Intracellular protozoan parasites enter cells within membrane-bound vacuoles, from which they must escape to multiply in the cytosol or to gain access to the extracellular environment. Mechanisms for inducing membrane disruption are therefore probably essential for the survival of these organisms. Our long term goal is to characterize at the molecular level the mechanism used by Trypanosoma cruzi (the causative agent of Chagas' disease in man) for disrupting the phagosome membrane. In recent studies we found that T. cruzi requires an acidic environment to escape from the phagolysosome, and secretes an acid pH-dependent lytic protein (TC-TOX) that is immunologically related to human C9, the terminal component of the complement system. C9 shares sequence homology with a group of eukaryotic pore-forming proteins that includes C6, C7 and C8 (other members of the complement membrane attack complex) and perforin, the cytotoxic lymphocyte lytic protein. We plan to: 1) clone and sequence the gene encoding TC-TOX to obtain definitive evidence for its homology with other eukaryotic pore- forming proteins. 2) verify if TC-TOX is functionally analogous to C9 by performing lytic assays using target cells carrying partially assembled C5b-8 complexes, and determine if it behaves like an integral membrane protein after membrane attack. 3) generate monoclonal antibodies to TC-TOX and map their epitopes, to obtain evidence for pH-induced conformational changes related to activity. 4) establish in vitro assays for phagosome disruption using isolated phagolysosomes and permeabilized infected cells, to study the mechanisms involved in clearance of membrane fragments and the requirement for cytosolic factors. The proposed experiments will indicate if TC-TOX is a pore-forming protein structurally and functionally similar to C9, provide information about its role in phagosome disruption, and establish a basis for future studies of its involvement in the pathogenesis of Chagas' disease.
Showing the most recent 10 out of 11 publications
