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A LysM and SH3-domain containing region of the Listeria monocytogenes p60 protein stimulates accessory cells to promote activation of host NK cells.

ABSTRACT: 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.

SUBMITTER: Schmidt RL

PROVIDER: S-EPMC3207947 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UNLABELLED:Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have ?-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (or dl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminal l-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCE:Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.

Project description:Inducing long-term protective memory CD8(+) T-cells is a desirable goal for vaccines against intracellular pathogens. However, the mechanisms of differentiation of CD8(+) T-cells into long-lived memory cells capable of mediating protection of immunized hosts remain incompletely understood. We have developed an experimental system using mice immunized with wild type (WT) or mutants of the intracellular bacterium Listeria monocytogenes (Lm) that either do or do not develop protective memory CD8(+) T-cells. We previously reported that mice immunized with Lm lacking functional SecA2, an auxiliary secretion system of gram-positive bacteria, did not differentiate functional memory CD8(+) T-cells that protected against a challenge infection with WT Lm. Herein we hypothesized that the p60 and NamA autolysins of Lm, which are major substrates of the SecA2 pathway, account for this phenotype. We generated Lm genetically deficient for genes encoding for the p60 and NamA proteins, ?iap?murA Lm, and further characterized this mutant. ?p60?NamA Lm exhibited a strong filamentous phenotype, inefficiently colonized host tissues, and grew mostly outside cells. When ?p60?NamA Lm was made single unit, cell invasion was restored to WT levels during vaccination, yet induced memory T-cells still did not protect immunized hosts against recall infection. Recruitment of blood phagocytes and antigen-presenting cell activation was close to that of mice immunized with ?ActA Lm, which develop protective memory. However, key inflammatory factors involved in optimal T-cell programming such as IL-12 and type I IFN (IFN-I) were lacking, suggesting that cytokine signals may largely account for the observed phenotype. Thus, altogether, these results establish that p60 and NamA secreted by Lm promote primary host cell invasion, the inflammatory response and the differentiation of functional memory CD8(+) T-cells, by preventing Lm filamentation during growth and subsequent triggering of innate sensing mechanisms.

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A LysM and SH3-domain containing region of the Listeria monocytogenes p60 protein stimulates accessory cells to promote activation of host NK cells.

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