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John-Demian (JD) Sauer

Picture of John-Demian (JD) SauerAssistant Professor of Medical Microbiology & Immunology
4203 Microbial Sciences Building
1550 Linden Drive
Office: (608) 263-1529
Laboratory: 263-4230
Email: sauer3@wisc.edu
Overview · Personnel · Publications · Lab Website
  • Chen GY, McDougal CE, D'Antonio MA, Portman JL, Sauer JD (2017) A Genetic Screen Reveals that Synthesis of 1,4-Dihydroxy-2-Naphthoate (DHNA), but Not Full-Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival. MBio 8(2): (PMC5362031) View Abstract · Pubmed Record

    Through unknown mechanisms, the host cytosol restricts bacterial colonization; therefore, only professional cytosolic pathogens are adapted to colonize this host environment. Listeria monocytogenes is a Gram-positive intracellular pathogen that is highly adapted to colonize the cytosol of both phagocytic and nonphagocytic cells. To identify L. monocytogenes determinants of cytosolic survival, we designed and executed a novel screen to isolate L. monocytogenes mutants with cytosolic survival defects. Multiple mutants identified in the screen were defective for synthesis of menaquinone (MK), an essential molecule in the electron transport chain. Analysis of an extensive set of MK biosynthesis and respiratory chain mutants revealed that cellular respiration was not required for cytosolic survival of L. monocytogenes but that, instead, synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an MK biosynthesis intermediate, was essential. Recent discoveries showed that modulation of the central metabolism of both host and pathogen can influence the outcome of host-pathogen interactions. Our results identify a potentially novel function of the MK biosynthetic intermediate DHNA and specifically highlight how L. monocytogenes metabolic adaptations promote cytosolic survival and evasion of host immunity.IMPORTANCE Cytosolic bacterial pathogens, such as Listeria monocytogenes and Francisella tularensis, are exquisitely evolved to colonize the host cytosol in a variety of cell types. Establishing an intracellular niche shields these pathogens from effectors of humoral immunity, grants access to host nutrients, and is essential for pathogenesis. Through yet-to-be-defined mechanisms, the host cytosol restricts replication of non-cytosol-adapted bacteria, likely through a combination of cell autonomous defenses (CADs) and nutritional immunity. Utilizing a novel genetic screen, we identified determinants of L. monocytogenes cytosolic survival and virulence and identified a role for the synthesis of the menaquinone precursor 1,4-dihydroxy-2-naphthoate (DHNA) in cytosolic survival. Together, these data begin to elucidate adaptations that allow cytosolic pathogens to survive in their intracellular niches.

  • Theisen E, Sauer JD (2017) Listeria monocytogenes-Induced Cell Death Inhibits the Generation of Cell-Mediated Immunity. Infect. Immun. 85(1): (PMC5203660) View Abstract · Pubmed Record

    The influence of cell death on adaptive immunity has been studied for decades. Despite these efforts, the intricacies of how various cell death pathways shape immune responses in the context of infection remain unclear, particularly with regard to more recently discovered pathways such as pyroptosis. The emergence of Listeria monocytogenes as a promising immunotherapeutic platform demands a thorough understanding of how cell death induced in the context of infection influences the generation of CD8(+) T-cell-mediated immune responses. To begin to address this question, we designed strains of L. monocytogenes that robustly activate necrosis, apoptosis, or pyroptosis. We hypothesized that proinflammatory cell death such as necrosis would be proimmunogenic while apoptosis would be detrimental, as has previously been reported in the context of sterile cell death. Surprisingly, we found that the activation of any host cell death in the context of L. monocytogenes infection inhibited the generation of protective immunity and specifically the activation of antigen-specific CD8(+) T cells. Importantly, the mechanism of attenuation was unique for each type of cell death, ranging from deficits in costimulation in the context of necrosis to a suboptimal inflammatory milieu in the case of pyroptosis. Our results suggest that cell death in the context of infection is different from sterile-environment-induced cell death and that inhibition of cell death or its downstream consequences is necessary for developing effective cell-mediated immune responses using L. monocytogenes-based immunotherapeutic platforms.

