MacAB-TolC efflux pump influences pathogenesis of invasive Salmonella isolates from Africa. PLoS pathogens Honeycutt, J. D., Wenner, N. n., Li, Y. n., Brewer, S. M., Massis, L. M., Brubaker, S. W., Chairatana, P. n., Owen, S. V., Canals, R. n., Hinton, J. C., Monack, D. M. 2020; 16 (8): e1008763

  Abstract

  The various sub-species of Salmonella enterica cause a range of disease in human hosts. The human-adapted Salmonella enterica serovar Typhi enters the gastrointestinal tract and invades systemic sites to cause enteric (typhoid) fever. In contrast, most non-typhoidal serovars of Salmonella are primarily restricted to gut tissues. Across Africa, invasive non-typhoidal Salmonella (iNTS) have emerged with an ability to spread beyond the gastrointestinal tract and cause systemic bloodstream infections with increased morbidity and mortality. To investigate this evolution in pathogenesis, we compared the genomes of African iNTS isolates with other Salmonella enterica serovar Typhimurium and identified several macA and macB gene variants unique to African iNTS. MacAB forms a tripartite efflux pump with TolC and is implicated in Salmonella pathogenesis. We show that macAB transcription is upregulated during macrophage infection and after antimicrobial peptide exposure, with macAB transcription being supported by the PhoP/Q two-component system. Constitutive expression of macAB improves survival of Salmonella in the presence of the antimicrobial peptide C18G. Furthermore, these macAB variants affect replication in macrophages and influence fitness during colonization of the murine gastrointestinal tract. Importantly, the infection outcome resulting from these macAB variants depends upon both the Salmonella Typhimurium genetic background and the host gene Nramp1, an important determinant of innate resistance to intracellular bacterial infection. The variations we have identified in the MacAB-TolC efflux pump in African iNTS may reflect evolution within human host populations that are compromised in their ability to clear intracellular Salmonella infections.

  View details for DOI 10.1371/journal.ppat.1008763

  View details for PubMedID 32834002

• Editorial overview: The fortunate students, a tribute to the fortunate professor. Current opinion in microbiology Monack, D. M., Brodsky, I. E. 2020

  View details for DOI 10.1016/j.mib.2020.02.001

  View details for PubMedID 32171689

• Retinoic Acid and Lymphotoxin Signaling Promote Differentiation of Human Intestinal M Cells. Gastroenterology Ding, S. n., Song, Y. n., Brulois, K. F., Pan, J. n., Co, J. Y., Ren, L. n., Feng, N. n., Yasukawa, L. L., Sánchez-Tacuba, L. n., Wosen, J. E., Mellins, E. D., Monack, D. M., Amieva, M. R., Kuo, C. J., Butcher, E. C., Greenberg, H. B. 2020

  Abstract

  Intestinal microfold (M) cells are a unique subset of intestinal epithelial cells in the Peyer's patches that regulate mucosal immunity, serving as portals for sampling and uptake of luminal antigens. The inability to efficiently develop human M cells in cell culture has impeded studies of the intestinal immune system. We aimed to identify signaling pathways required for differentiation of human M cells and establish a robust culture system using human ileum enteroids.We analyzed transcriptome data from mouse Peyer's Patches to identify cell populations in close proximity to M cells. We used the human enteroid system to determine which cytokines were required to induce M cell differentiation. We performed transcriptome, immunofluorescence, scanning electron microscope, and transcytosis experiments to validate the development of phenotypic and functional human M cells.A combination of retinoic acid and lymphotoxin induced differentiation of glycoprotein 2-positive human M cells, which lack apical microvilli structure. Upregulated expression of innate immune-related genes within M cells correlated with a lack of viral antigens after rotavirus infection. Human M cells, developed in the enteroid system, internalized and transported enteric viruses, such as rotavirus and reovirus, across the intestinal epithelium barrier in the enteroids.We identified signaling pathways required for differentiation of intestinal M cells, and used this information to create a robust culture method to develop human M cells with capacity for internalization and transport of viruses. Studies of this model might increase our understanding of antigen presentation and the systemic entry of enteric pathogens in the human intestine.

  View details for DOI 10.1053/j.gastro.2020.03.053

  View details for PubMedID 32247021

• Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition. mBio Tal, M. C., Torrez Dulgeroff, L. B., Myers, L. n., Cham, L. B., Mayer-Barber, K. D., Bohrer, A. C., Castro, E. n., Yiu, Y. Y., Lopez Angel, C. n., Pham, E. n., Carmody, A. B., Messer, R. J., Gars, E. n., Kortmann, J. n., Markovic, M. n., Hasenkrug, M. n., Peterson, K. E., Winkler, C. W., Woods, T. A., Hansen, P. n., Galloway, S. n., Wagh, D. n., Fram, B. J., Nguyen, T. n., Corey, D. n., Kalluru, R. S., Banaei, N. n., Rajadas, J. n., Monack, D. M., Ahmed, A. n., Sahoo, D. n., Davis, M. M., Glenn, J. S., Adomati, T. n., Lang, K. S., Weissman, I. L., Hasenkrug, K. J. 2020; 11 (3)

  Abstract

  It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.

  View details for DOI 10.1128/mBio.01293-20

  View details for PubMedID 32576678

• Salmonella Effector SteE Converts the Mammalian Serine/Threonine Kinase GSK3 into a Tyrosine Kinase to Direct Macrophage Polarization. Cell host & microbe Panagi, I., Jennings, E., Zeng, J., Gunster, R. A., Stones, C. D., Mak, H., Jin, E., Stapels, D. A., Subari, N. Z., Pham, T. H., Brewer, S. M., Ong, S. Y., Monack, D. M., Helaine, S., Thurston, T. L. 2019

  Abstract

  Many Gram-negative bacterial pathogens antagonize anti-bacterial immunity through translocated effector proteins that inhibit pro-inflammatory signaling. Inaddition, the intracellular pathogen Salmonella enterica serovar Typhimurium initiates an anti-inflammatorytranscriptional response in macrophages through its effector protein SteE. However, the target(s) and molecular mechanism of SteE remain unknown. Here, we demonstrate that SteE converts both the amino acid and substrate specificity of the host pleiotropic serine/threonine kinase GSK3. SteE itself is a substrate of GSK3, and phosphorylationof SteE is required for its activity. Remarkably, phosphorylated SteE then forces GSK3 to phosphorylate the non-canonical substrate signal transducer and activator of transcription 3 (STAT3) on tyrosine-705. This results in STAT3 activation, which along with GSK3 is required for SteE-mediated upregulation of the anti-inflammatory M2 macrophage marker interleukin-4Ralpha (IL-4Ralpha). Overall, the conversion of GSK3 to a tyrosine-directed kinase representsa tightly regulated event that enables a bacterial virulence protein to reprogram innate immune signaling and establish an anti-inflammatory environment.

  View details for DOI 10.1016/j.chom.2019.11.002

  View details for PubMedID 31862381

• Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathy JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY Haileselassie, B., Mukherjee, R., Joshi, A. U., Napier, B. A., Massis, L. M., Ostberg, N., Queliconi, B. B., Monack, D., Bernstein, D., Mochly-Rosen, D. 2019; 130: 160–69

  View details for DOI 10.1016/j.yjmcc.2019.04.006

  View details for Web of Science ID 000468257200016

• Escalating Threat Levels of Bacterial Infection Can Be Discriminated by Distinct MAPK and NF-kappa B Signaling Dynamics in Single Host Cells CELL SYSTEMS Lane, K., Andres-Terre, M., Kudo, T., Monack, D. M., Covert, M. W. 2019; 8 (3): 183-+

  View details for DOI 10.1016/j.cels.2019.02.008

  View details for Web of Science ID 000462516900004

• Escalating Threat Levels of Bacterial Infection Can Be Discriminated by Distinct MAPK and NF-kappaB Signaling Dynamics in Single Host Cells. Cell systems Lane, K., Andres-Terre, M., Kudo, T., Monack, D. M., Covert, M. W. 2019

  Abstract

  During an infection, immune cells must identify the specific level of threat posed by a given bacterial input in order to generate an appropriate response. Given that they use a general non-self-recognition system, known as Toll-like receptors (TLRs), to detect bacteria, it remains unclear how they transmit information about a particular threat. To determine whether host cells can use signaling dynamics to transmit contextual information about a bacterial stimulus, we use live-cell imaging to make simultaneous quantitative measurements of host MAPK and NF-kappaB signaling, two key pathways downstream of TLRs, and bacterial infection and load. This combined, single-cell approach reveals that NF-kappaB and MAPK signaling dynamics are sufficient to discriminate between (1) pathogen-associated molecular patterns (PAMPs) versus bacteria, (2) extracellular versus intracellular bacteria, (3) pathogenic versus non-pathogenic bacteria, and (4) the presence or absence of features indicating an active intracellular bacterial infection, such as replication and effector secretion.

  View details for PubMedID 30904375

• Western diet regulates immune status and the response to LPS-driven sepsis independent of diet-associated microbiome PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Napier, B. A., Andres-Terre, M., Massis, L. M., Hryckowian, A. J., Higginbottom, S. K., Cumnock, K., Casey, K. M., Haileselassie, B., Lugo, K. A., Schneider, D. S., Sonnenburg, J. L., Monack, D. M. 2019; 116 (9): 3688–94

  View details for DOI 10.1073/pnas.1814273116

  View details for Web of Science ID 000459694400055

• Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions CELL REPORTS Co, J. Y., Margalef-Catala, M., Li, X., Mah, A. T., Kuo, C. J., Monack, D. M., Amieva, M. R. 2019; 26 (9): 2509-+

  View details for DOI 10.1016/j.celrep.2019.01.108

  View details for Web of Science ID 000460279100022

• Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions. Cell reports Co, J. Y., Margalef-Catala, M., Li, X., Mah, A. T., Kuo, C. J., Monack, D. M., Amieva, M. R. 2019; 26 (9): 2509

  Abstract

  Human enteroids-epithelial spheroids derived from primary gastrointestinal tissue-are a promising model to study pathogen-epithelial interactions. However, accessing the apical enteroid surface ischallenging because it is enclosed within the spheroid. We developed a technique to reverse enteroid polarity such that the apical surface everts to face the media. Apical-out enteroids maintain proper polarity and barrier function, differentiate into the major intestinal epithelial cell (IEC) types, and exhibit polarized absorption of nutrients. We used this model to study host-pathogen interactions and identified distinct polarity-specific patterns of infection by invasive enteropathogens. Salmonella enterica serovar Typhimurium targets IEC apical surfaces for invasion via cytoskeletal rearrangements, and Listeria monocytogenes, which binds to basolateral receptors, invade apical surfaces at sites of cellextrusion. Despite different modes of entry, both pathogens exit the epithelium within apically extruding enteroid cells. This model will enable further examination of IECs in health and disease.

