A trypanosome-soluble factor induces IP₃ formation, intracellular Ca²⁺ mobilization and microfilament rearrangement in host cells

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 trypomastigotes, but not the noninfective epimastigotes, contain a proteolytically 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 after exposure to trypomastigote extracts is a result of Ca²⁺ release from intracellular stores. Removal of Ca²⁺ from the extracellular medium or inhibition of Ca²⁺ channels with NiCl₂ did not affect the response to PGTF, while depletion of intracellular stores with thapsigargin abolished it. [Ca²⁺](i) transients induced by PGTF were shown to be coupled to the activity of phospholipase C (PLC), since the specific inhibitor U73122 completely blocked the response, while its inactive analogue U73343 had no effect. In addition, polyphosphoinositide hydrolysis and inositol 1,4,5-trisphosphate (IP₃) were detected upon cell stimulation with PGTF, suggesting the participation of IP₃-sensitive intracellular 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 to be a direct consequence of increased [Ca²⁺](i), since thrombin and the Ca²⁺ ionophore ionomycin produced a similar effect, with a time course that corresponded to the kinetics of the elevation in [Ca²⁺](i). These observations support the hypothesis that PGTF-induced disassembly of the cortical actin cytoskeleton may play a role in T. cruzi invasion, by facilitating lysosome access to the invasion site. Taken together, our findings suggest that the proteolytically generated trypomastigote factor PGTF is a novel agonist that acts through the PLC/phosphoinositide signaling pathway of mammalian cells.