  • Pensinger DA, Boldon KM, Chen GY, Vincent WJ, Sherman K, Xiong M, Schaenzer AJ, Forster ER, Coers J, Striker R, Sauer JD (2016) The Listeria monocytogenes PASTA Kinase PrkA and Its Substrate YvcK Are Required for Cell Wall Homeostasis, Metabolism, and Virulence. PLoS Pathog. 12(11):e1006001 (PMC5091766) View Abstract · Pubmed Record

    Obstacles to bacterial survival and replication in the cytosol of host cells, and the mechanisms used by bacterial pathogens to adapt to this niche are not well understood. Listeria monocytogenes is a well-studied Gram-positive foodborne pathogen that has evolved to invade and replicate within the host cell cytosol; yet the mechanisms by which it senses and responds to stress to survive in the cytosol are largely unknown. To assess the role of the L. monocytogenes penicillin-binding-protein and serine/threonine associated (PASTA) kinase PrkA in stress responses, cytosolic survival and virulence, we constructed a ΔprkA deletion mutant. PrkA was required for resistance to cell wall stress, growth on cytosolic carbon sources, intracellular replication, cytosolic survival, inflammasome avoidance and ultimately virulence in a murine model of Listeriosis. In Bacillus subtilis and Mycobacterium tuberculosis, homologues of PrkA phosphorylate a highly conserved protein of unknown function, YvcK. We found that, similar to PrkA, YvcK is also required for cell wall stress responses, metabolism of glycerol, cytosolic survival, inflammasome avoidance and virulence. We further demonstrate that similar to other organisms, YvcK is directly phosphorylated by PrkA, although the specific site(s) of phosphorylation are not highly conserved. Finally, analysis of phosphoablative and phosphomimetic mutants of YvcK in vitro and in vivo demonstrate that while phosphorylation of YvcK is irrelevant to metabolism and cell wall stress responses, surprisingly, a phosphomimetic, nonreversible negative charge of YvcK is detrimental to cytosolic survival and virulence in vivo. Taken together our data identify two novel virulence factors essential for cytosolic survival and virulence of L. monocytogenes. Furthermore, our data demonstrate that regulation of YvcK phosphorylation is tightly controlled and is critical for virulence. Finally, our data suggest that yet to be identified substrates of PrkA are essential for cytosolic survival and virulence of L. monocytogenes and illustrate the importance of studying protein phosphorylation in the context of infection.

  • Felley LE, Sharma A, Theisen E, Romero-Masters JC, Sauer JD, Gumperz JE (2016) Human Invariant NKT Cells Induce IL-1β Secretion by Peripheral Blood Monocytes via a P2X7-Independent Pathway. J. Immunol. 197(6):2455-64 (PMC5011004) View Abstract · Pubmed Record

    The cytokine IL-1β plays a central role in inflammatory responses that are initiated by microbial challenges, as well as in those that are due to endogenous processes (often called sterile inflammation). IL-1β secretion that occurs independently of microbial stimulation is typically associated with the presence of endogenous alarmins, such as extracellular ATP (an indicator of cytopathic damage). In this study, we show that IL-2-activated human invariant NKT (iNKT) cells stimulate the secretion of IL-1β protein by human peripheral blood monocytes in a manner that requires neither the presence of microbial compounds nor signaling through the extracellular ATP receptor P2X7 Monocyte IL-1β production was specifically induced by iNKT cells, because similarly activated polyclonal autologous T cells did not have this effect. Secretion of IL-1β protein occurred rapidly (within 3-4 h) and required cell contact between the iNKT cells and monocytes. Similar to IL-1β production induced by TLR stimulation, the iNKT-induced pathway appeared to entail a two-step process involving NF-κB signaling and IL1B gene transcription, as well as assembly of the NLRP3 inflammasome and activation of caspase-1. However, in contrast to the classical inflammasome-mediated pathway of IL-1β production, activation of monocytes via P2X7 was dispensable for iNKT-induced IL-1β secretion, and potassium efflux was not required. Moreover, the iNKT-induced effect involved caspase-8 activity, yet it induced little monocyte death. These results suggest that IL-2-activated human iNKT cells induce monocytes to produce IL-1β through a distinctive pathway that does not require the presence of microbial danger signals or alarmins associated with cytopathic damage.

