Search

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.

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.

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.

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.

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.

We developed a competitive index assay for murine listeriosis that tests the virulence of Listeria monocytogenes strains in different organs and at various times postinoculation. Studies presented here demonstrate the reproducibility of this assay during primary and secondary infection of inbred and outbred mice. We verified the validity of this assay by performing competitive index analysis of a well-characterized strain of L. monocytogenes lacking the actA gene. In addition, we found that while L. monocytogenes strains unable to recruit vasodilator-stimulated phosphoprotein (VASP) to their surface exhibit a 10-fold virulence attenuation in the livers of naive animals, they display a 50-fold survival defect in the liver during secondary listeriosis.

Bone marrow (BM)-derived antigen-presenting cells (APCs) are potent stimulators of T cell immune responses. We investigated the requirements for antigen presentation by these cells in priming cytotoxic T lymphocyte (CTL) responses to intracellular bacterial and viral pathogens. [Parent-->F(1)] radiation BM chimeras were constructed using C57BL/6 donors and (C57BL/6 x BALB/c)F(1) recipients. Infection of chimeric mice with either Listeria monocytogenes or vaccinia virus expressing the nucleoprotein (NP) antigen from lymphocytic choriomeningitis virus (LCMV) primed H2-D(b)-restricted, but not H2-K(d)-restricted CTL responses, demonstrating the requirement for BM-derived APCs for successful priming of CTL responses to these pathogens. Surprisingly, this did not hold true for chimeric mice infected with LCMV itself. LCMV-infected animals developed strong CTL responses specific for both H2-D(b)- and H2-L(d)-restricted NP epitopes. These findings indicate that in vivo priming of CTL responses to LCMV is remarkably insensitive to deficiencies in antigen presentation by professional BM-derived APCs.

Protective immunity to infection by many intracellular pathogens requires recognition by cytotoxic T lymphocytes (CTLs) of antigens presented on major histocompatibility complex (MHC) class I molecules. To be presented for recognition by pathogen-specific CTLs, these antigens must gain access to the host cell class I processing pathway. In the case of intracellular bacterial pathogens, the majority of bacterial proteins are retained within the bacterial membrane and therefore remain inaccessible to the host cell for antigen processing. We have isolated a CTL clone from a C57BL/6 mouse infected with the intracellular gram-positive bacterium Listeria monocytogenes (LM) and have identified the source of the antigen. Using a genomic expression library, we determined that the clone recognizes an antigenic N-formyl peptide presented by the nonpolymorphic murine MHC class Ib molecule, H2-M3. Several lengths of this peptide were able to sensitize cells for lysis by this CTL clone. The source of this antigenic peptide is a 23-amino acid polypeptide encoded at the start of a polycistronic region. Analysis of mRNA secondary structure of this region suggests that this polypeptide may be a leader peptide encoded by a transcriptional attenuator.

Cytotoxic T cells (CTL) play a critical role in the murine immune response to Listeria monocytogenes (Listeria). Bacterial antigens are presented to Listeria-specific CTL by products of both conventional, polymorphic MHC class Ia and non-polymorphic MHC class Ib alleles. The H2-M3 class Ib gene product, M3, preferentially presents formylmethionine-initiating (fMet) peptides derived from the N termini of bacterial and mitochondrial proteins. Thus, M3 signals the presence of bacterial invaders to CTL effectors. Listeria-encoded fMet peptide epitopes for H2-M3-restricted CTL have recently been identified. These and other identified fMet peptides are predominantly comprised of hydrophobic residues and appear to be cleaved from membrane-bound proteins. The subcellular location and membrane topology of such proteins may be significant factors in their selection as target antigens for H2-M3-restricted CTL. Such rules may prove useful for prediction of candidate fMet peptide epitopes from other bacterial proteins and species. Studies using synthetic fMet peptides to stimulate CTL ex vivo are also discussed. These latter studies indicate that Listeria infection boosts H2-M3-restricted CTL responses. However, in contrast to MHC class Ia-restricted CTL responses, fMet peptide-specific CTL are observed in a large proportion of cultures from non-immunized, conventionally housed (non-SPF) mice. The CTL activity in these latter cultures may reflect priming in vivo on cross-reactive antigens, or may indicate that requirements for priming of H2-M3-restricted CTL are less stringent than for class Ia-restricted responses.

Recent studies have revealed the complexity of cytokine and cellular interactions required for resistance to primary Listeria monocytogenes infection and have illustrated that resistance to secondary infection may occur through multiple pathways. Analyses of Listeria epitope generation and the specificity of protective CD8(+) T cells have suggested that future research should focus on secreted protein antigens in specific resistance to infection and have increased our understanding of Listeria antigens presented by MHC class l-b molecules.

Using expression cloning, we have identified an H2-M3-restricted epitope of the intracellular bacterial pathogen Listeria monocytogenes. Picomolar concentrations of an amino-terminal N-formylated hexapeptide, fMIGWII, targeted cells for lysis by CD8+ cytotoxic T cells, while the nonformylated peptide was approximately 100-fold less active. The sequence of the 185 aa protein source of this epitope predicts a transmembrane protein that retains its N terminus and assumes an N(out)-C(in) topology. This membrane orientation offers an explanation for the protection of the epitope from deformylases present in the bacterial cell and suggests an explanation for the ability of phagocytes to present H2-M3-restricted bacterial epitopes via a vacuolar TAP-independent mechanism.