Identification of a Second Listeria SecA Gene Associated with Protein Secretion and the Rough Phenotype

Molecular Microbiology. Aug, 2002  |  Pubmed ID: 12180923

We describe the identification and characterization of a second secA gene in Listeria monocytogenes. This gene, termed secA2, is involved in smooth-rough phenotypic variation and secA2 expression contributes to bacterial virulence. Spontaneous rough (R-) variants of L. monocytogenes grow in chains and form rough colonies on solid media. A subset of R-variants, classified here as type I, also shows reduced secretion of an autolysin, p60. We find that disruptions and in frame deletions in secA2 confer phenotypes identical to those of spontaneous type I R-variants. Additionally, the secA2 genes from two spontaneous type I R-variants encoded truncated SecA2 proteins. Mutations were not found in the secA2 genes from the remaining five independent R-variants, four of which showed a distinct (type II) rough morphology and secreted wild-type levels of p60. Expression of an epitope-tagged SecA2 in the DeltasecA2 strain and a spontaneous R-variant restored normal cell septation and smooth colony morphology. These data suggest that mutations in both secA2 and other genes contribute to smooth-rough phase variation in L. monocytogenes. Expression of the full-length SecA2 also promotes secretion of p60 and a set of additional L. monocytogenes proteins. We hypothesize that SecA2-dependent protein secretion plays a role in the colonization of environmental and host surfaces.

SecA2-dependent Secretion of Autolytic Enzymes Promotes Listeria Monocytogenes Pathogenesis

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2003  |  Pubmed ID: 14527997

Pathogenic bacteria secrete proteins that promote invasion of host tissues and resistance to immune responses. However, secretion mechanisms that contribute to the enormous morbidity and mortality of Gram-positive bacteria are largely undefined. An auxiliary protein secretion system (SecA2) has recently emerged in Listeria monocytogenes and eight other Gram-positive pathogens. Here, a proteomics approach identified seventeen SecA2-dependent secreted and surface proteins of L. monocytogenes, the two most abundant of which [the p60 and N-acetylmuramidase (NamA) autolysins] hydrolyze bacterial peptidoglycan (PGN) and contribute to host colonization. SecA2-deficient (DeltaSecA2) bacteria were rapidly cleared after systemic infection of murine hosts, and in cultured cells showed reduced cell-cell spread. p60 or NamA deficiencies (Deltap60 and DeltaNamA) caused intermediate reductions in bacterial virulence in vivo, yet showed no defect for infection of cultured cells. Restoration of virulence in Deltap60 bacteria required full-length p60 with an intact catalytic domain, suggesting that PGN hydrolysis by p60 is crucial for L. monocytogenes virulence. Coordinated PGN hydrolysis by p60 and NamA activities is predicted to generate a muramyl glycopeptide, glucosaminylmuramyl dipeptide (GMDP), which is known to modify host inflammatory responses. Thus, SecA2-dependent secretion may promote release of muramyl peptides that subvert host pattern recognition.

Promiscuity of MHC Class Ib-restricted T Cell Responses

Journal of Immunology (Baltimore, Md. : 1950). Dec, 2003  |  Pubmed ID: 14634106

Murine infection with the Gram-positive intracellular bacterium Listeria monocytogenes activates CD8(+) T cells that recognize bacterially derived N-formyl methionine peptides in the context of H2-M3 MHC class Ib molecules. Three peptides, fMIGWII, fMIVIL, and fMIVTLF, are targets of L. monocytogenes-specific CD8(+) T cells. To investigate epitope cross-recognition by H2-M3-restricted CD8(+) T cells, we deleted the sequence encoding fMIGWII from a virulent strain of L. monocytogenes. Infection with fMIGWII-deficient L. monocytogenes unexpectedly primed CD8(+) T cells that stain with fMIGWII/H2-M3 tetramers and lyse fMIGWII-coated target cells in vivo. Because the fMIGWII sequence is nonredundant, we speculated that other bacterially derived Ags are priming these responses. HPLC peptide fractionation of bacterial culture supernatants revealed several distinct L. monocytogenes-derived peptides that are recognized by fMIGWII-specific T cells. Our results demonstrate that the dominant H2-M3-restricted CD8(+) T cell population, although reactive with fMIGWII, is primed by other, non-fMIGWII peptides derived from L. monocytogenes. Although this degree of Ag receptor promiscuity is unusual for the adaptive immune system, it may be a more common feature of T cell responses restricted by nonpolymorphic MHC class Ib molecules.