  • Theisen E, Sauer JD (2016) Listeria monocytogenes and the Inflammasome: From Cytosolic Bacteriolysis to Tumor Immunotherapy. Curr. Top. Microbiol. Immunol. 397:133-60 (PMC5027901) View Abstract · Pubmed Record

    Inflammasomes are cytosolic innate immune surveillance systems that recognize a variety of danger signals, including those from pathogens. Listeria monocytogenes is a Gram-positive intracellular bacterium evolved to live within the harsh environment of the host cytosol. Further, L. monocytogenes can activate a robust cell-mediated immune response that is being harnessed as an immunotherapeutic platform. Access to the cytosol is critical for both causing disease and inducing a protective immune response, and it is hypothesized that the cytosolic innate immune system, including the inflammasome, is critical for both host protection and induction of long-term immunity. L. monocytogenes can activate a variety of inflammasomes via its pore-forming toxin listeriolysin-O, flagellin, or DNA released through bacteriolysis; however, inflammasome activation attenuates L. monocytogenes, and as such, L. monocytogenes has evolved a variety of ways to limit inflammasome activation. Surprisingly, inflammasome activation also impairs the host cell-mediated immune response. Thus, understanding how L. monocytogenes activates or avoids detection by the inflammasome is critical to understand the pathogenesis of L. monocytogenes and improve the cell-mediated immune response generated to L. monocytogenes for more effective immunotherapies.

  • Vincent WJ, Freisinger CM, Lam PY, Huttenlocher A, Sauer JD (2016) Macrophages mediate flagellin induced inflammasome activation and host defense in zebrafish. Cell. Microbiol. 18(4):591-604 (PMC5027955) View Abstract · Pubmed Record

    The inflammasome is an innate immune complex whose rapid inflammatory outputs play a critical role in controlling infection; however, the host cells that mediate inflammasome responses in vivo are not well defined. Using zebrafish larvae, we examined the cellular immune responses to inflammasome activation during infection. We compared the host responses with two Listeria monocytogenes strains: wild type and Lm-pyro, a strain engineered to activate the inflammasome via ectopic expression of flagellin. Infection with Lm-pyro led to activation of the inflammasome, macrophage pyroptosis and ultimately attenuation of virulence. Depletion of caspase A, the zebrafish caspase-1 homolog, restored Lm-pyro virulence. Inflammasome activation specifically recruited macrophages to infection sites, whereas neutrophils were equally recruited to wild type and Lm-pyro infections. Similar to caspase A depletion, macrophage deficiency rescued Lm-pyro virulence to wild-type levels, while defective neutrophils had no specific effect. Neutrophils were, however, important for general clearance of L. monocytogenes, as both wild type and Lm-pyro were more virulent in larvae with defective neutrophils. This study characterizes a novel model for inflammasome studies in an intact host, establishes the importance of macrophages during inflammasome responses and adds importance to the role of neutrophils in controlling L. monocytogenes infections.

  • Berti AD, Theisen E, Sauer JD, Nonejuie P, Olson J, Pogliano J, Sakoulas G, Nizet V, Proctor RA, Rose WE (2015) Penicillin Binding Protein 1 Is Important in the Compensatory Response of Staphylococcus aureus to Daptomycin-Induced Membrane Damage and Is a Potential Target for β-Lactam-Daptomycin Synergy. Antimicrob. Agents Chemother. 60(1):451-8 (PMC4704217) View Abstract · Pubmed Record