  View details for PubMedID 30811997

• Western diet regulates immune status and the response to LPS-driven sepsis independent of diet-associated microbiome. Proceedings of the National Academy of Sciences of the United States of America Napier, B. A., Andres-Terre, M., Massis, L. M., Hryckowian, A. J., Higginbottom, S. K., Cumnock, K., Casey, K. M., Haileselassie, B., Lugo, K. A., Schneider, D. S., Sonnenburg, J. L., Monack, D. M. 2019; 116 (9): 3688–94

  Abstract

  Sepsis is a deleterious immune response to infection that leads to organ failure and is the 11th most common cause of death worldwide. Despite plaguing humanity for thousands of years, the host factors that regulate this immunological response and subsequent sepsis severity and outcome are not fully understood. Here we describe how the Western diet (WD), a diet high in fat and sucrose and low in fiber, found rampant in industrialized countries, leads to worse disease and poorer outcomes in an LPS-driven sepsis model in WD-fed mice compared with mice fed standard fiber-rich chow (SC). We find that WD-fed mice have higher baseline inflammation (metaflammation) and signs of sepsis-associated immunoparalysis compared with SC-fed mice. WD mice also have an increased frequency of neutrophils, some with an "aged" phenotype, in the blood during sepsis compared with SC mice. Importantly, we found that the WD-dependent increase in sepsis severity and higher mortality is independent of the microbiome, suggesting that the diet may be directly regulating the innate immune system through an unknown mechanism. Strikingly, we could predict LPS-driven sepsis outcome by tracking specific WD-dependent disease factors (e.g., hypothermia and frequency of neutrophils in the blood) during disease progression and recovery. We conclude that the WD is reprogramming the basal immune status and acute response to LPS-driven sepsis and that this correlates with alternative disease paths that lead to more severe disease and poorer outcomes.

  View details for PubMedID 30808756

• Host inflammasome defense mechanisms and bacterial pathogen evasion strategies. Current opinion in immunology Brewer, S. M., Brubaker, S. W., Monack, D. M. 2019; 60: 63–70

  Abstract

  Inflammasomes are a formidable armada of intracellular pattern recognition receptors. They recognize determinants of infection, such as foreign entities or danger signals within the host cell cytosol, rapidly executing innate immune defenses and initiating adaptive immune responses. Although inflammasomes are implicated in many diseases, they are especially critical in host protection against intracellular bacterial pathogens. Given this role, it is not surprising that many pathogens have evolved effective strategies to evade inflammasome activation. In this review, we will provide a brief summary of inflammasome activation during infection with the intent of highlighting recent advances in the field. Additionally, we will review known bacterial evasion strategies and countermeasures that impact pathogenesis.

  View details for DOI 10.1016/j.coi.2019.05.001

  View details for PubMedID 31174046

• Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580 PLOS BIOLOGY Canals, R., Hammarlof, D. L., Kroger, C., Owen, S. V., Fong, W., Lacharme-Lora, L., Zhu, X., Wenner, N., Carden, S. E., Honeycutt, J., Monack, D. M., Kingsley, R. A., Brownridge, P., Chaudhuri, R. R., Rowe, W. M., Predeus, A. V., Hokamp, K., Gordon, M. A., Hinton, J. D. 2019; 17 (1)

  View details for DOI 10.1371/journal.pbio.3000059

  View details for Web of Science ID 000457596000011

• Editorial: Protein Export and Secretion Among Bacterial Pathogens. Frontiers in cellular and infection microbiology Sana, T. G., Voulhoux, R., Monack, D. M., Ize, B., Bleves, S. 2019; 9: 473

  View details for DOI 10.3389/fcimb.2019.00473

  View details for PubMedID 32039049

• ROLE OF DRP1/FIS1-MEDIATED MITOCHONDRIAL FRAGMENTATION IN SEPSIS-INDUCED MYOCARDIAL DYSFUNCTION Haileselassie, B., Joshi, A., Mukherjee, R., Napier, B., Massis, L., Ostberg, N., Monack, D., Bernstein, D., Mochly-Rosen, D. LIPPINCOTT WILLIAMS & WILKINS. 2019

  View details for Web of Science ID 000498593400042

• Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathy. Journal of molecular and cellular cardiology Haileselassie, B. n., Mukherjee, R. n., Joshi, A. U., Napier, B. A., Massis, L. M., Ostberg, N. P., Queliconi, B. B., Monack, D. n., Bernstein, D. n., Mochly-Rosen, D. n. 2019

  Abstract

  Mitochondrial dysfunction is a key contributor to septic cardiomyopathy. Although recent literature implicates dynamin related protein 1 (Drp1) and its mitochondrial adaptor fission 1 (Fis1) in the development of pathologic fission and mitochondrial failure in neurodegenerative disease, little is known about the role of Drp1/Fis1 interaction in the context of sepsis-induced cardiomyopathy. Our study tests the hypothesis that Drp1/Fis1 interaction is a major driver of sepsis-mediated pathologic fission, leading to mitochondrial dysfunction in the heart.H9C2 cardiomyocytes were treated with lipopolysaccharide (LPS) to evaluate changes in mitochondrial membrane potential, oxidative stress, cellular respiration, and mitochondrial morphology. Balb/c mice were treated with LPS, cardiac function was measured by echocardiogaphy, and mitochondrial morphology determined by electron microscopy (EM). Drp1/Fis1 interaction was inhibited by P110 to determine whether limiting mitochondrial fission can reduce LPS-induced oxidative stress and cardiac dysfunction.LPS-treated H9C2 cardiomyocytes demonstrated a decrease in mitochondrial respiration followed by an increase in mitochondrial oxidative stress and a reduction in membrane potential. Inhibition of Drp1/Fis1 interaction with P110 attenuated LPS-mediated cellular oxidative stress and preserved membrane potential. In vivo, cardiac dysfunction in LPS-treated mice was associated with increased mitochondrial fragmentation. Treatment with P110 reduced cardiac mitochondrial fragmentation, prevented decline in cardiac function, and reduced mortality.Sepsis decreases cardiac mitochondrial respiration and membrane potential while increasing oxidative stress and inducing pathologic fission. Treatment with P110 was protective in both in vitro and in vivo models of septic cardiomyopathy, suggesting a key role of Drp1/Fis1 interaction, and a potential target to reduce its morbidity and mortality.

  View details for PubMedID 30981733

• Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580. PLoS biology Canals, R., Hammarlof, D. L., Kroger, C., Owen, S. V., Fong, W. Y., Lacharme-Lora, L., Zhu, X., Wenner, N., Carden, S. E., Honeycutt, J., Monack, D. M., Kingsley, R. A., Brownridge, P., Chaudhuri, R. R., Rowe, W. P., Predeus, A. V., Hokamp, K., Gordon, M. A., Hinton, J. C. 2019; 17 (1): e3000059

  Abstract

  Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

  View details for PubMedID 30645593

• A Gut Commensal-Produced Metabolite Mediates Colonization Resistance to Salmonella Infection CELL HOST & MICROBE Jacobson, A., Lam, L., Rajendram, M., Tamburini, F., Honeycutt, J., Trung Pham, Van Treuren, W., Pruss, K., Stabler, S., Lugo, K., Bouley, D. M., Vilches-Moure, J. G., Smith, M., Sonnenburg, J. L., Bhatt, A. S., Huang, K., Monack, D. 2018; 24 (2): 296-+

  View details for DOI 10.1016/j.chom.2018.07.002

  View details for Web of Science ID 000441083900016

• Stanley Falkow (1934-2018) Obituary CELL HOST & MICROBE Monack, D. 2018; 23 (6): 687–88

  View details for DOI 10.1016/j.chom.2018.05.014

  View details for Web of Science ID 000438408600001

• Stanley Falkow (1934-2018) SCIENCE Monack, D., Strauss, E. 2018; 360 (6393): 1077

  View details for DOI 10.1126/science.aau2284

  View details for Web of Science ID 000434635500031

• The oxidized phospholipid oxPAPC ameliorates septic shock by targeting the non-canonical inflammasome in macrophages Chu, L. H., Indramohan, M., Ratsimandresy, R. A., Gangopadhyay, A., Morris, E. P., Monack, D. M., Dorfleutner, A., Stehlik, C. AMER ASSOC IMMUNOLOGISTS. 2018

  View details for Web of Science ID 000459977701281

• LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens JOURNAL OF BIOLOGICAL CHEMISTRY Yokoyama, C. C., Baldridge, M. T., Leung, D. W., Zhao, G., Desai, C., Liu, T., Diaz-Ochoa, V. E., Huynh, J. P., Kimmey, J. M., Sennott, E. L., Hole, C. R., Idol, R. A., Park, S., Storek, K. M., Wang, C., Hwang, S., Milam, A., Chen, E., Kerrinnes, T., Starnbach, M. N., Handley, S. A., Mysorekar, I. U., Allen, P. M., Monack, D. M., Dinauer, M. C., Doering, T. L., Tsolis, R. M., Dworkin, J. E., Stallings, C. L., Amarasinghe, G. K., Micchelli, C. A., Virgin, H. W. 2018; 293 (16): 6022–38

  Abstract

  Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.

  View details for PubMedID 29496999

  View details for PubMedCentralID PMC5912457

• The oxidized phospholipid oxPAPC protects from septic shock by targeting the non-canonical inflammasome in macrophages NATURE COMMUNICATIONS Chu, L. H., Indramohan, M., Ratsimandresy, R. A., Gangopadhyay, A., Morris, E. P., Monack, D. M., Dorfleutner, A., Stehlik, C. 2018; 9: 996

  Abstract

  Lipopolysaccharide (LPS) of Gram-negative bacteria can elicit a strong immune response. Although extracellular LPS is sensed by TLR4 at the cell surface and triggers a transcriptional response, cytosolic LPS binds and activates non-canonical inflammasome caspases, resulting in pyroptotic cell death, as well as canonical NLRP3 inflammasome-dependent cytokine release. Contrary to the highly regulated multiprotein platform required for caspase-1 activation in the canonical inflammasomes, the non-canonical mouse caspase-11 and the orthologous human caspase-4 function simultaneously as innate sensors and effectors, and their regulation is unclear. Here we show that the oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) inhibits the non-canonical inflammasome in macrophages, but not in dendritic cells. Aside from a TLR4 antagonistic role, oxPAPC binds directly to caspase-4 and caspase-11, competes with LPS binding, and consequently inhibits LPS-induced pyroptosis, IL-1β release and septic shock. Therefore, oxPAPC and its derivatives might provide a basis for therapies that target non-canonical inflammasomes during Gram-negative bacterial sepsis.

  View details for PubMedID 29520027

• T6SS: The bacterial "fight club" in the host gut. PLoS pathogens Sana, T. G., Lugo, K. A., Monack, D. M. 2017; 13 (6): e1006325

  View details for DOI 10.1371/journal.ppat.1006325

  View details for PubMedID 28594921

  View details for PubMedCentralID PMC5464660

• Cell-Intrinsic Defense at the Epithelial Border Wall: Salmonella Pays the Price IMMUNITY Brubaker, S. W., Monack, D. M. 2017; 46 (4): 522–24

  Abstract

  Within the gut, Salmonella-infected enterocytes are expelled into the lumen, limiting pathogen replication. In this issue of Immunity, Rauch et al. (2017) expand our understanding of this cell-intrinsic response by characterizing the genetic determinants that control the expulsion and death of epithelial cells.

  View details for PubMedID 28423331

• Editorial: The sum of all defenses: tolerance plus resistance PATHOGENS AND DISEASE Napier, B. A., Monack, D. M. 2017; 75 (2)

  View details for DOI 10.1093/femspd/ftx015

  View details for Web of Science ID 000398086900007

  View details for PubMedID 28175285

• Pseudogenization of the Secreted Effector Gene sseI Confers Rapid Systemic Dissemination of S. Typhimurium ST313 within Migratory Dendritic Cells. Cell host & microbe Carden, S. E., Walker, G. T., Honeycutt, J., Lugo, K., Pham, T., Jacobson, A., Bouley, D., Idoyaga, J., Tsolis, R. M., Monack, D. 2017; 21 (2): 182-194

  Abstract

  Genome degradation correlates with host adaptation and systemic disease in Salmonella. Most lineages of the S. enterica subspecies Typhimurium cause gastroenteritis in humans; however, the recently emerged ST313 lineage II pathovar commonly causes systemic bacteremia in sub-Saharan Africa. ST313 lineage II displays genome degradation compared to gastroenteritis-associated lineages; yet, the mechanisms and causal genetic differences mediating these infection phenotypes are largely unknown. We find that the ST313 isolate D23580 hyperdisseminates from the gut to systemic sites, such as the mesenteric lymph nodes (MLNs), via CD11b(+) migratory dendritic cells (DCs). This hyperdissemination was facilitated by the loss of sseI, which encodes an effector that inhibits DC migration in gastroenteritis-associated isolates. Expressing functional SseI in D23580 reduced the number of infected migratory DCs and bacteria in the MLN. Our study reveals a mechanism linking pseudogenization of effectors with the evolution of niche adaptation in a bacterial pathogen.