TRAIL-R As a Negative Regulator of Innate Immune Cell Responses

Immunity. Dec, 2004  |  Pubmed ID: 15589175

TRAIL receptor (TRAIL-R) signaling has been implicated in inducing apoptosis in tumor cells, but little is understood about its physiological function. Here, we report the generation and characterization of TRAIL-R(-/-) mice, which develop normal lymphocyte populations but possess enhanced innate immune responses. TRAIL-R(-/-) mice exhibited increased clearance of murine cytomegalovirus that correlated with increased levels of IL-12, IFN-alpha, and IFN-gamma. Stimulation of macrophages with Mycobacterium and Toll-like receptor (TLR)-2, -3, and -4, but not TLR9, ligands resulted in high levels of TRAIL upregulation and enhanced cytokine production in TRAIL-R(-/-) cells. The immediate-early TLR signaling events in TRAIL-R(-/-) macrophages and dendritic cells are normal, but I kappa B-alpha homeostatic regulation and NF-kappa B activity at later time points is perturbed. These data suggest that TRAIL-R negatively regulates innate immune responses.

Cytosolic Expression of SecA2 is a Prerequisite for Long-term Protective Immunity

Cellular Microbiology. Jun, 2007  |  Pubmed ID: 17244189

Induction of efficient adaptive T cell-mediated immunity against the intracellular bacterium Listeria monocytogenes requires its successful invasion of host cell cytosol. However, it is not clear whether its cytosolic escape and growth are sufficient to induce T cell-mediated clearance and protection upon secondary infection. To investigate this issue, we have searched for mutants that do not induce long-term protective immunity yet invade the cytosol of infected cells. We found that mice immunized with L. monocytogenes lacking the SecA2 ATPase, an auxiliary protein secretion system present in several Gram-positive pathogenic bacteria, mounted a robust cytolytic IFN-gamma-secreting CD8+ T cell response but were not protected against a secondary challenge with wild-type (wt) bacteria. Furthermore, CD8+ T cells from mice immunized with secA2- bacteria failed to transfer protection when injected into recipient mice demonstrating that they were unable to confer protection. Also, secA2- and wt L. monocytogenes spread to the same myeloid-derived cell types in vivo and SecA2 deficiency does not interfere with intracytosolic bacteria multiplication. Therefore, cytosol invasion is not sufficient for inducing secondary protective responses and induction of memory CD8+ T cells mediating long-term antibacterial protective immunity is dependent upon SecA2 expression inside the cytosol of host cells in vivo.

Expression of the P60 Autolysin Enhances NK Cell Activation and is Required for Listeria Monocytogenes Expansion in IFN-gamma-responsive Mice

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2007  |  Pubmed ID: 17277147