    The activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA) is enhanced in the presence of β-lactam antibiotics. This effect is more pronounced with β-lactam antibiotics that exhibit avid binding to penicillin binding protein 1 (PBP1). Here, we present evidence that PBP1 has a significant role in responding to DAP-induced stress on the cell. Expression of the pbpA transcript, encoding PBP1, was specifically induced by DAP exposure whereas expression of pbpB, pbpC, and pbpD, encoding PBP2, PBP3, and PBP4, respectively, remained unchanged. Using a MRSA COL strain with pbpA under an inducible promoter, increased pbpA transcription was accompanied by reduced susceptibility to, and killing by, DAP in vitro. Exposure to β-lactams that preferentially inactivate PBP1 was not associated with increased DAP binding, suggesting that synergy in the setting of anti-PBP1 pharmacotherapy results from increased DAP potency on a per-molecule basis. Combination exposure in an in vitro pharmacokinetic/pharmacodynamic model system with β-lactams that preferentially inactivate PBP1 (DAP-meropenem [MEM] or DAP-imipenem [IPM]) resulted in more-rapid killing than did combination exposure with DAP-nafcillin (NAF) (nonselective), DAP-ceftriaxone (CRO) or DAP-cefotaxime (CTX) (PBP2 selective), DAP-cefaclor (CEC) (PBP3 selective), or DAP-cefoxitin (FOX) (PBP4 selective). Compared to β-lactams with poor PBP1 binding specificity, exposure of S. aureus to DAP plus PBP1-selective β-lactams resulted in an increased frequency of septation and cell wall abnormalities. These data suggest that PBP1 activity may contribute to survival during DAP-induced metabolic stress. Therefore, targeted inactivation of PBP1 may enhance the antimicrobial efficiency of DAP, supporting the use of DAP-β-lactam combination therapy for serious MRSA infections, particularly when the β-lactam undermines the PBP1-mediated compensatory response.

  • Huynh TN, Luo S, Pensinger D, Sauer JD, Tong L, Woodward JJ (2015) An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence. Proc. Natl. Acad. Sci. U.S.A. 112(7):E747-56 (PMC4343097) View Abstract · Pubmed Record

    The nucleotide cyclic di-3',5'- adenosine monophosphate (c-di-AMP) was recently identified as an essential and widespread second messenger in bacterial signaling. Among c-di-AMP-producing bacteria, altered nucleotide levels result in several physiological defects and attenuated virulence. Thus, a detailed molecular understanding of c-di-AMP metabolism is of both fundamental and practical interest. Currently, c-di-AMP degradation is recognized solely among DHH-DHHA1 domain-containing phosphodiesterases. Using chemical proteomics, we identified the Listeria monocytogenes protein PgpH as a molecular target of c-di-AMP. Biochemical and structural studies revealed that the PgpH His-Asp (HD) domain bound c-di-AMP with high affinity and specifically hydrolyzed this nucleotide to 5'-pApA. PgpH hydrolysis activity was inhibited by ppGpp, indicating a cross-talk between c-di-AMP signaling and the stringent response. Genetic analyses supported coordinated regulation of c-di-AMP levels in and out of the host. Intriguingly, a L. monocytogenes mutant that lacks c-di-AMP phosphodiesterases exhibited elevated c-di-AMP levels, hyperinduced a host type-I IFN response, and was significantly attenuated for infection. Furthermore, PgpH homologs, which belong to the 7TMR-HD family, are widespread among hundreds of c-di-AMP synthesizing microorganisms. Thus, PgpH represents a broadly conserved class of c-di-AMP phosphodiesterase with possibly other physiological functions in this crucial signaling network.

  • Sureka K, Choi PH, Precit M, Delince M, Pensinger DA, Huynh TN, Jurado AR, Goo YA, Sadilek M, Iavarone AT, Sauer JD, Tong L, Woodward JJ (2014) The cyclic dinucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function. Cell 158(6):1389-401 (PMC4166403) View Abstract · Pubmed Record

    Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP-interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected catalytic activity of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic dinucleotide.