  View details for DOI 10.1016/j.chom.2017.01.009

  View details for PubMedID 28182950

  View details for PubMedCentralID PMC5325708

• Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library. Bio-protocol Napier, B. A., Monack, D. M. 2017; 7 (10)

  Abstract

  The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, and the PAM sequence that links the Cas9 to the gRNA. CRISPR-Cas9 genome wide libraries are used to screen the effect of each gene in the genome on the cellular phenotype of interest, in an unbiased high-throughput manner. In this protocol, we describe our method of creating a CRISPR-Cas9 genome wide library in a transformed murine macrophage cell-line (RAW264.7). We have employed this library to identify novel mediators in the caspase-11 cell death pathway (Napier et al., 2016); however, this library can then be used to screen the importance of specific genes in multiple murine macrophage cellular pathways.

  View details for PubMedID 28868328

  View details for PubMedCentralID PMC5580966

• T6SS: The bacterial "fight club" in the host gut. Plos Pathogens Sana, T. G., Lugo, K. A., Monack, D. M. 2017: e1006325

  View details for DOI 10.1371/journal.ppat.1006325

  View details for PubMedCentralID PMC5464660

• Complement pathway amplifies caspase-11-dependent cell death and endotoxin-induced sepsis severity. journal of experimental medicine Napier, B. A., Brubaker, S. W., Sweeney, T. E., Monette, P., Rothmeier, G. H., Gertsvolf, N. A., Puschnik, A., Carette, J. E., Khatri, P., Monack, D. M. 2016; 213 (11): 2365-2382

  Abstract

  Cell death and release of proinflammatory mediators contribute to mortality during sepsis. Specifically, caspase-11-dependent cell death contributes to pathology and decreases in survival time in sepsis models. Priming of the host cell, through TLR4 and interferon receptors, induces caspase-11 expression, and cytosolic LPS directly stimulates caspase-11 activation, promoting the release of proinflammatory cytokines through pyroptosis and caspase-1 activation. Using a CRISPR-Cas9-mediated genome-wide screen, we identified novel mediators of caspase-11-dependent cell death. We found a complement-related peptidase, carboxypeptidase B1 (Cpb1), to be required for caspase-11 gene expression and subsequent caspase-11-dependent cell death. Cpb1 modifies a cleavage product of C3, which binds to and activates C3aR, and then modulates innate immune signaling. We find the Cpb1-C3-C3aR pathway induces caspase-11 expression through amplification of MAPK activity downstream of TLR4 and Ifnar activation, and mediates severity of LPS-induced sepsis (endotoxemia) and disease outcome in mice. We show C3aR is required for up-regulation of caspase-11 orthologues, caspase-4 and -5, in primary human macrophages during inflammation and that c3aR1 and caspase-5 transcripts are highly expressed in patients with severe sepsis; thus, suggesting that these pathways are important in human sepsis. Our results highlight a novel role for complement and the Cpb1-C3-C3aR pathway in proinflammatory signaling, caspase-11 cell death, and sepsis severity.

  View details for PubMedID 27697835

• Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut. Proceedings of the National Academy of Sciences of the United States of America Sana, T. G., Flaugnatti, N., Lugo, K. A., Lam, L. H., Jacobson, A., Baylot, V., durand, e., Journet, L., Cascales, E., Monack, D. M. 2016; 113 (34): E5044-51

  Abstract

  The mammalian gastrointestinal tract is colonized by a high-density polymicrobial community where bacteria compete for niches and resources. One key competition strategy includes cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), a multiprotein needle-like apparatus that injects effector proteins into prokaryotic and/or eukaryotic target cells. However, the contribution of T6SS antibacterial activity during pathogen invasion of the gut has not been demonstrated. We report that successful establishment in the gut by the enteropathogenic bacterium Salmonella enterica serovar Typhimurium requires a T6SS encoded within Salmonella pathogenicity island-6 (SPI-6). In an in vitro setting, we demonstrate that bile salts increase SPI-6 antibacterial activity and that S Typhimurium kills commensal bacteria in a T6SS-dependent manner. Furthermore, we provide evidence that one of the two T6SS nanotube subunits, Hcp1, is required for killing Klebsiella oxytoca in vitro and that this activity is mediated by the specific interaction of Hcp1 with the antibacterial amidase Tae4. Finally, we show that K. oxytoca is killed in the host gut in an Hcp1-dependent manner and that the T6SS antibacterial activity is essential for Salmonella to establish infection within the host gut. Our findings provide an example of pathogen T6SS-dependent killing of commensal bacteria as a mechanism to successfully colonize the host gut.

  View details for DOI 10.1073/pnas.1608858113

  View details for PubMedID 27503894

  View details for PubMedCentralID PMC5003274

• Bacterial Exotoxins: How Bacteria Fight the Immune System FRONTIERS IN IMMUNOLOGY Sastalla, I., Monack, D. M., Kubatzky, K. F. 2016; 7: 300

  View details for PubMedID 27532004

• MICROBIOLOGY The dark side of antibiotics NATURE Sana, T. G., Monack, D. M. 2016; 534 (7609): 624–25

  View details for Web of Science ID 000378676000022

  View details for PubMedID 27309812

• IMMUNOLOGY. A lipid arsenal to control inflammation. Science Napier, B. A., Monack, D. M. 2016; 352 (6290): 1173-1174

  View details for DOI 10.1126/science.aag0366

  View details for PubMedID 27257241

• Disruption of glycolytic flux is a signal for inflammasome signaling and pyroptotic cell death ELIFE Sanman, L. E., Qian, Y., Eisle, N. A., Ng, T. M., van der Linden, W. A., Monack, D. M., Weerapana, E., Bogyo, M. 2016; 5

  Abstract

  When innate immune cells such as macrophages are challenged with environmental stresses or infection by pathogens, they trigger the rapid assembly of multi-protein complexes called inflammasomes that are responsible for initiating pro-inflammatory responses and a form of cell death termed pyroptosis. We describe here the identification of an intracellular trigger of NLRP3-mediated inflammatory signaling, IL-1β production and pyroptosis in primed murine bone marrow-derived macrophages that is mediated by the disruption of glycolytic flux. This signal results from a drop of NADH levels and induction of mitochondrial ROS production and can be rescued by addition of products that restore NADH production. This signal is also important for host-cell response to the intracellular pathogen Salmonella typhimurium, which can disrupt metabolism by uptake of host-cell glucose. These results reveal an important inflammatory signaling network used by immune cells to sense metabolic dysfunction or infection by intracellular pathogens.

  View details for DOI 10.7554/eLife.13663

  View details for Web of Science ID 000387452200001

  View details for PubMedCentralID PMC4846378

• Coordinate actions of innate immune responses oppose those of the adaptive immune system during Salmonella infection of mice SCIENCE SIGNALING Hotson, A. N., Gopinath, S., Nicolau, M., Khasanova, A., Finck, R., Monack, D., Nolan, G. P. 2016; 9 (410)

  Abstract

  The immune system enacts a coordinated response when faced with complex environmental and pathogenic perturbations. We used the heterogeneous responses of mice to persistent Salmonella infection to model system-wide coordination of the immune response to bacterial burden. We hypothesized that the variability in outcomes of bacterial growth and immune response across genetically identical mice could be used to identify immune elements that serve as integrators enabling co-regulation and interconnectedness of the innate and adaptive immune systems. Correlation analysis of immune response variation to Salmonella infection linked bacterial load with at least four discrete, interacting functional immune response "cassettes." One of these, the innate cassette, in the chronically infected mice included features of the innate immune system, systemic neutrophilia, and high serum concentrations of the proinflammatory cytokine interleukin-6. Compared with mice with a moderate bacterial load, mice with the highest bacterial burden exhibited high activity of this innate cassette, which was associated with a dampened activity of the adaptive T cell cassette-with fewer plasma cells and CD4(+) T helper 1 cells and increased numbers of regulatory T cells-and with a dampened activity of the cytokine signaling cassette. System-wide manipulation of neutrophil numbers revealed that neutrophils regulated signal transducer and activator of transcription (STAT) signaling in B cells during infection. Thus, a network-level approach demonstrated unappreciated interconnections that balanced innate and adaptive immune responses during the dynamic course of disease and identified signals associated with pathogen transmission status, as well as a regulatory role for neutrophils in cytokine signaling.

  View details for DOI 10.1126/scisignal.aaa9303

  View details for Web of Science ID 000368662000001

• Coordinate actions of innate immune responses oppose those of the adaptive immune system during Salmonella infection of mice. Science signaling Hotson, A. N., Gopinath, S., Nicolau, M., Khasanova, A., Finck, R., Monack, D., Nolan, G. P. 2016; 9 (410): ra4

  Abstract

  The immune system enacts a coordinated response when faced with complex environmental and pathogenic perturbations. We used the heterogeneous responses of mice to persistent Salmonella infection to model system-wide coordination of the immune response to bacterial burden. We hypothesized that the variability in outcomes of bacterial growth and immune response across genetically identical mice could be used to identify immune elements that serve as integrators enabling co-regulation and interconnectedness of the innate and adaptive immune systems. Correlation analysis of immune response variation to Salmonella infection linked bacterial load with at least four discrete, interacting functional immune response "cassettes." One of these, the innate cassette, in the chronically infected mice included features of the innate immune system, systemic neutrophilia, and high serum concentrations of the proinflammatory cytokine interleukin-6. Compared with mice with a moderate bacterial load, mice with the highest bacterial burden exhibited high activity of this innate cassette, which was associated with a dampened activity of the adaptive T cell cassette-with fewer plasma cells and CD4(+) T helper 1 cells and increased numbers of regulatory T cells-and with a dampened activity of the cytokine signaling cassette. System-wide manipulation of neutrophil numbers revealed that neutrophils regulated signal transducer and activator of transcription (STAT) signaling in B cells during infection. Thus, a network-level approach demonstrated unappreciated interconnections that balanced innate and adaptive immune responses during the dynamic course of disease and identified signals associated with pathogen transmission status, as well as a regulatory role for neutrophils in cytokine signaling.

  View details for DOI 10.1126/scisignal.aaa9303

  View details for PubMedID 26758212

• Disruption of glycolytic flux is a signal for inflammasome signaling and pyroptotic cell death. eLife Sanman, L. E., Qian, Y., Eisele, N. A., Ng, T. M., van der Linden, W. A., Monack, D. M., Weerapana, E., Bogyo, M. 2016; 5

  Abstract

  When innate immune cells such as macrophages are challenged with environmental stresses or infection by pathogens, they trigger the rapid assembly of multi-protein complexes called inflammasomes that are responsible for initiating pro-inflammatory responses and a form of cell death termed pyroptosis. We describe here the identification of an intracellular trigger of NLRP3-mediated inflammatory signaling, IL-1β production and pyroptosis in primed murine bone marrow-derived macrophages that is mediated by the disruption of glycolytic flux. This signal results from a drop of NADH levels and induction of mitochondrial ROS production and can be rescued by addition of products that restore NADH production. This signal is also important for host-cell response to the intracellular pathogen Salmonella typhimurium, which can disrupt metabolism by uptake of host-cell glucose. These results reveal an important inflammatory signaling network used by immune cells to sense metabolic dysfunction or infection by intracellular pathogens.