Both peptidoglycan and muropeptides potently modulate inflammatory and innate immune responses. The secreted Listeria monocytogenes p60 autolysin digests peptidoglycan and promotes bacterial infection in vivo. Here, we report that p60 contributes to bacterial subversion of NK cell activation and innate IFN-gamma production. L. monocytogenes deficient for p60 (Deltap60) competed well for expansion in mice doubly deficient for IFNAR1 and IFN-gammaR1 or singly deficient for IFN-gammaR1, but not in wild-type, IFNAR1(-/-), or TLR2(-/-) mice. The restored competitiveness of p60-deficient bacteria suggested a specific role for p60 in bacterial subversion of IFN-gamma-mediated immune responses, since in vivo expansion of three other mutant L. monocytogenes strains (DeltaActA, DeltaNamA, and DeltaPlcB) was not complemented in IFN-gammaR1(-/-) mice. Bacterial expression of p60 was not required to induce socs1, socs3, and il10 expression in infected mouse bone marrow macrophages but did correlate with enhanced production of IL-6, IL-12p70, and most strikingly IFN-gamma. The primary source of p60-dependent innate IFN-gamma was NK cells, whereas bacterial p60 expression did not significantly alter innate IFN-gamma production by T cells. The mechanism for p60-dependent NK cell stimulation was also indirect, given that treatment with purified p60 protein failed to directly activate NK cells for IFN-gamma production. These data suggest that p60 may act on infected cells to indirectly enhance NK cell activation and increase innate IFN-gamma production, which presumably promotes early bacterial expansion through its immunoregulatory effects on bystander cells. Thus, the simultaneous induction of IFN-gamma production and factors that inhibit IFN-gamma signaling may be a common strategy for misdirection of early antibacterial immunity.

Activation of Cytosolic Phospholipase A2alpha in Resident Peritoneal Macrophages by Listeria Monocytogenes Involves Listeriolysin O and TLR2

The Journal of Biological Chemistry. Feb, 2008  |  Pubmed ID: 18083708

Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2alpha). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (DeltahlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and DeltahlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and DeltahlyLM correlates with diminished MAPK activation. WTLM but not DeltahlyLM increases intracellular calcium, which is implicated in regulation of cPLA2alpha. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2alpha+/+ but not cPLA2alpha-/- macrophages in response to WTLM and DeltahlyLM. Tumor necrosis factor (TNF)-alpha production is significantly lower in cPLA2alpha+/+ than in cPLA2alpha-/- macrophages infected with WTLM and DeltahlyLM. Treatment of infected cPLA2alpha+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFalpha production to the level produced by cPLA2alpha-/- macrophages implicating prostaglandins in TNFalpha down-regulation. Therefore activation of cPLA2alpha in macrophages may impact immune responses to L. monocytogenes.

Silencing the Alarm: Insights into the Interaction Between Host and Pathogen. Conference on Microbial Pathogenesis: Mechanisms of Infectious Disease

EMBO Reports. Jan, 2008  |  Pubmed ID: 18084188

Stable Integration Vector for Nutrient Broth-based Selection of Attenuated Listeria Monocytogenes Strains with Recombinant Antigen Expression

Clinical and Vaccine Immunology : CVI. Sep, 2008  |  Pubmed ID: 18650400

Recombinant Listeria monocytogenes strains induce strong cellular immune responses and may prove useful for antigen delivery for the vaccination of humans. However, the genetic systems currently available for the stable expression of recombinant antigens by L. monocytogenes rely on the use of antibiotic resistance genes. We report on a derivative, pPL2dalGlnA, of the Listeria monocytogenes pPL2 integration vector that completely lacks drug resistance genes. The selectable markers in pPL2dalGlnA are glutamine synthetase (GlnA) and alanine racemase (Dal). This novel vector was stably maintained in auxotropic L. monocytogenes strains that normally require d-alanine. The pPL2dalGlnA vector also partially restored the ability of an L. monocytogenes Deltadal Deltadat strain to colonize the spleens and livers of infected mice. A novel, highly attenuated strain of L. monocytogenes with quadruple deletions was also engineered by deleting the L. monocytogenes actA and plcB virulence genes from a Deltadal Deltadat strain. Infection of mice with recombinants of this mutant strain that express the antigen from pPL2dalGlnA were shown to elicit CD8(+) T-cell responses to human immunodeficiency virus Tat. This vector system is thus useful for stable antigen expression and vaccination studies.