  • Pensinger DA, Aliota MT, Schaenzer AJ, Boldon KM, Ansari IU, Vincent WJ, Knight B, Reniere ML, Striker R, Sauer JD (2014) Selective pharmacologic inhibition of a PASTA kinase increases Listeria monocytogenes susceptibility to β-lactam antibiotics. Antimicrob. Agents Chemother. 58(8):4486-94 (PMC4135996) View Abstract · Pubmed Record

    While β-lactam antibiotics are a critical part of the antimicrobial arsenal, they are frequently compromised by various resistance mechanisms, including changes in penicillin binding proteins of the bacterial cell wall. Genetic deletion of the penicillin binding protein and serine/threonine kinase-associated protein (PASTA) kinase in methicillin-resistant Staphylococcus aureus (MRSA) has been shown to restore β-lactam susceptibility. However, the mechanism remains unclear, and whether pharmacologic inhibition would have the same effect is unknown. In this study, we found that deletion or pharmacologic inhibition of the PASTA kinase in Listeria monocytogenes by the nonselective kinase inhibitor staurosporine results in enhanced susceptibility to both aminopenicillin and cephalosporin antibiotics. Resistance to vancomycin, another class of cell wall synthesis inhibitors, or antibiotics that inhibit protein synthesis was unaffected by staurosporine treatment. Phosphorylation assays with purified kinases revealed that staurosporine selectively inhibited the PASTA kinase of L. monocytogenes (PrkA). Importantly, staurosporine did not inhibit a L. monocytogenes kinase without a PASTA domain (Lmo0618) or the PASTA kinase from MRSA (Stk1). Finally, inhibition of PrkA with a more selective kinase inhibitor, AZD5438, similarly led to sensitization of L. monocytogenes to β-lactam antibiotics. Overall, these results suggest that pharmacologic targeting of PASTA kinases can increase the efficacy of β-lactam antibiotics.

  • Fonseca MV, Sauer JD, Crepin S, Byrne B, Swanson MS (2014) The phtC-phtD locus equips Legionella pneumophila for thymidine salvage and replication in macrophages. Infect. Immun. 82(2):720-30 (PMC3911408) View Abstract · Pubmed Record

    The phagosomal transporter (Pht) family of the major facilitator superfamily (MFS) is encoded by phylogenetically related intracellular gammaproteobacteria, including the opportunistic pathogen Legionella pneumophila. The location of the pht genes between the putative thymidine kinase (tdk) and phosphopentomutase (deoB) genes suggested that the phtC and phtD loci contribute to thymidine salvage in L. pneumophila. Indeed, a phtC(+) allele in trans restored pyrimidine uptake to an Escherichia coli mutant that lacked all known nucleoside transporters, whereas a phtD(+) allele did not. The results of phenotypic analyses of L. pneumophila strains lacking phtC or phtD strongly indicate that L. pneumophila requires PhtC and PhtD function under conditions where sustained dTMP synthesis is compromised. First, in broth cultures that mimicked thymidine limitation or starvation, L. pneumophila exhibited a marked requirement for PhtC function. Conversely, mutation of phtD conferred a survival advantage. Second, in medium that lacked thymidine, multicopy phtC(+) or phtD(+) alleles enhanced the survival of L. pneumophila thymidylate synthase (thyA)-deficient strains, which cannot synthesize dTMP endogenously. Third, under conditions in which transport of the pyrimidine nucleoside analog 5-fluorodeoxyuridine (FUdR) would inhibit growth, PhtC and PhtD conferred a growth advantage to L. pneumophila thyA(+) strains. Finally, when cultured in macrophages, L. pneumophila required the phtC-phtD locus to replicate. Accordingly, we propose that PhtC and PhtD contribute to protect L. pneumophila from dTMP starvation during its intracellular life cycle.

