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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032056-04
Application #
2066952
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1992-02-01
Project End
1997-01-31
Budget Start
1995-02-01
Budget End
1996-01-31
Support Year
4
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Yale University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Leite, M F; Moyer, M S; Andrews, N W (1998) Expression of the mammalian calcium signaling response to Trypanosoma cruzi in Xenopus laevis oocytes. Mol Biochem Parasitol 92:1-13
Caler, E V; Vaena de Avalos, S; Haynes, P A et al. (1998) Oligopeptidase B-dependent signaling mediates host cell invasion by Trypanosoma cruzi. EMBO J 17:4975-86
Burleigh, B A; Caler, E V; Webster, P et al. (1997) A cytosolic serine endopeptidase from Trypanosoma cruzi is required for the generation of Ca2+ signaling in mammalian cells. J Cell Biol 136:609-20
Rodriguez, A; Rioult, M G; Ora, A et al. (1995) A trypanosome-soluble factor induces IP3 formation, intracellular Ca2+ mobilization and microfilament rearrangement in host cells. J Cell Biol 129:1263-73
Burleigh, B A; Andrews, N W (1995) A 120-kDa alkaline peptidase from Trypanosoma cruzi is involved in the generation of a novel Ca(2+)-signaling factor for mammalian cells. J Biol Chem 270:5172-80
Burleigh, B A; Andrews, N W (1995) The mechanisms of Trypanosoma cruzi invasion of mammalian cells. Annu Rev Microbiol 49:175-200
Tardieux, I; Nathanson, M H; Andrews, N W (1994) Role in host cell invasion of Trypanosoma cruzi-induced cytosolic-free Ca2+ transients. J Exp Med 179:1017-22
Andrews, N W (1994) From lysosomes into the cytosol: the intracellular pathway of Trypanosoma cruzi. Braz J Med Biol Res 27:471-5
Andrews, N W (1993) Living dangerously: how Trypanosoma cruzi uses lysosomes to get inside host cells, and then escapes into the cytoplasm. Biol Res 26:65-7
Hall, B F (1993) Trypanosoma cruzi: mechanisms for entry into host cells. Semin Cell Biol 4:323-33

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