CD5 Sweetens Lymphocyte Responses

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2009  |  Pubmed ID: 19174525

Bacterial Peptidoglycan Degrading Enzymes and Their Impact on Host Muropeptide Detection

Journal of Innate Immunity. 2009  |  Pubmed ID: 19319201

Peptidoglycan (PGN) is a major component of the bacterial cell envelope in both Gram-positive and Gram-negative bacteria. These muropeptides can be produced or modified by the activity of bacterial glycolytic and peptidolytic enzymes referred to as PGN hydrolases and autolysins. Some of these bacterial enzymes are crucial for bacterial pathogenicity and have been shown to modulate muropeptide release and/or host innate immune responses. The ability of muropeptides to modulate host responses is due to the fact that eukaryotes do not produce PGN and have instead evolved numerous strategies to detect intact PGN and PGN fragments (muropeptides). Here we review the structure of PGN and introduce the various bacterial enzymes known to degrade or modify bacterial PGN. Host factors involved in PGN and muropeptide detection are also briefly discussed, as are examples of how specific bacterial pathogens use PGN degradation and modification to subvert host innate immunity.

Induction of IFN-alphabeta Enables Listeria Monocytogenes to Suppress Macrophage Activation by IFN-gamma

The Journal of Experimental Medicine. Feb, 2010  |  Pubmed ID: 20123961

Production of type I interferon (IFN; IFN-alphabeta) increases host susceptibility to Listeria monocytogenes, whereas type II IFN (IFN-gamma) activates macrophages to resist infection. We show that these opposing immunological effects of IFN-alphabeta and IFN-gamma occur because of cross talk between the respective signaling pathways. We found that cultured macrophages infected with L. monocytogenes were refractory to IFN-gamma treatment as a result of down-regulation of the IFN-gamma receptor (IFNGR). The soluble factor responsible for these effects was identified as host IFN-alphabeta. Accordingly, macrophages and dendritic cells (DCs) showed reduced IFNGR1 expression and reduced responsiveness to IFN-gamma during systemic infection of IFN-alphabeta-responsive mice. Furthermore, the increased resistance of mice lacking the IFN-alphabeta receptor (IFNAR(-/-)) to L. monocytogenes correlated with increased expression of IFN-gamma-dependent activation markers by macrophages and DCs and was reversed by depletion of IFN-gamma. Thus, IFN-alphabeta produced in response to bacterial infection and other stimuli antagonizes the host response to IFN-gamma by down-regulating the IFNGR. Such cross talk permits prioritization of IFN-alphabeta-type immune responses and may contribute to the beneficial effects of IFN-beta in treatment of inflammatory diseases such as multiple sclerosis.

Activation of Naive NK Cells in Response to Listeria Monocytogenes Requires IL-18 and Contact with Infected Dendritic Cells

Journal of Immunology (Baltimore, Md. : 1950). May, 2010  |  Pubmed ID: 20351186

The mechanisms for NK cell activation during infection by intracellular bacterial pathogens are not clearly defined. To dissect how Listeria monocytogenes infection elicits NK cell activation, we evaluated the requirements for activation of naive splenic NK cells by infected bone marrow-derived dendritic cells (BMDCs). We found that NK cell activation in this setting required infection of BMDCs by live wild type bacteria. NK cells were not activated when BMDCs were infected with a live hemolysin deficient (Deltahly) strain. Neutralization of IL-12, TNF-alpha, or caspase-1 each dramatically reduced NK cell IFN-gamma production in response to live wt L. monocytogenes infection. Addition of recombinant IL-18, but not IL-1beta, reversed the effects of caspase-1 inhibition. Recombinant IL-18 also restored NK cell activation by BMDCs infected with Deltahly L. monocytogenes, which produced IL-12 but not IL-18. IL-18 acted on NK cells because MyD88 expression was required in responding NK cells, but not infected BMDC. However, secreted cytokines were not sufficient for activation of naive NK cells by infected BMDCs. Rather, NK cell activation additionally required contact between infected BMDCs and NK cells. These data suggest that the activation of NK cells during L. monocytogenes infection requires both secreted cytokines and ligation of NK activating receptors during direct contact with infected DCs.