  • Williams CR, Dustin ML, Sauer JD (2013) Inflammasome-mediated inhibition of Listeria monocytogenes-stimulated immunity is independent of myelomonocytic function. PLoS ONE 8(12):e83191 (PMC3857309) View Abstract · Pubmed Record

    Activation of the Nlrc4 inflammasome results in the secretion of IL-1β and IL-18 through caspase-1 and induction of pyroptosis. L. monocytogenes engineered to activate Nlrc4 by expression of Legionella pneumophilia flagellin (L. monocytogenes L.p.FlaA) are less immunogenic for CD8(+) T cell responses than wt L. monocytogenes. It is also known that IL-1β orchestrates recruitment of myelomonocytic cells (MMC), which have been shown to interfere with T cell-dendritic cells (DC) interactions in splenic white pulp (WP), limiting T cell priming and protective immunity. We have further analyzed the role of MMCs in the immunogenicity of L. monocytogenes L.p.FlaA. We confirmed that MMCs infiltrate the WP between 24-48 hours in response to wt L. monocytogenes infection and that depletion of MMCs enhances CD8(+) T cell priming and protective memory. L. monocytogenes L.p.FlaA elicited accelerated recruitment of MMCs into the WP. While MMCs contribute to control of L. monocytogenes L.p.FlaA, MMC depletion did not increase immunogenicity of L.p.FlaA expressing strains. There was a significant decrease in L. monocytogenes L.p.FlaA in CD8α(+) DCs independent of MMCs. These findings suggest that limiting inflammasome activation is important for bacterial accumulation in CD8α(+) DCs, which are known to be critical for T cell response to L. monocytogenes.

  • Witte CE, Whiteley AT, Burke TP, Sauer JD, Portnoy DA, Woodward JJ (2013) Cyclic di-AMP is critical for Listeria monocytogenes growth, cell wall homeostasis, and establishment of infection. MBio 4(3):e00282-13 (PMC3663569) View Abstract · Pubmed Record

    Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection.

  • Witte CE, Archer KA, Rae CS, Sauer JD, Woodward JJ, Portnoy DA (2012) Innate immune pathways triggered by Listeria monocytogenes and their role in the induction of cell-mediated immunity. Adv. Immunol. 113:135-56 View Abstract · Pubmed Record

    Acquired cell-mediated immunity to Listeria monocytogenes is induced by infection with live, replicating bacteria that grow in the host cell cytosol, whereas killed bacteria, or those trapped in a phagosome, fail to induce protective immunity. In this chapter, we focus on how L. monocytogenes is sensed by the innate immune system, with the presumption that innate immunity affects the development of acquired immunity. Infection by L. monocytogenes induces three innate immune pathways: an MyD88-dependent pathway emanating from a phagosome leading to expression of inflammatory cytokines; a STING/IRF3-dependent pathway emanating from the cytosol leading to the expression of IFN-β and coregulated genes; and very low levels of a Caspase-1-dependent, AIM2-dependent inflammasome pathway resulting in proteolytic activation and secretion of IL-1β and IL-18 and pyroptotic cell death. Using a combination of genetics and biochemistry, we identified the listerial ligand that activates the STING/IRF3 pathway as secreted cyclic diadenosine monophosphate, a newly discovered conserved bacterial signaling molecule. We also identified L. monocytogenes mutants that caused robust inflammasome activation due to bacteriolysis in the cytosol, release of DNA, and activation of the AIM2 inflammasome. A strain was constructed that ectopically expressed and secreted a fusion protein containing Legionella pneumophila flagellin that robustly activated the Nlrc4-dependent inflammasome and was highly attenuated in mice, also in an Nlrc4-dependent manner. Surprisingly, this strain was a poor inducer of adaptive immunity, suggesting that inflammasome activation is not necessary to induce cell-mediated immunity and may even be detrimental under some conditions. To the best of our knowledge, no single innate immune pathway is necessary to mount a robust acquired immune response to L. monocytogenes infection.

