Proteolytic modulation of toxoplasma invasion proteins

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DESCRIPTION (provided by applicant): The obligate intracellular protozoan Toxoplasma gondii is a leading cause of posterior uveitis in the general population, encephalitis in immunodeficient individuals, and cognitive impairment in congenitally infected children. We seek to understand how Toxoplasma prepares for and achieves cell invasion. Although it has been well recognized that many Toxoplasma invasion proteins undergo proteolytic maturation en route to apical secretory organelles, little is known about the proteases involved or where and why maturation occurs. We hypothesize that T. gondii uses a cathepsin proteases to proteolytically activate and stabilize invasion proteins within a novel multivesicular endosome (MVE), based on preliminary immunolocalization, gene knockout, and inhibitor studies, along with protease specificity analysis and in vitro processing of a putative substrate. To test this hypothesis we will: (1) Characterize defects and compensatory mechanisms in CPL knockout parasites; (2) Confirm the overlapping roles of cathepsins in proMIC maturation by genetic and inhibitor studies; and (3) Test the "lock and load" hypothesis that proteolytic maturation stabilizes proMIC protein complexes. This work will illuminate basic mechanisms of secretory protein trafficking and proteolytic modulation of protein function that may be shared with related apicomplexan parasites and other eukaryotic cells that utilize regulated secretion. PUBLIC HEALTH RELEVANCE: Our goal is to find new ways of interfering with Toxoplasma invasion of human cells as a strategy of controlling infections caused by this parasite. Toxoplasma proteases may be good targets for new anti-parasitic drugs; this work is designed to test the function and significance of two such proteases.