Cellular Reactive Oxygen Species Inhibit MPYS Induction of IFNβ

PloS One. 2010  |  Pubmed ID: 21170271

Many inflammatory diseases, as well as infections, are accompanied by elevation in cellular levels of Reactive Oxygen Species (ROS). Here we report that MPYS, a.k.a. STING, which was recently shown to mediate activation of IFNβ expression during infection, is a ROS sensor. ROS induce intermolecular disulfide bonds formation in MPYS homodimer and inhibit MPYS IFNβ stimulatory activity. Cys-64, -148, -292, -309 and the potential C₈₈xxC₉₁ redox motif in MPYS are indispensable for IFNβ stimulation and IRF3 activation. Thus, our results identify a novel mechanism for ROS regulation of IFNβ stimulation.

Antagonistic Crosstalk Between Type I and II Interferons and Increased Host Susceptibility to Bacterial Infections

Virulence. Sep-Oct, 2010  |  Pubmed ID: 21178482

Type I and II interferons (IFNs αβ and γ) have opposing effects on immune resistance to certain pathogenic bacteria. While IFNγ generally plays a protective role, IFNαβ exacerbates Listeria monocytogenes and Mycobacterium tuberculosis infections. Our findings provided evidence that this increased susceptibility reflects a novel antagonistic cross talk between IFNαβ and IFNγ. Macrophages infected with L. monocytogenes strains that induce IFNαβ production responded poorly to IFNγ, as measured by reduced phosphorylation of STAT1 and reduced IFNγ-dependent gene expression. The impaired responsiveness to IFNγ correlated with reduced expression of its receptor, IFNGR, by both infected and bystander macrophages. Down regulation of IFNGR was dependent on responsiveness to IFNγ and mimicked by recombinant IFNβ. Mice lacking responsiveness to IFNαβ (IFNAR1 (-/-)) retained high IFNGR expression, developed higher expression of MHC-II on macrophages and DCs, and were more resistant to systemic L. monocytogenes infection--but only in the presence of IFNγ. Thus, the ability of IFNαβ to down regulate IFNGR provides an explanation for its ability to reduce responsiveness to IFNγ and to increase host susceptibility to bacterial infection. It remains to be determined whether and how such antagonistic interferon crosstalk benefits the host.

IL-15 and Type I Interferon Are Required for Activation of Tumoricidal NK Cells by Virus-infected Dendritic Cells

Cancer Research. Apr, 2011  |  Pubmed ID: 21307131

There is increasing evidence that natural killer (NK) cells play an important role in antitumor immunity following dendritic cell (DC) vaccination. Little is known, however, about the optimal stimulation of DCs that favors NK activation in tumor-bearing hosts. In this study, we demonstrate that treatment with toll-like receptor (TLR) ligands and infection with a mutant vesicular stomatitis virus (VSV-ΔM51) both induced DC maturation. Further, inoculation of these DCs led to robust NK-mediated protection against tumor challenge. Strikingly, only VSV-ΔM51-infected DCs were capable of suppressing the growth of established tumors, suggesting that additional signals provided by viral infection may be required to activate tumoricidal NK cells in tumor-bearing hosts. VSV-ΔM51 infection of DCs induced greater type I interferon (IFN I) production than TLR ligand treatment, and disruption of the IFN I pathway in DCs eliminated their ability to induce NK activation and tumor protection. However, further studies indicated that IFN I alone was not sufficient to activate NK cells, especially in the presence of a tumor, and DC-derived IL-15 was additionally required for tumoricidal NK activation. These results suggest that induction of IFN I by VSV-ΔM51 allows DCs to overcome tumor-associated immunosuppression and facilitate IL-15-mediated priming of tumoricidal NK cells. Thus, the mode of DC maturation should be carefully considered when designing DC-based cancer immunotherapies.

