The cellular and immunological consequences of LLO mediated vacuolar escape

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen capable of causing severe food born illness. Escape from the phagosome is essential for the intracellular life cycle of L. monocytogenes and has tremendous consequences on many biological processes including protective immunity and autophagy.;Wild-type L. monocytogenes express the gene hly that results in the production of the cholesterol dependent cytolysin listeriolysin O (LLO). Expression of LLO is required for phagosomal escape and for the induction of a cell-mediated immune response to L. monocytogenes . A mutant of L. monocytogenes that does not express LLO is avirulent, remains trapped in the phagosome, and is unable induce cell-mediated immunity. The reason that L. monocytogenes mutants, lacking LLO, do not induce a cell-mediated immune response is not understood.;Using a mixed inoculum of cytosolic and phagosome-trapped L. monocytogenes we found that when L. monocytogenes was confined to the vacuole, it suppressed the immunizing capability of L. monocytogenes in the cytosol. Further, as we increased the concentration of the bacteria trapped in the phagosome with an inoculum containing an immunizing dose of cytosolic L. monocytogenes, a concomitant decrease in the expression of pro-inflammatory cytokines and L. monocytogenes specific T cells was observed. Additionally, immunosuppressive cytokines, including IL10, were made in response to bacteria trapped in the phagosome, and blockage of the IL10R restored the immunizing capability of cytosolic L. monocytogenes even in the presence of phagosome-trapped bacteria.;L. monocytogenes escape from the phagosome also induced autophagy. Autophagy is cellular bulk degradation pathway used to degrade long-lived proteins and organelles. We found L. monocytogenes mutants lacking LLO did not induce autophagy, whereas wild-type and other mutants induced relatively equal amounts of autophagy early in an infection of bone marrow derived macrophages. Bacillus subtilis expressing LLO also induced autophagy, indicating that the induction was not specific to L. monocytogenes. Lastly, we found that liposomes containing LLO were sufficient to induce autophagy in the absence of infection. Thus, LLO was necessary and sufficient to induce autophagy in an L. monocytogenes infection.;In summary, we found that LLO expression by L. monocytogenes was required for both the proper induction of the cell-mediated immune response to L. monocytogenes and was also required for the induction of autophagy in response to infection with L. monocytogenes .