A Cytosolic Serine Endopeptidase from Trypanosoma cruzi Is Required for the Generation of Ca2+ Signaling in Mammalian Cells

Abstract

An early event in the Trypanosoma cruzi cell invasion process, the recruitment of host lysosomes, led us to investigate the involvement of signal transduction. Infective trypomastigotes were found to contain a soluble Ca2+-signaling activity for mammalian cells that is sensitive to protease inhibitors. Inhibitor and substrate utilization profiles were used to purify a candidate peptidase for involvement in this process, from which we isolated a full-length cDNA clone. The sequence revealed a novel enzyme, denominated T. cruzi oligopeptidase B, which is homologous to members of the prolyl oligopeptidase family of serine hydrolases, known to participate in the maturation of biologically active peptides. The T. cruzi oligopeptidase B was expressed as a fully active product in Escherichia coli, and antibodies to the recombinant enzyme inhibited both peptidase activity and Ca2+ signaling induced in normal rat kidney cells by trypomastigote extracts. Our data suggest that the T. cruzi oligopeptidase B participates in processing events in the cytoplasm of the parasites, generating a factor with Ca2+-signaling activity for mammalian cells.

Dual Role of Signaling Pathways Leading to Ca2+and Cyclic AMP Elevation in Host Cell Invasion byTrypanosoma cruzi

“…Here we show that host cell pretreatment with PTx, a condition previously shown to inhibit infection by WT T. cruzi (31), also does not affect invasion by OPBnull trypomastigotes. These findings reinforce the hypothesis that OPBnull trypomastigotes lost the capacity to activate the G protein-dependent host cell signaling pathway that leads to IP 3 formation and Ca 2ϩ mobilization from intracellular stores and that has been directly linked to lysosome recruitment (5,6). Such observations raised the possibility that the residual invasion capacity of OPBnull mutants might be dependent on a completely different mechanism, not involving lysosome recruitment.…”

“…Recent studies have provided extensive evidence that activation of signaling cascades in host cells plays a central role in the T. cruzi cell invasion mechanism (5,20,34). Mobilization of Ca 2ϩ from intracellular stores has been specifically implicated, since cell loading with Ca 2ϩ chelators and Ca 2ϩ store depletion by thapsigargin effectively inhibit trypomastigote entry (7,24,31).…”

“…Mobilization of Ca 2ϩ from intracellular stores has been specifically implicated, since cell loading with Ca 2ϩ chelators and Ca 2ϩ store depletion by thapsigargin effectively inhibit trypomastigote entry (7,24,31). An investigation of the T. cruzi Ca 2ϩ signaling capacity revealed that the infective trypomastigotes forms contain a soluble factor capable of triggering intracellular free Ca 2ϩ transients in several mammalian cell types and that production of this factor requires the activity of a parasite serine peptidase, OPB (4,5,6,24,31). Deletion of the T. cruzi OPB gene by targeted replacement resulted in a significant attenuation of the parasite's virulence for mice and in a 60 to 70% reduction in their ability to invade mammalian cells.…”

“…This hypothesis was reinforced when trypomastigotes, the infective T. cruzi life cycle stages, were shown to activate phospholipase C and to trigger IP 3 -mediated Ca 2ϩ release from host cell intracellular stores (24,31). Characterization of this signaling pathway revealed that a parasite serine peptidase oligopeptidase B (OPB), is required for the generation of a soluble factor that triggers intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) transients in mammalian cells (4)(5)(6).…”

Biological Roles of Peptidases in Trypanosomatids~!2009-11-26~!2010-02-15~!2010-03-18~!

“…Oligopeptidases only hydrolyze peptides smaller than 30 amino acid residues and as a result have no naturally occurring inhibitors. It was proposed that the Ca 2+ agonist generated by oligopeptidase B is exported from the parasite and binds to a receptor on the surface of cells, activating phospholipase C and generating inositol phosphate, which binds to its receptor on the membrane of endoplasmic reticulum and promotes Ca 2+ release [111][112][113]. Oligopeptidase B null mutant trypomastigotes are defective in mobilizing Ca 2+ from thapsigargin-sensitive stores in mammalian cells and in establishing infection in vitro and in vivo [113].…”