  View details for DOI 10.7554/eLife.13663

  View details for PubMedID 27011353

  View details for PubMedCentralID PMC4846378

• A quantitative analysis of single-cell TLR signaling dynamics in response to Salmonella infection. Lane, K., Terre, M., Kudo, T., Monack, D., Covert, M. W. AMER SOC CELL BIOLOGY. 2016

  View details for Web of Science ID 000396047100631

• Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin JOURNAL OF IMMUNOLOGY Kortmann, J., Brubaker, S. W., Monack, D. M. 2015; 195 (3): 815-819

  Abstract

  Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6.

  View details for DOI 10.4049/jimmunol.1403100

  View details for Web of Science ID 000358070400011

  View details for PubMedCentralID PMC4505955

• Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin. Journal of immunology (Baltimore, Md. : 1950) Kortmann, J., Brubaker, S. W., Monack, D. M. 2015; 195 (3): 815-9

  Abstract

  Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6.

  View details for DOI 10.4049/jimmunol.1403100

  View details for PubMedID 26109648

  View details for PubMedCentralID PMC4505955

• IMMUNOLOGY. Microbial metabolite triggers antimicrobial defense. Science Brubaker, S. W., Monack, D. M. 2015; 348 (6240): 1207-1208

  View details for DOI 10.1126/science.aac5835

  View details for PubMedID 26068833

• Non-typhoidal Salmonella Typhimurium ST313 isolates that cause bacteremia in humans stimulate less inflammasome activation than ST19 isolates associated with gastroenteritis. Pathogens and disease Carden, S., Okoro, C., Dougan, G., Monack, D. 2015; 73 (4)

  Abstract

  Salmonella is an enteric pathogen that causes a range of diseases in humans. Non-typhoidal Salmonella (NTS) serovars such as Salmonella enterica serovar Typhimurium generally cause a self-limiting gastroenteritis whereas typhoidal serovars cause a systemic disease, typhoid fever. However, S. Typhimurium isolates within the multi-locus sequence type ST313 have emerged in sub-Saharan Africa as a major cause of bacteremia in humans. The S. Typhimurium ST313 lineage is phylogenetically distinct from classical S. Typhimurium lineages, such as ST19, that cause zoonotic gastroenteritis worldwide. Previous studies have shown that the ST313 lineage has undergone genome degradation when compared to the ST19 lineage, similar to that observed for typhoidal serovars. Currently, little is known about phenotypic differences between ST313 isolates and other NTS isolates. We find that representative ST313 isolates invade non-phagocytic cells less efficiently than the classical ST19 isolates that are more commonly associated with gastroenteritis. In addition, ST313 isolates induce less Caspase-1-dependent macrophage death and IL-1β release than ST19 isolates. ST313 isolates also express relatively lower levels of mRNA of the genes encoding the SPI-1 effector sopE2 and the flagellin, fliC, providing possible explanations for the decrease in invasion and inflammasome activation. The ST313 isolates have invasion and inflammatory phenotypes that are intermediate; more invasive and inflammatory than Salmonella enterica serovar Typhi and less than ST19 isolates associated with gastroenteritis. This suggests that both phenotypically and at the genomic level ST313 isolates are evolving signatures that facilitate a systemic lifestyle in humans.

  View details for DOI 10.1093/femspd/ftu023

  View details for PubMedID 25808600

• Non-typhoidal Salmonella Typhimurium ST313 isolates that cause bacteremia in humans stimulate less inflammasome activation than ST19 isolates associated with gastroenteritis. Pathogens and disease Carden, S., Okoro, C., Dougan, G., Monack, D. 2015; 73 (4)

  View details for DOI 10.1093/femspd/ftu023

  View details for PubMedID 25808600

• Bacterial recognition pathways that lead to inflammasome activation IMMUNOLOGICAL REVIEWS Storek, K. M., Monack, D. M. 2015; 265 (1): 112-129

  Abstract

  Inflammasomes are multi-protein signaling platforms that upon activation trigger the maturation of the pro-inflammatory cytokines, interleukin-1β (IL-1β) and IL-18, and cell death. Inflammasome sensors detect microbial and host-derived molecules. Here, we review the mechanisms of inflammasome activation triggered by bacterial infection, primarily focusing on two model intracellular bacterial pathogens, Francisella novicida and Salmonella typhimurium. We discuss the complex relationship between bacterial recognition through direct and indirect detection by inflammasome sensors. We highlight regulation mechanisms that potentiate or limit inflammasome activation. We discuss the importance of caspase-1 and caspase-11 in host defense, and we examine the downstream consequences of inflammasome activation within the context of bacterial infections.

  View details for DOI 10.1111/imr.12289

  View details for Web of Science ID 000353882900010

  View details for PubMedID 25879288

  View details for PubMedCentralID PMC4437016

• cGAS and Ifi204 Cooperate To Produce Type I IFNs in Response to Francisella Infection JOURNAL OF IMMUNOLOGY Storek, K. M., Gertsvolf, N. A., Ohlson, M. B., Monack, D. M. 2015; 194 (7): 3236-3245

  Abstract

  Type I IFN production is an important host immune response against viral and bacterial infections. However, little is known about the ligands and corresponding host receptors that trigger type I IFN production during bacterial infections. We used a model intracellular pathogen, Francisella novicida, to begin characterizing the type I IFN response to bacterial pathogens. F. novicida replicates in the cytosol of host cells and elicits a robust type I IFN response that is largely TLR independent, but is dependent on the adapter molecule STING, suggesting that the type I IFN stimulus during F. novicida infection is cytosolic. In this study, we report that the cytosolic DNA sensors, cyclic GMP-AMP synthase (cGAS) and Ifi204, are both required for the STING-dependent type I IFN response to F. novicida infection in both primary and immortalized murine macrophages. We created cGAS, Ifi204, and Sting functional knockouts in RAW264.7 macrophages and demonstrated that cGAS and Ifi204 cooperate to sense dsDNA and activate the STING-dependent type I IFN pathway. In addition, we show that dsDNA from F. novicida is an important type I IFN stimulating ligand. One outcome of cGAS-STING signaling is the activation of the absent in melanoma 2 inflammasome in response to F. novicida infection. Whereas the absent in melanoma 2 inflammasome is beneficial to the host during F. novicida infection, type I IFN signaling by STING and IFN regulatory factor 3 is detrimental to the host during F. novicida infection. Collectively, our studies indicate that cGAS and Ifi204 cooperate to sense cytosolic dsDNA and F. novicida infection to produce a strong type I IFN response.

  View details for DOI 10.4049/jimmunol.1402764

  View details for Web of Science ID 000351663400029

  View details for PubMedID 25710914

  View details for PubMedCentralID PMC4367159

• Intraspecies Competition for Niches in the Distal Gut Dictate Transmission during Persistent Salmonella Infection. PLoS pathogens Lam, L. H., Monack, D. M. 2014; 10 (12)

  View details for DOI 10.1371/journal.ppat.1004527

  View details for PubMedID 25474319

• Role of disease-associated tolerance in infectious superspreaders. Proceedings of the National Academy of Sciences of the United States of America Gopinath, S., Lichtman, J. S., Bouley, D. M., Elias, J. E., Monack, D. M. 2014; 111 (44): 15780-15785

  Abstract

  Natural populations show striking heterogeneity in their ability to transmit disease. For example, a minority of infected individuals known as superspreaders carries out the majority of pathogen transmission events. In a mouse model of Salmonella infection, a subset of infected hosts becomes superspreaders, shedding high levels of bacteria (>10(8) cfu per g of feces) but remain asymptomatic with a dampened systemic immune state. Here we show that superspreader hosts remain asymptomatic when they are treated with oral antibiotics. In contrast, nonsuperspreader Salmonella-infected hosts that are treated with oral antibiotics rapidly shed superspreader levels of the pathogen but display signs of morbidity. This morbidity is linked to an increase in inflammatory myeloid cells in the spleen followed by increased production of acute-phase proteins and proinflammatory cytokines. The degree of colonic inflammation is similar in antibiotic-treated superspreader and nonsuperspreader hosts, indicating that the superspreader hosts are tolerant of antibiotic-mediated perturbations in the intestinal tract. Importantly, neutralization of acute-phase proinflammatory cytokines in antibiotic-induced superspreaders suppresses the expansion of inflammatory myeloid cells and reduces morbidity. We describe a unique disease-associated tolerance to oral antibiotics in superspreaders that facilitates continued transmission of the pathogen.

  View details for DOI 10.1073/pnas.1409968111

  View details for PubMedID 25331868

• Host recognition of intracellular bacterial pathogens Monack, D. ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. 2014: 24

  View details for DOI 10.1016/j.cyto.2014.07.237

  View details for Web of Science ID 000343021700020

• Structure and Function of REP34 Implicates Carboxypeptidase Activity in Francisella tularensis Host Cell Invasion JOURNAL OF BIOLOGICAL CHEMISTRY Feld, G. K., El-Etr, S., Corzett, M. H., Hunter, M. S., Belhocine, K., Monack, D. M., Frank, M., Segelke, B. W., Rasley, A. 2014; 289 (44): 30668-30679

  Abstract

  Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essential to the pathogen's long term intracellular survival in the amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1' recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells.

  View details for DOI 10.1074/jbc.M114.599381

  View details for Web of Science ID 000344549700043

  View details for PubMedID 25231992

  View details for PubMedCentralID PMC4215245

• Toll-like Receptor and Inflammasome Signals Converge to Amplify the Innate Bactericidal Capacity of T Helper 1 Cells. Immunity O'Donnell, H., Pham, O. H., Li, L., Atif, S. M., Lee, S., Ravesloot, M. M., Stolfi, J. L., Nuccio, S., Broz, P., Monack, D. M., Baumler, A. J., McSorley, S. J. 2014; 40 (2): 213-224

  Abstract

  T cell effector functions can be elicited by noncognate stimuli, but the mechanism and contribution of this pathway to the resolution of intracellular macrophage infections have not been defined. Here, we show that CD4(+) T helper 1 (Th1) cells could be rapidly stimulated by microbe-associated molecular patterns during active infection with Salmonella or Chlamydia. Further, maximal stimulation of Th1 cells by lipopolysaccharide (LPS) did not require T-cell-intrinsic expression of toll-like receptor 4 (TLR4), interleukin-1 receptor (IL-1R), or interferon-γ receptor (IFN-γR) but instead required IL-18R, IL-33R, and adaptor protein MyD88. Innate stimulation of Th1 cells also required host expression of TLR4 and inflammasome components that together increased serum concentrations of IL-18. Finally, the elimination of noncognate Th1 cell stimulation hindered the resolution of primary Salmonella infection. Thus, the in vivo bactericidal capacity of Th1 cells is regulated by the response to noncognate stimuli elicited by multiple innate immune receptors.

  View details for DOI 10.1016/j.immuni.2013.12.013

  View details for PubMedID 24508233

• The Battle in the Gut IMMUNITY Monack, D. M. 2014; 40 (2): 173–75

  Abstract

  Our molecular understanding of how pathogen-microbiota-immune system interactions influence disease outcomes is limited. In this issue of Immunity, Behnsen et al. (2014) report that the cytokine interleukin-22, which usually plays a protective role, promotes pathogen colonization by suppressing related commensal bacteria.