  • Sauer JD, Pereyre S, Archer KA, Burke TP, Hanson B, Lauer P, Portnoy DA (2011) Listeria monocytogenes engineered to activate the Nlrc4 inflammasome are severely attenuated and are poor inducers of protective immunity. Proc. Natl. Acad. Sci. U.S.A. 108(30):12419-24 (PMC3145703) View Abstract · Pubmed Record

    Inflammasomes are intracellular multiprotein signaling complexes that activate Caspase-1, leading to the cleavage and secretion of IL-1β and IL-18, and ultimately host cell death. Inflammasome activation is a common cellular response to infection; however, the consequences of inflammasome activation during acute infection and in the development of long-term protective immunity is not well understood. To investigate the role of the inflammasome in vivo, we engineered a strain of Listeria monocytogenes that ectopically expresses Legionella pneumophila flagellin, a potent activator of the Nlrc4 inflammasome. Compared with wild-type L. monocytogenes, strains that ectopically secreted flagellin induced robust host cell death and IL-1β secretion. These strains were highly attenuated both in bone marrow-derived macrophages and in vivo compared with wild-type L. monocytogenes. Attenuation in vivo was dependent on Nlrc4, but independent of IL-1β/IL-18 or neutrophil activity. L. monocytogenes strains that activated the inflammasome generated significantly less protective immunity, a phenotype that correlated with decreased induction of antigen-specific T cells. Our data suggest that avoidance of inflammasome activation is a critical virulence strategy for intracellular pathogens, and that activation of the inflammasome leads to decreased long-term protective immunity and diminished T-cell responses.

  • Lightfield KL, Persson J, Trinidad NJ, Brubaker SW, Kofoed EM, Sauer JD, Dunipace EA, Warren SE, Miao EA, Vance RE (2011) Differential requirements for NAIP5 in activation of the NLRC4 inflammasome. Infect. Immun. 79(4):1606-14 (PMC3067536) View Abstract · Pubmed Record

    Inflammasomes are cytosolic multiprotein complexes that assemble in response to infectious or noxious stimuli and activate the CASPASE-1 protease. The inflammasome containing the nucleotide binding domain-leucine-rich repeat (NBD-LRR) protein NLRC4 (interleukin-converting enzyme protease-activating factor [IPAF]) responds to the cytosolic presence of bacterial proteins such as flagellin or the inner rod component of bacterial type III secretion systems (e.g., Salmonella PrgJ). In some instances, such as infection with Legionella pneumophila, the activation of the NLRC4 inflammasome requires the presence of a second NBD-LRR protein, NAIP5. NAIP5 also is required for NLRC4 activation by the minimal C-terminal flagellin peptide, which is sufficient to activate NLRC4. However, NLRC4 activation is not always dependent upon NAIP5. In this report, we define the molecular requirements for NAIP5 in the activation of the NLRC4 inflammasome. We demonstrate that the N terminus of flagellin can relieve the requirement for NAIP5 during the activation of the NLRC4 inflammasome. We also demonstrate that NLRC4 responds to the Salmonella protein PrgJ independently of NAIP5. Our results indicate that NAIP5 regulates the apparent specificity of the NLRC4 inflammasome for distinct bacterial ligands.

  • Sauer JD, Sotelo-Troha K, von Moltke J, Monroe KM, Rae CS, Brubaker SW, Hyodo M, Hayakawa Y, Woodward JJ, Portnoy DA, Vance RE (2011) The N-ethyl-N-nitrosourea-induced Goldenticket mouse mutant reveals an essential function of Sting in the in vivo interferon response to Listeria monocytogenes and cyclic dinucleotides. Infect. Immun. 79(2):688-94 (PMC3028833) View Abstract · Pubmed Record

