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.
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