  View details for PubMedID 24560194

• A microfluidic-based genetic screen to identify microbial virulence factors that inhibit dendritic cell migration INTEGRATIVE BIOLOGY McLaughlin, L. M., Xu, H., Carden, S. E., Fisher, S., Reyes, M., Heilshorn, S. C., Monack, D. M. 2014; 6 (4): 438-449

  Abstract

  Microbial pathogens are able to modulate host cells and evade the immune system by multiple mechanisms. For example, Salmonella injects effector proteins into host cells and evades the host immune system in part by inhibiting dendritic cell (DC) migration. The identification of microbial factors that modulate normal host functions should lead to the development of new classes of therapeutics that target these pathways. Current screening methods to identify either host or pathogen genes involved in modulating migration towards a chemical signal are limited because they do not employ stable, precisely controlled chemical gradients. Here, we develop a positive selection microfluidic-based genetic screen that allows us to identify Salmonella virulence factors that manipulate DC migration within stable, linear chemokine gradients. Our screen identified 7 Salmonella effectors (SseF, SifA, SspH2, SlrP, PipB2, SpiC and SseI) that inhibit DC chemotaxis toward CCL19. This method is widely applicable for identifying novel microbial factors that influence normal host cell chemotaxis as well as revealing new mammalian genes involved in directed cell migration.

  View details for DOI 10.1039/c3ib40177d

  View details for Web of Science ID 000333331800007

  View details for PubMedID 24599496

• Helicobacter and Salmonella Persistent Infection Strategies COLD SPRING HARBOR PERSPECTIVES IN MEDICINE Monack, D. M. 2013; 3 (12)

  Abstract

  Some host-adapted bacterial pathogens are capable of causing persistent infections in humans. For example, Helicobacter pylori inhabits the human gastric mucosa and persistence can be lifelong. Salmonella enterica serovar Typhi causes systemic infections that involve colonization of the reticuloendothelial system and some individuals become lifelong carriers. In this review, I compare and contrast the different lifestyles of Helicobacter and Salmonella within the host and the strategies they have evolved to persist in mammalian hosts. Persistently infected carriers serve as the reservoirs for these pathogens, and the carrier state is an essential feature that is required for survival of the bacteria within a restricted host population. Therefore, investigating the chronic carrier state should provide insight into bacterial survival strategies, as well as new therapeutic approaches for treatments.

  View details for DOI 10.1101/cshperspect.a010348

  View details for Web of Science ID 000328277800004

  View details for PubMedID 24296347

• Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens NATURE Ng, K. M., Ferreyra, J. A., Higginbottom, S. K., Lynch, J. B., Kashyap, P. C., Gopinath, S., Naidu, N., Choudhury, B., Weimer, B. C., Monack, D. M., Sonnenburg, J. L. 2013; 502 (7469): 96-?

  Abstract

  The human intestine, colonized by a dense community of resident microbes, is a frequent target of bacterial pathogens. Undisturbed, this intestinal microbiota provides protection from bacterial infections. Conversely, disruption of the microbiota with oral antibiotics often precedes the emergence of several enteric pathogens. How pathogens capitalize upon the failure of microbiota-afforded protection is largely unknown. Here we show that two antibiotic-associated pathogens, Salmonella enterica serovar Typhimurium (S. typhimurium) and Clostridium difficile, use a common strategy of catabolizing microbiota-liberated mucosal carbohydrates during their expansion within the gut. S. typhimurium accesses fucose and sialic acid within the lumen of the gut in a microbiota-dependent manner, and genetic ablation of the respective catabolic pathways reduces its competitiveness in vivo. Similarly, C. difficile expansion is aided by microbiota-induced elevation of sialic acid levels in vivo. Colonization of gnotobiotic mice with a sialidase-deficient mutant of Bacteroides thetaiotaomicron, a model gut symbiont, reduces free sialic acid levels resulting in C. difficile downregulating its sialic acid catabolic pathway and exhibiting impaired expansion. These effects are reversed by exogenous dietary administration of free sialic acid. Furthermore, antibiotic treatment of conventional mice induces a spike in free sialic acid and mutants of both Salmonella and C. difficile that are unable to catabolize sialic acid exhibit impaired expansion. These data show that antibiotic-induced disruption of the resident microbiota and subsequent alteration in mucosal carbohydrate availability are exploited by these two distantly related enteric pathogens in a similar manner. This insight suggests new therapeutic approaches for preventing diseases caused by antibiotic-associated pathogens.

  View details for DOI 10.1038/nature12503

  View details for Web of Science ID 000325106000038

  View details for PubMedID 23995682

• Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens. Nature Ng, K. M., Ferreyra, J. A., Higginbottom, S. K., Lynch, J. B., Kashyap, P. C., Gopinath, S., Naidu, N., Choudhury, B., Weimer, B. C., Monack, D. M., Sonnenburg, J. L. 2013; 502 (7469): 96-99

  Abstract

  The human intestine, colonized by a dense community of resident microbes, is a frequent target of bacterial pathogens. Undisturbed, this intestinal microbiota provides protection from bacterial infections. Conversely, disruption of the microbiota with oral antibiotics often precedes the emergence of several enteric pathogens. How pathogens capitalize upon the failure of microbiota-afforded protection is largely unknown. Here we show that two antibiotic-associated pathogens, Salmonella enterica serovar Typhimurium (S. typhimurium) and Clostridium difficile, use a common strategy of catabolizing microbiota-liberated mucosal carbohydrates during their expansion within the gut. S. typhimurium accesses fucose and sialic acid within the lumen of the gut in a microbiota-dependent manner, and genetic ablation of the respective catabolic pathways reduces its competitiveness in vivo. Similarly, C. difficile expansion is aided by microbiota-induced elevation of sialic acid levels in vivo. Colonization of gnotobiotic mice with a sialidase-deficient mutant of Bacteroides thetaiotaomicron, a model gut symbiont, reduces free sialic acid levels resulting in C. difficile downregulating its sialic acid catabolic pathway and exhibiting impaired expansion. These effects are reversed by exogenous dietary administration of free sialic acid. Furthermore, antibiotic treatment of conventional mice induces a spike in free sialic acid and mutants of both Salmonella and C. difficile that are unable to catabolize sialic acid exhibit impaired expansion. These data show that antibiotic-induced disruption of the resident microbiota and subsequent alteration in mucosal carbohydrate availability are exploited by these two distantly related enteric pathogens in a similar manner. This insight suggests new therapeutic approaches for preventing diseases caused by antibiotic-associated pathogens.

  View details for DOI 10.1038/nature12503

  View details for PubMedID 23995682

• Salmonella Require the Fatty Acid Regulator PPARd for the Establishment of a Metabolic Environment Essential for Long-Term Persistence. Cell host & microbe Eisele, N. A., Ruby, T., Jacobson, A., Manzanillo, P. S., Cox, J. S., Lam, L., Mukundan, L., Chawla, A., Monack, D. M. 2013; 14 (2): 171-182

  Abstract

  Host-adapted Salmonella strains are responsible for a number of disease manifestations in mammals, including an asymptomatic chronic infection in which bacteria survive within macrophages located in systemic sites. However, the host cell physiology and metabolic requirements supporting bacterial persistence are poorly understood. In a mouse model of long-term infection, we found that S. typhimurium preferentially associates with anti-inflammatory/M2 macrophages at later stages of infection. Further, PPARδ, a eukaryotic transcription factor involved in sustaining fatty acid metabolism, is upregulated in Salmonella-infected macrophages. PPARδ deficiency dramatically inhibits Salmonella replication, which is linked to the metabolic state of macrophages and the level of intracellular glucose available to bacteria. Pharmacological activation of PPARδ increases glucose availability and enhances bacterial replication in macrophages and mice, while Salmonella fail to persist in Pparδ null mice. These data suggest that M2 macrophages represent a unique niche for long-term intracellular bacterial survival and link the PPARδ-regulated metabolic state of the host cell to persistent bacterial infection.

  View details for DOI 10.1016/j.chom.2013.07.010

  View details for PubMedID 23954156

  View details for PubMedCentralID PMC3785333

• PPAR?-mediated increase in glucose availability sustains chronic Brucella abortus infection in alternatively activated macrophages. Cell host & microbe Xavier, M. N., Winter, M. G., Spees, A. M., den Hartigh, A. B., Nguyen, K., Roux, C. M., Silva, T. M., Atluri, V. L., Kerrinnes, T., Keestra, A. M., Monack, D. M., Luciw, P. A., Eigenheer, R. A., Bäumler, A. J., Santos, R. L., Tsolis, R. M. 2013; 14 (2): 159-170

  Abstract

  Eradication of persistent intracellular bacterial pathogens with antibiotic therapy is often slow or incomplete. However, strategies to augment antibiotics are hampered by our poor understanding of the nutritional environment that sustains chronic infection. Here we show that the intracellular pathogen Brucella abortus survives and replicates preferentially in alternatively activated macrophages (AAMs), which are more abundant during chronic infection. A metabolic shift induced by peroxisome proliferator-activated receptor γ (PPARγ), which increases intracellular glucose availability, is identified as a causal mechanism promoting enhanced bacterial survival in AAMs. Glucose uptake was crucial for increased replication of B. abortus in AAMs, and for chronic infection, as inactivation of the bacterial glucose transporter gluP reduced both intracellular survival in AAMs and persistence in mice. Thus, a shift in intracellular nutrient availability induced by PPARγ promotes chronic persistence of B. abortus within AAMs, and targeting this pathway may aid in eradicating chronic infection.

  View details for DOI 10.1016/j.chom.2013.07.009

  View details for PubMedID 23954155

• PPAR gamma-Mediated Increase in Glucose Availability Sustains Chronic Brucella abortus Infection in Alternatively Activated Macrophages CELL HOST & MICROBE Xavier, M. N., Winter, M. G., Spees, A. M., den Hartigh, A. B., Nguyen, K., Roux, C. M., Silva, T. M., Atluri, V. L., Kerrinnes, T., Keestra, A. M., Monack, D. M., Luciw, P. A., Eigenheer, R. A., Baeumler, A. J., Santos, R. L., Tsolis, R. M. 2013; 14 (2): 159-170

  Abstract

  Eradication of persistent intracellular bacterial pathogens with antibiotic therapy is often slow or incomplete. However, strategies to augment antibiotics are hampered by our poor understanding of the nutritional environment that sustains chronic infection. Here we show that the intracellular pathogen Brucella abortus survives and replicates preferentially in alternatively activated macrophages (AAMs), which are more abundant during chronic infection. A metabolic shift induced by peroxisome proliferator-activated receptor γ (PPARγ), which increases intracellular glucose availability, is identified as a causal mechanism promoting enhanced bacterial survival in AAMs. Glucose uptake was crucial for increased replication of B. abortus in AAMs, and for chronic infection, as inactivation of the bacterial glucose transporter gluP reduced both intracellular survival in AAMs and persistence in mice. Thus, a shift in intracellular nutrient availability induced by PPARγ promotes chronic persistence of B. abortus within AAMs, and targeting this pathway may aid in eradicating chronic infection.

  View details for DOI 10.1016/j.chom.2013.07.009

  View details for Web of Science ID 000330851600007

  View details for PubMedCentralID PMC3777723

• Newly described pattern recognition receptors team up against intracellular pathogens NATURE REVIEWS IMMUNOLOGY Broz, P., Monack, D. M. 2013; 13 (8): 551-565

  Abstract

  Recognizing the presence of invading pathogens is key to mounting an effective innate immune response. Mammalian cells express different classes of germline-encoded pattern recognition receptors that monitor the extracellular and intracellular compartments of host cells for signs of infection and that activate several conserved signalling pathways. An efficient immune response often requires the sequential detection of a pathogen by different receptors in different subcellular compartments, which results in a complex interplay of downstream signalling pathways. In this Review, we discuss the recent identification of previously unknown pattern recognition receptors and how they complement the repertoire of established receptors.