    Type I interferons (IFNs) are central regulators of the innate and adaptive immune responses to viral and bacterial infections. Type I IFNs are induced upon cytosolic detection of microbial nucleic acids, including DNA, RNA, and the bacterial second messenger cyclic-di-GMP (c-di-GMP). In addition, a recent study demonstrated that the intracellular bacterial pathogen Listeria monocytogenes stimulates a type I IFN response due to cytosolic detection of bacterially secreted c-di-AMP. The transmembrane signaling adaptor Sting (Tmem173, Mita, Mpys, Eris) has recently been implicated in the induction of type I IFNs in response to cytosolic DNA and/or RNA. However, the role of Sting in response to purified cyclic dinucleotides or during in vivo L. monocytogenes infection has not been addressed. In order to identify genes important in the innate immune response, we have been conducting a forward genetic mutagenesis screen in C57BL/6 mice using the mutagen N-ethyl-N-nitrosourea (ENU). Here we describe a novel mutant mouse strain, Goldenticket (Gt), that fails to produce type I IFNs upon L. monocytogenes infection. By genetic mapping and complementation experiments, we found that Gt mice harbor a single nucleotide variant (T596A) of Sting that functions as a null allele and fails to produce detectable protein. Analysis of macrophages isolated from Gt mice revealed that Sting is absolutely required for the type I interferon response to both c-di-GMP and c-di-AMP. Additionally, Sting is required for the response to c-di-GMP and L. monocytogenes in vivo. Our results provide new functions for Sting in the innate interferon response to pathogens.

  • Sauer JD, Witte CE, Zemansky J, Hanson B, Lauer P, Portnoy DA (2010) Listeria monocytogenes triggers AIM2-mediated pyroptosis upon infrequent bacteriolysis in the macrophage cytosol. Cell Host Microbe 7(5):412-9 (PMC2947455) View Abstract · Pubmed Record

    A host defense strategy against pathogens is the induction of cell death, thereby eliminating the pathogen's intracellular niche. Pyroptosis, one such form of cell death, is dependent on inflammasome activation. In a genetic screen to identify Listeria monocytogenes mutants that induced altered levels of host cell death, we identified a mutation in lmo2473 that caused hyperstimulation of IL-1beta secretion and pyroptosis following bacteriolysis in the macrophage cytosol. In addition, strains engineered to lyse in the cytosol by expression of both bacteriophage holin and lysin or induced to lyse by treatment with ampicillin stimulated pyroptosis. Pyroptosis was independent of the Nlrp3 and Nlrc4 inflammasome receptors but dependent on the inflammasome adaptor ASC and the cytosolic DNA sensor AIM2. Importantly, wild-type L. monocytogenes were also found to lyse, albeit at low levels, and trigger AIM2-dependent pyroptosis. These data suggested that pyroptosis is triggered by bacterial DNA released during cytosolic lysis.

  • Henry T, Kirimanjeswara GS, Ruby T, Jones JW, Peng K, Perret M, Ho L, Sauer JD, Iwakura Y, Metzger DW, Monack DM (2010) Type I IFN signaling constrains IL-17A/F secretion by gammadelta T cells during bacterial infections. J. Immunol. 184(7):3755-67 (PMC2879132) View Abstract · Pubmed Record

    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.

  • Chen DE, Podell S, Sauer JD, Swanson MS, Saier MH (2008) The phagosomal nutrient transporter (Pht) family. Microbiology (Reading, Engl.) 154(Pt 1):42-53 View Abstract · Pubmed Record

    Phagosomal transporters (Phts), required for intracellular growth of Legionella pneumophila, comprise a novel family of multispanning alpha-helical proteins within the major facilitator superfamily (MFS). The members of this family derive exclusively from bacteria. Multiple paralogues are present in a restricted group of Alpha- and Gammaproteobacteria, but single members were also found in Chlamydia and Cyanobacteria. Their protein sequences were aligned, yielding a phylogenetic tree showing the relations of the proteins to each other. Topological analyses revealed a probable 12 alpha-helical transmembrane segment (TMS) topology. Motif identification and statistical analyses provided convincing evidence that these proteins arose from a six TMS precursor by intragenic duplication. The phylogenetic tree revealed some potential orthologous relationships, suggestive of common function. However, several probable examples of lateral transfer of the encoding genetic material between bacteria were identified and analysed. The Pht family most closely resembles a smaller MFS family (the UMF9 family) with no functionally characterized members. However, the UMF9 family occurs in a broader range of prokaryotic organism types, including Archaea. These two families differ in that organisms bearing members of the Pht family often have numerous paralogues, whereas organisms bearing members of the UMF9 family never have more than two. This work serves to characterize two novel families within the MFS and provides compelling evidence for horizontal transfer of some of the family members.