The protozoan parasite Trypanosoma cruzi invades mammalian cells by a mechanism not dependent on actin polymerization, and thus distinct from classical phagocytosis. There is no pseudopod extention or any sign of membrane activity during host cell entry by this large parasite. These observations have raised the following question: what is the origin of the large amount of membrane required to form the T. cruzi parasitophorous vacuole, if it is not derived from the host cell plasma membrane? Recent observations demonstrated that intracellular vacuoles containing T. cruzi acquire lysosomal markers prior to complete parasite internalization, while the trypanosomes are still at the host cell periphery. Lysosomes were found to aggregate at the sites of parasite attachment, and to fuse with vacuoles at early stages of their formation. Depletion in peripheral lysosomes or impaired lysosomal fusion capacity inhibited T. cruzi entry, indicating that lysosomal membranes contribute to formation of the parasitophorous vacuole. These are novel findings both for lysosome function and for entry of a parasite into host cells. Our working hypothesis is that upon binding to host cells the infective stages of T. cruzi transduce signals capable of triggering local disassembly of the cytoskeleton and lysosome fusion. In order to test this hypothesis, we plan to: 1) determine the kinetics of lysosome recruitment and fusion during the invasion of live cells by trypanosomes, by video-enhanced light microscopy image recording. 2) Investigate the role of host cell intracellular Ca2+ fluxes in the lysosome recruitment and fusion process, using specific agonists, chelators, channel blockers, microspectrofluorometry and laser confocal microscopy. 3) verify if a localized disruption of the cortical actin cytoskeleton facilitating lysosome access to the plasma membrane occurs at the site of parasite attachment, and if this event can be correlated with phosphorylation and/or redistribution of specific cytoskeletal-associated proteins. 4) verify if T. cruzi has to bind to specific host cell receptors for transducing signals leading to lysosome recruitment and fusion, or if receptors not normally present in non-phagocytic cells, such as the Fc receptor, can perform the same function. 5) If a specific host cell receptor proves not to be necessary, we plan to investigate the involvement of soluble trypanosome metabolic products in the lysosome recruitment process. The proposed experiments will increase our understanding of a novel mechanism promoting cell invasion by this important human pathogen, in addition to providing basic new information on cytoskeletal rearrangements and lysosome function.
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