  View details for DOI 10.1038/nri3479

  View details for Web of Science ID 000322217500010

  View details for PubMedID 23846113

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

  Abstract

  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

• A coupled protein and probe engineering approach for selective inhibition and activity-based probe labeling of the caspases. Journal of the American Chemical Society Xiao, J., Broz, P., Puri, A. W., Deu, E., Morell, M., Monack, D. M., Bogyo, M. 2013; 135 (24): 9130-9138

  Abstract

  Caspases are cysteine proteases that play essential roles in apoptosis and inflammation. Unfortunately, their highly conserved active sites and overlapping substrate specificities make it difficult to use inhibitors or activity-based probes to study the function, activation, localization, and regulation of individual members of this family. Here we describe a strategy to engineer a caspase to contain a latent nucleophile that can be targeted by a probe containing a suitably placed electrophile, thereby allowing specific, irreversible inhibition and labeling of only the engineered protease. To accomplish this, we have identified a non-conserved residue on the small subunit of all caspases that is near the substrate-binding pocket and that can be mutated to a non-catalytic cysteine residue. We demonstrate that an active-site probe containing an irreversible binding acrylamide electrophile can specifically target this cysteine residue. Here we validate the approach using the apoptotic mediator, caspase-8, and the inflammasome effector, caspase-1. We show that the engineered enzymes are functionally identical to the wild-type enzymes and that the approach allows specific inhibition and direct imaging of the engineered targets in cells. Therefore, this method can be used to image localization and activation as well as the functional contributions of individual caspase proteases to the process of cell death or inflammation.

  View details for DOI 10.1021/ja403521u

  View details for PubMedID 23701470

  View details for PubMedCentralID PMC3722599

• 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)

  Abstract

  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

  View details for PubMedCentralID PMC3675027

• Innate amplification of CD4 T cell responses during infection: NLRs and TLRs converge O'Donnell, H., Monack, D., McSorley, S. AMER ASSOC IMMUNOLOGISTS. 2013

  View details for Web of Science ID 000322987104017

• 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

  Abstract

  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-γ (IFN-γ) 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-γ locus. Thus, this lncRNA regulates epigenetic marking of IFN-γ-encoding chromatin, expression of IFN-γ, and susceptibility to a viral and a bacterial pathogen.

  View details for DOI 10.1016/j.cell.2013.01.015

  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

  Abstract

  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

• Noncanonical Inflammasomes: Caspase-11 Activation and Effector Mechanisms PLOS PATHOGENS Broz, P., Monack, D. M. 2013; 9 (2)

  View details for DOI 10.1371/journal.ppat.1003144

  View details for Web of Science ID 000315648900006

  View details for PubMedID 23468620

  View details for PubMedCentralID PMC3585133

• 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

  Abstract

  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

• Phosphorylation of NLRC4 is critical for inflammasome activation NATURE Qu, Y., Misaghi, S., Izrael-Tomasevic, A., Newton, K., Gilmour, L. L., Lamkanfi, M., Louie, S., Kayagaki, N., Liu, J., Koemueves, L., Cupp, J. E., Arnott, D., Monack, D., Dixit, V. M. 2012; 490 (7421): 539-?

  Abstract

  NLRC4 is a cytosolic member of the NOD-like receptor family that is expressed in innate immune cells. It senses indirectly bacterial flagellin and type III secretion systems, and responds by assembling an inflammasome complex that promotes caspase-1 activation and pyroptosis. Here we use knock-in mice expressing NLRC4 with a carboxy-terminal 3×Flag tag to identify phosphorylation of NLRC4 on a single, evolutionarily conserved residue, Ser 533, following infection of macrophages with Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium). Western blotting with a NLRC4 phospho-Ser 533 antibody confirmed that this post-translational modification occurs only in the presence of stimuli known to engage NLRC4 and not the related protein NLRP3 or AIM2. Nlrc4(-/-) macrophages reconstituted with NLRC4 mutant S533A, unlike those reconstituted with wild-type NLRC4, did not activate caspase-1 and pyroptosis in response to S. typhimurium, indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors identified PRKCD (PKCδ) as a candidate NLRC4 kinase. Recombinant PKCδ phosphorylated NLRC4 S533 in vitro, immunodepletion of PKCδ from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro, and Prkcd(-/-) macrophages exhibited greatly attenuated caspase-1 activation and IL-1β secretion specifically in response to S. typhimurium. Phosphorylation-defective NLRC4 S533A failed to recruit procaspase-1 and did not assemble inflammasome specks during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity.

  View details for DOI 10.1038/nature11429

  View details for Web of Science ID 000310196200042

  View details for PubMedID 22885697

• 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-?

  Abstract

  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)-1β 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-β (TRIF)-dependent interferon-β 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

  View details for PubMedCentralID PMC3470772

• 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

  Abstract

  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

  View details for PubMedCentralID PMC3461347

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

  Abstract

  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

  Abstract

  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

  Abstract

  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

  View details for PubMedCentralID PMC3372747

• 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

  Abstract

  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

  View details for PubMedCentralID PMC3370950

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

  Abstract

  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

  Abstract

  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-1β 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-β is upregulated in BMDC following Francisella infection, and the IFN-β 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

  View details for PubMedCentralID PMC3240688

• 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

  View details for DOI 10.4161/gmic.19141

  View details for Web of Science ID 000525667600002

• Dermacentor andersoni Transmission of Francisella tularensis subsp novicida Reflects Bacterial Colonization, Dissemination, and Replication Coordinated with Tick Feeding INFECTION AND IMMUNITY Reif, K. E., Palmer, G. H., Ueti, M. W., Scoles, G. A., Margolis, J. J., Monack, D. M., Noh, S. M. 2011; 79 (12): 4941-4946

  Abstract

  Ticks serve as biological vectors for a wide variety of bacterial pathogens which must be able to efficiently colonize specific tick tissues prior to transmission. The bacterial determinants of tick colonization are largely unknown, a knowledge gap attributed in large part to the paucity of tools to genetically manipulate these pathogens. In this study, we demonstrated that Francisella tularensis subsp. novicida, for which a complete two-allele transposon mutant library has been constructed, initially infects the midguts of 100% of acquisition-fed Dermacentor andersoni nymphs, with stable colonization and replication during a subsequent molt. Increased dissemination to and marked replication within the salivary gland was closely linked to a second (transmission) feed and culminated in secretion of bacteria into the saliva and successful transmission. Simultaneous testing of multiple mutants resulted in total bacterial levels similar to those observed for single mutants. However, there was evidence of a bottleneck during colonization, resulting in a founder effect in which the most successful mutant varied when comparing individual ticks. Thus, it is essential to assess mutant success at the level of the tick population rather than in individual ticks. The ability of F. tularensis subsp. novicida to recapitulate the key physiological events by which bacteria colonize and are transmitted by ixodid ticks provides a new genome-wide approach to identify the required pathogen molecules and pathways. The identification of specific genes and, more importantly, conserved pathways that function at the tick-pathogen interface will accelerate the development of new methods to block transmission.

  View details for DOI 10.1128/IAI.05676-11

  View details for Web of Science ID 000296920800019

  View details for PubMedID 21930762

  View details for PubMedCentralID PMC3232653

• 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

  Abstract

  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-1β, 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 (ΔFTT0584, ΔmviN, ΔripA, ΔfopA and ΔFTN1217) or deficient for genes involved in O-antigen or LPS biosynthesis (ΔwbtA and ΔlpxH) 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

  View details for PubMedCentralID PMC3173570

• 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

  Abstract

  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

  Abstract

  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-1β (IL-1β) 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

  View details for PubMedCentralID PMC3170129

• TLR Signaling Is Required for Salmonella typhimurium Virulence CELL Arpaia, N., Godec, J., Lau, L., Sivick, K. E., McLaughlin, L. M., Jones, M. B., Dracheva, T., Peterson, S. N., Monack, D. M., Barton, G. M. 2011; 144 (5): 675-688

  Abstract

  Toll-like receptors (TLRs) contribute to host resistance to microbial pathogens and can drive the evolution of virulence mechanisms. We have examined the relationship between host resistance and pathogen virulence using mice with a functional allele of the nramp-1 gene and lacking combinations of TLRs. Mice deficient in both TLR2 and TLR4 were highly susceptible to the intracellular bacterial pathogen Salmonella typhimurium, consistent with reduced innate immune function. However, mice lacking additional TLRs involved in S. typhimurium recognition were less susceptible to infection. In these TLR-deficient cells, bacteria failed to upregulate Salmonella pathogenicity island 2 (SPI-2) genes and did not form a replicative compartment. We demonstrate that TLR signaling enhances the rate of acidification of the Salmonella-containing phagosome, and inhibition of this acidification prevents SPI-2 induction. Our results indicate that S. typhimurium requires cues from the innate immune system to regulate virulence genes necessary for intracellular survival, growth, and systemic infection.

  View details for DOI 10.1016/j.cell.2011.01.031

  View details for Web of Science ID 000288007100008

  View details for PubMedID 21376231

  View details for PubMedCentralID PMC3063366

• 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

  Abstract

  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

  View details for PubMedCentralID PMC3028865

• 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-?

  Abstract

  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

  View details for PubMedCentralID PMC3042314

• 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

  Abstract

  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 Web of Science ID 000208863500026

  View details for PubMedCentralID PMC3109290

• 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-?

  Abstract

  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

  View details for PubMedCentralID PMC3109290

• 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

  Abstract

  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-1β 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-1β/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-1β/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

  View details for PubMedCentralID PMC3016200

• Genome wide transcriptome profiling of a murine acute melioidosis model reveals new insights into how Burkholderia pseudomallei overcomes host innate immunity BMC GENOMICS Chin, C., Monack, D. M., Nathan, S. 2010; 11

  Abstract

  At present, very little is known about how Burkholderia pseudomallei (B. pseudomallei) interacts with its host to elicit melioidosis symptoms. We established a murine acute-phase melioidosis model and used DNA microarray technology to investigate the global host/pathogen interaction. We compared the transcriptome of infected liver and spleen with uninfected tissues over an infection period of 42 hr to identify genes whose expression is altered in response to an acute infection.Viable B. pseudomallei cells were consistently detected in the blood, liver and spleen during the 42 hr course of infection. Microarray analysis of the liver and spleen over this time course demonstrated that genes involved in immune response, stress response, cell cycle regulation, proteasomal degradation, cellular metabolism and signal transduction pathways were differentially regulated. Up regulation of toll-like receptor 2 (TLR2) gene expression suggested that a TLR2-mediated signalling pathway is responsible for recognition and initiation of an inflammatory response to the acute B. pseudomallei infection. Most of the highly elevated inflammatory genes are a cohort of "core host immune response" genes commonly seen in general inflammation infections. Concomitant to this initial inflammatory response, we observed an increase in transcripts associated with cell-death, caspase activation and peptidoglysis that ultimately promote tissue injury in the host. The complement system responsible for restoring host cellular homeostasis and eliminating intracellular bacteria was activated only after 24 hr post-infection. However, at this time point, diverse host nutrient metabolic and cellular pathways including glycolysis, fatty acid metabolism and tricarboxylic acid (TCA) cycle were repressed.This detailed picture of the host transcriptional response during acute melioidosis highlights a broad range of innate immune mechanisms that are activated in the host within 24 hrs, including the core immune response commonly seen in general inflammatory infections. Nevertheless, this activation is suppressed at 42 hr post-infection and in addition, suboptimal activation and function of the downstream complement system promotes uncontrolled spread of the bacteria.

