FIGURE 1.

Trypanosoma cruzi induces a calcium‐dependent lEV release from epithelial HeLa cells which can be reduced by blocking mechanosensitive surface channels. (a), HeLa cells (1.0 × 10⁶) were loaded with Fura 2‐AM at 37°C in nominal Ca²⁺‐free buffer and for all experiments left to equilibrate by preincubating at 37°C for 5 min, from which point t = 0 s was taken. After 60 s, metacyclic trypomastigotes (Mtr) (5.0 × 10⁶) were added (red arrow) and [Ca²⁺]_cyt was monitored using a spectrophotometer over the ensuing 90 s. Mtr infected cells were also treated with EGTA (5 mM) and BAPTA‐AM (10 µM) and epimastigote and non‐infected (NI) controls were included. (b), HeLa cells harvested by trypsinization (1 × 10⁶/well in triplicate) were stimulated at 37°C for 30 min with T. cruzi Mtr (5:1, parasites‐to‐cell ratio) also in the presence of calpeptin, CPT (10 µg/mL). The dose‐response curves were fitted with a 4‐parameter logistic equation. Additional analysis used 2‐way ANOVA with Sidak's post‐test. ***p < 0.001, **p < 0.01 (n = 6). lEVs released were isolated and analysed as described in Materials and Methods. (c), Transmission electron microscopy of HeLa cell‐derived lEVs being released after stimulation with T. cruzi Mtr 5:1 (parasites‐to‐cell) ratio at 37°C for 30 min or of resting, uninfected cells, (d). HeLa cells showing increased injury (%PI⁺), in representative histogram following 30 min exposure of HeLa to metacyclic trypomastigotes (Mtr) (red curves), compared to uninfected cells (green curves), in the presence of propidium iodide, (e). In (f), HeLa cells were injured by a 5:1 ratio of T. cruzi metacyclic forms (30 min/37°C) in the presence or absence of Ca²⁺. (g), HeLa cells (1.0 × 10⁶) were loaded with Fura 2‐AM at 37°C in nominal Ca²⁺‐free buffer and left to equilibrate for 5 min, from which point t = 0 s was taken. After 60 s, in calcium‐free buffer, thapsigargin (TG) was added and [Ca²⁺]_cyt monitored using a spectrophotometer over the ensuing 600 s, whereupon cells were incubated in 1.8 mM Ca²⁺ to stimulate SOCE (green line). HeLa were also infected with Mtr, (with no TG) but in the presence of extracellular Ca²⁺ (1.8 mM) (blue line). Also in the absence of TG, cells were also infected and then 240 s later, in the presence of Ca²⁺, treated with 2‐APB (5 µM) (red line). In (h), the effect on lEV release from infected HeLa cells of removing extracellular Ca²⁺ with EGTA was monitored. Other treatments included 2‐APB (SOCE modulator), YM‐58483 (CRAC channel inhibitor), U‐73122 (PLC inhibitor) and BAPTA‐AM (cell‐permeant Ca²⁺ chelator). HeLa cells were also stimulated with sublytic MAC (C5b‐9) to release lEVs. HeLa cells (1 × 10⁵/well) when pretreated with the Stretch‐Activated Calcium TRPC blocker, GsMTx‐4, showed marked reduction in infection with Mtr. HeLa cells (1 × 10⁶/well were infected with Mtr and showed reduced invasion with GsMTx‐4 (i)). To measure the G‐actin to F‐actin ratios in uninfected versus T. cruzi‐infected HeLa, G‐actin was measured by flow cytometry with DNase 1‐Alexa Fluor 488 conjugate (j) and F‐actin by Phalloidin Alex Fluor 660 (k) as described in Materials and Methods. Results in (l) show mean ± S.D from a representative experiment performed in triplicate, for the G/F‐actin ratios of HeLa cells infected (60 mpi), with or without GsMTx‐4. Upon infection of HeLa cells (1 × 10⁶/well) with Mtr (m), the greatest reduction in host cell mEV release was for GdCl₃ and GsMTx‐4. EGTA (5 mM) was used as a negative control for parasite‐stimulated Ca²⁺‐mediated mEV release. The inhibitors used to limit T. cruzi‐mediated lEV release did not work when the stimulus for lEV release was sublytic complement (n). Data represents the mean ± SD of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant.