Infective forms of the intracellular parasite Trypanosoma cruzi contain a soluble factor that triggers intracellular free Ca2+ transients in mammalian cells. Prevention of these transients by buffering or depleting intracellular Ca2+ stores blocks trypomastigote entry, indicating a central role for this signaling event in invasion. Ca2+ signaling by trypomastigote extracts is closely linked to the activity of a novel parasite endopeptidase, the T. cruzi peptidase A, recently cloned and expressed in our laboratory. The peptidase A sequence shows significant homology to the recently emerged prolyl oligopeptidase family of serine hydrolases, several of which are processing enzymes which remove dipeptides from N-terminal extentions of immature precursor peptides. Recent data obtained with neutralizing antibodies reinforce the hypothesis that peptidase A acts on a trypomastigote precursor molecule, to generate a peptide with Ca2+ signaling activity for mammalian cells. The first part of this proposal focuses on peptidase A and its role in generation of the trypomastigote Ca2+ signaling factor, and specific aims are: (1) to generate T. cruzi peptidase A null mutants and evaluate their capacity for Ca2+ signaling and invasion of mammalian cells; (2) utilize the null mutants and/or neutralizing antibodies as tools to identify candidate substrate molecules for the peptidase in trypomastigotes. A second part of the project focuses on the mammalian cell receptor for the T. cruzi signaling factor, and the specific aims are: (3) to expression clone the receptor in Xenopus laevis oocytes, and (4) to utilize the information derived from the receptor sequence to search for its ligand molecule in trypomastigotes. In addition to greatly expanding our understanding of T. cruzi/host cell interactions, these studies will significantly advance our knowledge in a broader and novel area: receptor-mediated signal transduction induced in host cells by secreted pathogen factors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032056-07
Application #
2672100
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1992-02-01
Project End
2000-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
7
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Yale University
Department
Anatomy/Cell Biology
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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