  View details for DOI 10.1186/1471-2164-11-672

  View details for Web of Science ID 000285386000001

  View details for PubMedID 21110886

  View details for PubMedCentralID PMC3017868

• 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

  Abstract

  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

  View details for PubMedCentralID PMC2916133

• 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)

  Abstract

  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

  View details for PubMedCentralID PMC2928790

• 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

  Abstract

  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

  View details for PubMedCentralID PMC2876573

• 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

  Abstract

  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 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

  Abstract

  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 PubMedID 20176744

• 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

  Abstract

  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

  View details for PubMedCentralID PMC2823920

• 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

  Abstract

  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

  View details for PubMedCentralID PMC2805214

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

  Abstract

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

  View details for DOI 10.1073/pnas.0912386106

  View details for Web of Science ID 000273178700071

  View details for PubMedID 20018727

  View details for PubMedCentralID PMC2792164

• Francisella tularensis Type A Strains Cause the Rapid Encystment of Acanthamoeba castellanii and Survive in Amoebal Cysts for Three Weeks Postinfection APPLIED AND ENVIRONMENTAL MICROBIOLOGY El-Etr, S. H., Margolis, J. J., Monack, D., Robison, R. A., Cohen, M., Moore, E., Rasley, A. 2009; 75 (23): 7488-7500

  Abstract

  Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown and to the organism's potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype is caused by factor(s) secreted by amoebae and/or F. tularensis into the coculture medium. Further, our results indicate that in contrast to the live vaccine strain LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks postinfection and that the induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that interactions between F. tularensis strains and amoebae may play a role in the environmental persistence of F. tularensis.

  View details for DOI 10.1128/AEM.01829-09

  View details for Web of Science ID 000271944800023

  View details for PubMedID 19820161

  View details for PubMedCentralID PMC2786426

• 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)

  Abstract

  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

  View details for PubMedCentralID PMC2777311

• NLR-mediated control of inflammasome assembly in the host response against bacterial pathogens SEMINARS IN IMMUNOLOGY Brodsky, I. E., Monack, D. 2009; 21 (4): 199-207

  Abstract

  The host response against diverse bacterial pathogens involves activation of specialized immune cells and elaboration of pro-inflammatory cytokines that help to coordinate appropriate host defense. Members of the interleukin-1 (IL-1) cytokine family, IL-1beta and IL-18, are central players in this process. Extracellular release of the mature, active form of these cytokines requires their processing by the cysteine protease caspase-1, which therefore serves as a key regulator of the inflammatory response. In addition to its role in secretion of pro-inflammatory cytokines, caspase-1 is also required for a form of cell death, recently termed pyroptosis, that occurs in macrophages infected by certain bacterial pathogens. Caspase-1 itself is synthesized as a pro-enzyme, which must first be activated by autocatalytic cleavage. This activation requires recruitment of caspase-1 into multiprotein complexes known as inflammasomes. The Nod-like receptor (NLR) family of cytosolic proteins play an important role in detecting inflammatory stimuli and subsequently mediate inflammasome assembly. A common feature of NLR proteins that trigger inflammasome assembly in response to bacterial infection is that they appear to sense membrane perturbation or delivery of bacterial components into the cytosol through bacterial pore-forming toxins or bacterial secretion systems. This review will discuss the recent developments regarding caspase-1 activation in response to bacterial infection, cross-talk between caspase-1 and other pathways involved in regulating cell death, and recent findings that a number of bacterial pathogens possess mechanisms to inhibit caspase-1 activation.

  View details for DOI 10.1016/j.smim.2009.05.007

  View details for Web of Science ID 000274725100005

  View details for PubMedID 19539499

• 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

  Abstract

  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

  View details for PubMedCentralID PMC2681779

• Intracytosolic Sensing of Pathogens: Nucleic Acid Receptors, NLRs, and the Associated Responses during Infections and Autoinflammatory Diseases PHAGOCYTE-PATHOGEN INTERACTIONS: MACROPHAGES AND THE HOST RESPONSE TO INFECTION Henry, T., Monack, D. M., Russell, D. G., Gordon, S. 2009: 153–69

  View details for Web of Science ID 000268445000011

• Critical function for Naip5 in inflammasome activation by a conserved carboxy-terminal domain of flagellin NATURE IMMUNOLOGY Lightfield, K. L., Persson, J., Brubaker, S. W., Witte, C. E., von Moltke, J., Dunipace, E. A., Henry, T., Sun, Y., Cado, D., Dietrich, W. F., Monack, D. M., Tsolis, R. M., Vance, R. E. 2008; 9 (10): 1171-1178

  Abstract

  Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.

  View details for DOI 10.1038/ni.1646

  View details for Web of Science ID 000259315600016

  View details for PubMedID 18724372

  View details for PubMedCentralID PMC2614210

• 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

  Abstract

  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

  View details for PubMedCentralID PMC2493208

• 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

• 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

  Abstract

  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

  View details for PubMedCentralID PMC2223630

• 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

  Abstract

  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 PubMedID 17662071

• 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

  Abstract

  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 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

  Abstract

  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 PubMedID 17389372

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

  Abstract

  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

  Abstract

  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 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

  Abstract

  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

  View details for PubMedCentralID PMC1698089

• Caspase-1-mediated activation of interleukin-1 beta (IL-1 beta) and IL-18 contributes to innate immune defenses against Salmonella enterica serovar typhimurium infection INFECTION AND IMMUNITY Raupach, B., Peuschel, S., Monack, D. M., Zychlinsky, A. 2006; 74 (8): 4922-4926

  Abstract

  Caspase-1 (Casp-1) mediates the processing of the proinflammatory cytokines interleukin-1beta (IL-1beta) and IL-18 to their mature forms. Casp-1-deficient mice succumb more rapidly to Salmonella challenge than do wild-type animals. Both Casp-1 substrates, IL-18 and IL-1beta, are relevant for control of Salmonella enterica serovar Typhimurium. We used IL-18-/- and IL-1beta-/- mice in addition to administration of recombinant IL-18 to Casp-1-/- mice to demonstrate that IL-18 is important for resistance to the systemic infection but not for resistance to the intestinal phase of the infection. This suggests that IL-1beta is critical for the intestinal phase of the disease. Thus, we show that Casp-1 is essential for host innate immune defense against S. enterica serovar Typhimurium and that Casp-1 substrates are required at distinct times and anatomical sites.

  View details for DOI 10.1128/IAI.00417-06

  View details for Web of Science ID 000239381400060

  View details for PubMedID 16861683

  View details for PubMedCentralID PMC1539628

• Cryopyrin activates the inflammasome in response to toxins and ATP NATURE Mariathasan, S., Weiss, D. S., Newton, K., McBride, J., O'Rourke, K., Roose-Girma, M., Lee, W. P., Weinrauch, Y., Monack, D. M., Dixit, V. M. 2006; 440 (7081): 228-232

  Abstract

  A crucial part of the innate immune response is the assembly of the inflammasome, a cytosolic complex of proteins that activates caspase-1 to process the proinflammatory cytokines interleukin (IL)-1beta and IL-18. The adaptor protein ASC is essential for inflammasome function, binding directly to caspase-1 (refs 3, 4), but the triggers of this interaction are less clear. ASC also interacts with the adaptor cryopyrin (also known as NALP3 or CIAS1). Activating mutations in cryopyrin are associated with familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, diseases that are characterized by excessive production of IL-1beta. Here we show that cryopyrin-deficient macrophages cannot activate caspase-1 in response to Toll-like receptor agonists plus ATP, the latter activating the P2X7 receptor to decrease intracellular K+ levels. The release of IL-1beta in response to nigericin, a potassium ionophore, and maitotoxin, a potent marine toxin, was also found to be dependent on cryopyrin. In contrast to Asc-/- macrophages, cells deficient in the gene encoding cryopyrin (Cias1-/-) activated caspase-1 and secreted normal levels of IL-1beta and IL-18 when infected with Gram-negative Salmonella typhimurium or Francisella tularensis. Macrophages exposed to Gram-positive Staphylococcus aureus or Listeria monocytogenes, however, required both ASC and cryopyrin to activate caspase-1 and secrete IL-1beta. Therefore, cryopyrin is essential for inflammasome activation in response to signalling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L. monocytogenes.

  View details for DOI 10.1038/nature04515

  View details for Web of Science ID 000235839500050

  View details for PubMedID 16407890

• 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

  Abstract

  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

  View details for PubMedCentralID PMC1383486

• Yersinia virulence factor YopJ acts as a deubiquitinase to inhibit NF-kappa B activation JOURNAL OF EXPERIMENTAL MEDICINE Zhou, H. L., Monack, D. M., Kayagaki, N., Wertz, I., Yin, J. P., Wolf, B., Dixit, V. M. 2005; 202 (10): 1327-1332

  Abstract

  The bacterial pathogens of the genus Yersinia, the causative agents of plague, septicemia, and gastrointestinal syndromes, use a type III secretion system to inject virulence factors into host target cells. One virulence factor, YopJ, is essential for the death of infected macrophages and can block host proinflammatory responses by inhibiting both the nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinase pathways, which might be important for evasion of the host immune response and aid in establishing a systemic infection. Here, we show that YopJ is a promiscuous deubiquitinating enzyme that negatively regulates signaling by removing ubiquitin moieties from critical proteins, such as TRAF2, TRAF6, and IkappaBalpha. In contrast to the cylindromatosis tumor suppressor CYLD, which attenuates NF-kappaB signaling by selectively removing K63-linked polyubiquitin chains that activate IkappaB kinase, YopJ also cleaves K48-linked chains and thereby inhibits proteasomal degradation of IkappaBalpha. YopJ, but not a catalytically inactive YopJ mutant, promoted deubiquitination of cellular proteins and cleaved both K48- and K63-linked polyubiquitin. Moreover, an in vitro assay was established to demonstrate directly the deubiquitinating activity of purified YopJ.

  View details for Web of Science ID 000233429500003

  View details for PubMedID 16301742

  View details for PubMedCentralID PMC2212976

• Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis JOURNAL OF EXPERIMENTAL MEDICINE Mariathasan, S., Weiss, D. S., DIXIT, V. M., Monack, D. M. 2005; 202 (8): 1043-1049

  Abstract

  Francisella tularensis is a highly infectious gram-negative coccobacillus that causes the zoonosis tularemia. This bacterial pathogen causes a plague-like disease in humans after exposure to as few as 10 cells. Many of the mechanisms by which the innate immune system fights Francisella are unknown. Here we show that wild-type Francisella, which reach the cytosol, but not Francisella mutants that remain localized to the vacuole, induced a host defense response in macrophages, which is dependent on caspase-1 and the death-fold containing adaptor protein ASC. Caspase-1 and ASC signaling resulted in host cell death and the release of the proinflammatory cytokines interleukin (IL)-1beta and IL-18. F. tularensis-infected caspase-1- and ASC-deficient mice showed markedly increased bacterial burdens and mortality as compared with wild-type mice, demonstrating a key role for caspase-1 and ASC in innate defense against infection by this pathogen.

