A TRYPANOSOME-SOLUBLE FACTOR INDUCES IP3 FORMATION, INTRACELLULAR CA2-CELLS( MOBILIZATION AND MICROFILAMENT REARRANGEMENT IN HOST)

Lysosomes are recruited to the invasion site during host cell entry by Trypanosoma cruzi, an unusual process suggestive of the triggering of signal transduction mechanisms. Previous studies showed that trypomas tigotes, but not the noninfective epimastigotes, contain a proteolytic ally generated trypomastigote factor (PGTF) that induces intracellular free Ca2+ transients in several mammalian cell types. Using confocal time-lapse imaging of normal rat kidney (NRK) fibroblasts loaded with the Ca2+-sensitive dye fluo-3, we show that the initial intracellular free Ca2+ concentration ([Ca2+](i)) transient detected a few seconds a fter exposure to trypomastigote extracts is a result of Ca2+ release f rom intracellular stores, Removal of Ca2+ from the extracellular mediu m or inhibition of Ca2+ channels with NiCl2 did not affect the respons e to PGTF, while depletion of intracellular stores with thapsigargin a bolished it, [Ca2+](i) transients induced by PGTF were shown to be cou pled to the activity of phospholipase C (PLC), since the specific inhi bitor U73122 completely blocked the response, while its inactive analo gue U73343 had no effect, In addition, polyphosphoinositide hydrolysis and inositol 1,4,5-trisphosphate (IP3) were detected upon cell stimul ation with PGTF, suggesting the participation of IP3-sensitive intrace llular Ca2+ channels. An immediate effect of the signaling induced by PGTF and live trypomastigotes was a rapid and transient reorganization of host cell microfilaments. The redistribution of F-actin appeared t o be a direct consequence of increased [Ca2+](i), since thrombin and t he Ca2+ ionophore ionomycin produced a similar effect, with a time cou rse that corresponded to the kinetics of the elevation in [Ca2+](i). T hese observations support the hypothesis that PGTF-induced disassembly of the cortical actin cytoskeleton may play a role in T. cruzi invasi on, by facilitating lysosome access to the invasion site. Taken togeth er, our findings suggest that the proteolytically generated trypomasti gote factor PGTF is a novel agonist that acts through the PLC/phosphoi nositide signaling pathway of mammalian cells.