Transient Receptor Potential Melastatin 2 (TRPM2) Ion Channel is Required for Innate Immunity Against Listeria Monocytogenes

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2011  |  Pubmed ID: 21709234

The generation of reactive oxygen species (ROS) is inherent to immune responses. ROS are crucially involved in host defense against pathogens by promoting bacterial killing, but also as signaling agents coordinating the production of cytokines. Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable channel gated via binding of ADP-ribose, a metabolite formed under conditions of cellular exposure to ROS. Here, we show that TRPM2-deficient mice are extremely susceptible to infection with Listeria monocytogenes (Lm), exhibiting an inefficient innate immune response. In a comparison with IFNγR-deficient mice, TRPM2(-/-) mice shared similar features of uncontrolled bacterial replication and reduced levels of inducible (i)NOS-expressing monocytes, but had intact IFNγ responsiveness. In contrast, we found that levels of cytokines IL-12 and IFNγ were diminished in TRPM2(-/-) mice following Lm infection, which correlated with their reduced innate activation. Moreover, TRPM2(-/-) mice displayed a higher degree of susceptibility than IL-12-unresponsive mice, and supplementation with recombinant IFNγ was sufficient to reverse the unrestrained bacterial growth and ultimately the lethal phenotype of Lm-infected TRPM2(-/-) mice. The severity of listeriosis we observed in TRPM2(-/-) mice has not been reported for any other ion channel. These findings establish an unsuspected role for ADP-ribose and ROS-mediated cation flux for innate immunity, opening up unique possibilities for immunomodulatory intervention through TRPM2.

MPYS is Required for IFN Response Factor 3 Activation and Type I IFN Production in the Response of Cultured Phagocytes to Bacterial Second Messengers Cyclic-di-AMP and Cyclic-di-GMP

Journal of Immunology (Baltimore, Md. : 1950). Sep, 2011  |  Pubmed ID: 21813776

Cyclic-di-GMP and cyclic-di-AMP are second messengers produced by bacteria and influence bacterial cell survival, differentiation, colonization, biofilm formation, virulence, and bacteria-host interactions. In this study, we show that in both RAW264.7 macrophage cells and primary bone marrow-derived macrophages, the production of IFN-β and IL-6, but not TNF, in response to cyclic-di-AMP and cyclic-di-GMP requires MPYS (also known as STING, MITA, and TMEM173). Furthermore, expression of MPYS was required for IFN response factor 3 but not NF-κB activation in response to these bacterial metabolites. We also confirm that MPYS is required for type I IFN production by cultured macrophages infected with the intracellular pathogens Listeria monocytogenes and Francisella tularensis. However, during systemic infection with either pathogen, MPYS deficiency did not impact bacterial burdens in infected spleens. Serum IFN-β and IL-6 concentrations in the infected control and MPYS(-/-) mice were also similar at 24 h postinfection, suggesting that these pathogens stimulate MPYS-independent cytokine production during in vivo infection. Our findings indicate that bifurcating MPYS-dependent and -independent pathways mediate sensing of cytosolic bacterial infections.

A LysM and SH3-domain Containing Region of the Listeria Monocytogenes P60 Protein Stimulates Accessory Cells to Promote Activation of Host NK Cells

PLoS Pathogens. Nov, 2011  |  Pubmed ID: 22072975

Listeria monocytogenes (Lm) infection induces rapid and robust activation of host natural killer (NK) cells. Here we define a region of the abundantly secreted Lm endopeptidase, p60, that potently but indirectly stimulates NK cell activation in vitro and in vivo. Lm expression of p60 resulted in increased IFNγ production by naïve NK cells co-cultured with treated dendritic cells (DCs). Moreover, recombinant p60 protein stimulated activation of naive NK cells when co-cultured with TLR or cytokine primed DCs in the absence of Lm. Intact p60 protein weakly digested bacterial peptidoglycan (PGN), but neither muropeptide recognition by RIP2 nor the catalytic activity of p60 was required for NK cell activation. Rather, the immune stimulating activity mapped to an N-terminal region of p60, termed L1S. Treatment of DCs with a recombinant L1S polypeptide stimulated them to activate naïve NK cells in a cell culture model. Further, L1S treatment activated NK cells in vivo and increased host resistance to infection with Francisella tularensis live vaccine strain (LVS). These studies demonstrate an immune stimulating function for a bacterial LysM domain-containing polypeptide and suggest that recombinant versions of L1S or other p60 derivatives can be used to promote NK cell activation in therapeutic contexts.