  View details for DOI 10.1084/jem.20050977

  View details for Web of Science ID 000232929500005

  View details for PubMedID 16230474

  View details for PubMedCentralID PMC2213215

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

  Abstract

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

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

  View details for Web of Science ID 000226457800024

  View details for PubMedID 15661016

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

  Abstract

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

  View details for DOI 10.1038/nrmicro955

  View details for Web of Science ID 000223627200019

  View details for PubMedID 15372085

• Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf NATURE Mariathasan, S., Newton, K., Monack, D. M., Vucic, D., French, D. M., Lee, W. P., Roose-Girma, M., Erickson, S., Dixit, V. M. 2004; 430 (6996): 213-218

  Abstract

  Specific adaptors regulate the activation of initiator caspases; for example, FADD and Apaf-1 engage caspases 8 and 9, respectively. The adaptors ASC, Ipaf and RIP2 have each been proposed to regulate caspase-1 (also called interleukin (IL)-1 converting enzyme), which is activated within the 'inflammasome', a complex comprising several adaptors. Here we show the impact of ASC-, Ipaf- or RIP2-deficiency on inflammasome function. ASC was essential for extracellular ATP-driven activation of caspase-1 in toll-like receptor (TLR)-stimulated macrophages. Accordingly, ASC-deficient macrophages exhibited defective maturation of IL-1beta and IL-18, and ASC-null mice were resistant to lipopolysaccharide-induced endotoxic shock. Furthermore, activation of caspase-1 in response to an intracellular pathogen (Salmonella typhimurium) was abrogated severely in ASC-null macrophages. Unexpectedly, Ipaf-deficient macrophages activated caspase-1 in response to TLR plus ATP stimulation but not S. typhimurium. Caspase-1 activation was not compromised by loss of RIP2. These data show that whereas ASC is key to caspase-1 activation within the inflammasome, Ipaf provides a special conduit to the inflammasome for signals triggered by intracellular pathogens. Notably, cell death triggered by stimuli that engage caspase-1 was ablated in macrophages lacking either ASC or Ipaf, suggesting a coupling between the inflammatory and cell death pathways.

  View details for DOI 10.1038/nature02664

  View details for Web of Science ID 000222470600045

  View details for PubMedID 15190255

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

  Abstract

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

  View details for DOI 10.1084/jem.20031319

  View details for Web of Science ID 000188369700009

  View details for PubMedID 14734525

  View details for PubMedCentralID PMC2211772

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

  Abstract

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

  View details for DOI 10.1128/IAI.71.6.3196-3205.2003

  View details for Web of Science ID 000183116300027

  View details for PubMedID 12761099

  View details for PubMedCentralID PMC155741

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

  Abstract

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

  View details for Web of Science ID 000182042800009

  View details for PubMedID 12675799

• The Salmonella-containing vacuole is a major site of intracellular cholesterol accumulation and recruits the GPI-anchored protein CD55 CELLULAR MICROBIOLOGY Catron, D. M., Sylvester, M. D., Lange, Y., Kadekoppala, M., Jones, B. D., Monack, D. M., FALKOW, S., Haldar, K. 2002; 4 (6): 315-328

  Abstract

  Intracellular, pathogenic Salmonella typhimurium avoids phago-lysosome fusion, and exists within a unique vacuolar niche that resembles a late endosome. This model has emerged from studying the trafficking of host proteins to the Salmonella-containing vacuole (SCV). Very little is known about the role of major host lipids during infection. Here, we show using biochemical analyses as well as fluorescence microscopy, that intracellular infection perturbs the host sterol biosynthetic pathway and induces cholesterol accumulation in the SCV. Cholesterol accumulation is seen in both macrophages and epithelial cells: at the terminal stages of infection, as much as 30% of the total cellular cholesterol resides in the SCV. We find that accumulation of cholesterol in the SCV is linked to intracellular bacterial replication and may be dependent on Salmonella pathogenicity island 2 (SPI-2). Furthermore, the construction of a three-dimensional space-filling model yields novel insights into the structure of the SCV: bacteria embedded in cholesterol-rich membranes. Finally, we show that the glycosylphosphatidylinositol (GPI)-anchored protein CD55 is recruited to the SCV. These data suggest that, in contrast to prevailing models, the SCV accumulates components of cholesterol-rich early endocytic pathways during intracellular bacterial replication.

  View details for Web of Science ID 000176214100001

  View details for PubMedID 12067317

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

  Abstract

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

  View details for Web of Science ID 000172601200005

  View details for PubMedID 11736994

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

  Abstract

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

  View details for Web of Science ID 000173168500004

  View details for PubMedID 11755408

• 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

  Abstract

  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

  Abstract

  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

• Differential regulation of Toll-like receptor mRNAs by microbes, their products & cytokines Zarember, K., Monack, D., Falkow, S., Godowski, P. FEDERATION AMER SOC EXP BIOL. 2001: 32

  View details for Web of Science ID 000171482200061

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

  Abstract

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

  View details for Web of Science ID 000088261100011

  View details for PubMedID 10899911

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

  Abstract

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

  View details for Web of Science ID 000086862900002

  View details for PubMedID 11043977

• Polarized localization of the Shigella flexneri IcsA protein McCallum, S. J., Vegas, A. J., Monack, D., Theriot, J. A. AMER SOC CELL BIOLOGY. 1999: 312A

  View details for Web of Science ID 000083673501805

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

  Abstract

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

  View details for Web of Science ID 000078956600106

  View details for PubMedID 10051653

  View details for PubMedCentralID PMC26795

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

  Abstract

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

  View details for Web of Science ID 000077484700016

  View details for PubMedID 9841926

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

  Abstract

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

  View details for Web of Science ID 000076538500015

  View details for PubMedID 9786194

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

  Abstract

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

  View details for Web of Science ID A1997XX39900070

  View details for PubMedID 9294220

• Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella Pathogenicity Island 2 MOLECULAR MICROBIOLOGY Hensel, M., Shea, J. E., Raupach, B., Monack, D., FALKOW, S., Gleeson, C., Kubo, T., Holden, D. W. 1997; 24 (1): 155-167

  Abstract

  We have investigated the structure and transcriptional organization of 13 genes of Salmonella Pathogenicity Island 2 (SPI2) that encode components of the second type III secretion apparatus of Salmonella typhimurium. ssaK, L, M, V, N, O, P, Q, R, S, T, U constitute one operon of 10 kb. ssaJ lles upstream of ssaK and is the terminal gene of another operon. The deduced products of ssaJ, ssaK, ssaV, ssaN, ssaO, ssaQ, ssaR, ssaS, ssaT, and ssaU show greatest similarity to the Yersinia spp. genes yscJ, yscL, lcrD, yscN, yscO, yscQ, yscR, yscS, yscT, and yscU, respectively. The products of the ssaL, ssaM and ssaP genes do not have significant similarity to products of other type III secretion systems, and might be important for the specific function of the SPI2 type III secretion system. Bacterial strains carrying different ssa mutations display minor alterations in terms of serum sensitivity when compared with the wild-type strain, but none are defective in replication within macrophage-like RAW 264.7 cells. However, some of the ssa mutant strains invade HEp2 cells less efficiently and are less cytotoxic to RAW 264.7 macrophages than the wild-type strain. We show that the invasion defect is correlated with a lack of SipC in culture supernatants of these mutant strains. SipC is a product of the SPI1 type III secretion system of S. typhimurium, and is important for epithelial cell invasion. Therefore, mutations in SPI2 can affect the SPI1 secretion system, which raises the possibility of an interaction between the two type III secretion systems.

  View details for Web of Science ID A1997WV18800014

  View details for PubMedID 9140973

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

  Abstract

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

  View details for Web of Science ID A1996VF61400093

  View details for PubMedID 8790417

• HOST RESTRICTION PHENOTYPES OF SALMONELLA-TYPHI AND SALMONELLA-GALLINARUM INFECTION AND IMMUNITY Pascopella, L., Raupach, B., Ghori, N., Monack, D., FALKOW, S., Small, P. L. 1995; 63 (11): 4329-4335

  Abstract

  Salmonella typhi and Salmonella gallinarum phenotypes correlated with mouse host restriction have been identified by using in vitro and in vivo systems. S. typhi is capable of entering the murine intestinal epithelium via M cells, as is Salmonella typhimurium, which causes systemic infection in the mouse. But, unlike S. typhimurium, S. typhi does not destroy the epithelium and is cleared from the Peyer's patches soon after M-cell entry. S. gallinarum appears to be incapable of entering the murine Peyer's patch epithelium. Our in vitro evidence suggests that S. gallinarum is taken up in murine phagocytic cells by a mechanism different from that of S. typhimurium. S. typhimurium is taken up at a higher frequency and is maintained at higher viable counts throughout a 24-h time course in a murine macrophage-like cell line than are S. gallinarum and S. typhi.

  View details for Web of Science ID A1995TB40000019

  View details for PubMedID 7591067

  View details for PubMedCentralID PMC173616

• THE BVGAS LOCUS NEGATIVELY CONTROLS MOTILITY AND SYNTHESIS OF FLAGELLA IN BORDETELLA-BRONCHISEPTICA JOURNAL OF BACTERIOLOGY Akerley, B. J., Monack, D. M., FALKOW, S., Miller, J. F. 1992; 174 (3): 980-990

  Abstract

  The products of the bvgAS locus coordinately regulate the expression of Bordetella virulence factors in response to environmental conditions. We have identified a phenotype in Bordetella bronchiseptica that is negatively controlled by bvg. Environmental signals which decrease (modulate) the expression of bvg-activated genes lead to flagellum production and motility in B. bronchiseptica. Wild-type (Bvg+) strains are motile and produce peritrichous flagella only in the presence of modulating signals, whereas Bvg- (delta bvgAS or delta bvgS) strains are motile in the absence of modulators. The bvgS-C3 mutation, which confers signal insensitivity and constitutive activation of positively controlled loci, eliminates the induction of motility and production of flagellar organelles. The response to environmental signals is conserved in a diverse set of clinical isolates of both B. bronchiseptica and B. avium, another motile Bordetella species; however, nicotinic acid induced motility only in B. bronchiseptica. Purification of flagellar filaments from B. bronchiseptica strains by differential centrifugation followed by CsCl equilibrium density gradient centrifugation revealed two classes of flagellins of Mr 35,000 and 40,000. A survey of clinical isolates identified only these two flagellin isotypes, and coexpression of the two forms was not detected in any strain. All B. avium strains tested expressed a 42,000-Mr flagellin. Amino acid sequence analysis of the two B. bronchiseptica flagellins revealed 100% identity in the N-terminal region and 80% identity with Salmonella typhimurium flagellin. Monoclonal antibody 15D8, which recognizes a conserved epitope in flagellins in members of the family Enterobacteriaceae, cross-reacted with flagellins from B. bronchiseptica and B. avium. Our results highlight the biphasic nature of the B. bronchiseptica bvg regulon and provide a preliminary characterization of the Bvg-regulated motility phenotype.

  View details for Web of Science ID A1992HE45900040

  View details for PubMedID 1370665

  View details for PubMedCentralID PMC206178

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

  Abstract

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

  View details for Web of Science ID A1991GL95400020

  View details for PubMedID 1791760

• PATHOGENESIS IN BORDETELLA SPECIES - REPLY JOURNAL OF INFECTIOUS DISEASES MONACK, D., FALKOW, S. 1990; 161 (3): 582–83

  View details for DOI 10.1093/infdis/161.3.582

  View details for Web of Science ID A1990CT66300036

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

  Abstract

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

  View details for Web of Science ID A1989AP19100049

  View details for PubMedID 2549542

  View details for PubMedCentralID PMC297907

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

  Abstract

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

  View details for Web of Science ID A1989T690600065

  View details for PubMedID 2537932

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

  Abstract

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

  View details for PubMedID 2908523