Listeria monocytogenes is a facultative intracellular pathogen that provides an extremely amenable model for basic studies on host-pathogen interactions. Importantly, L. monocytogenes is also a clinically relevant food-borne pathogen that causes a high rate of mortality in pregnant women and the immunocompromised. A primary determinant of L. monocytogenes pathogenesis and a target of the host's immune response is Listeriolysin O (LLO). LLO is a member of a large family of pore-forming cytolysins that is largely responsible for mediating escape of L. monocytogenes from a phagosome and for virulence;LLO-minus mutants are 5-logs less virulent in animal models of infection. LLO activity is a double-edged sword as its activity must be restricted to an acidic phagosome or the host cell will die due to LLO-mediated cell death (referred to as compartmentalization).
In Aim I of this proposal the molecular determinants mediating compartmentalization will be determined by using a combination of mutagenesis, cell biology and biochemistry. LLO mutants will be characterized with respect to phosphorylation, ubiquitylation, proteolysis, half-life, and aggregation.
In Aim II, the role played by autophagy will be examined with respect to escape from a phagosome and the fate of LLO secreted into the host cytosol. Macrophages that are defective for autophagy will provide an excellent system with which to examine these questions.
In Aim III, a newly developed, mariner-based, transposon mutagenesis system will be used to identify the role played by gene-products, other than LLO, on LLO expression, synthesis, secretion and toxicity. These studies should identify the hypothetical host protein(s) that interact with LLO and LLO mRNA to prevent toxicity, and open up new areas of investigations pertaining to LLO expression, synthesis, secretion and toxicity. Lastly, the role of post-translational modifications and autophagy will be examined with respect to the presentation of LLO epitopes in both the MHC Class I and Class II pathways of antigen processing and presentation. Novel in vivo assays will be used to select LLO mutants that are not recognized by the host's acquired immunity, thereby providing basic information on properties of foreign proteins that leads to immunogenicity.
Diseases caused by intracellular pathogens, for example, tuberculosis, AIDS and Malaria, remain one of the largest challenges facing the international biomedical community. The proposed studies on Listeria monocytogenes will provide insight into the molecular biology, cell biology and immunology relevant to the treatment and prevention of diseases caused by intracellular pathogens.
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