The ecotin‐like peptidase inhibitor of Trypanosoma cruzi prevents TMPRSS2‐PAR2‐TLR4 crosstalk downmodulating infection and inflammation

Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in virtually all nucleated cells, using a variety of host cell receptors for infection. T. cruzi has a gene that encodes an ecotin‐like inhibitor of serine peptidases, ISP2. We generated ISP2 ‐null mutants (Δ isp 2) in T. cruzi Dm28c using CRISPR/Cas9. Epimastigotes of Δ isp 2 grew normally in vitro but were more susceptible to lysis by human serum compared to parental and ISP2 add‐back lines. Tissue culture trypomastigotes of Δ isp 2 were more infective to human muscle cells in vitro, which was reverted by the serine peptidase inhibitors aprotinin and camostat, suggesting that host cell epitheliasin/TMPRSS2 is the target of ISP2. Pretreatment of host cells with an antagonist to the protease‐activated receptor 2 (PAR2) or an inhibitor of Toll‐like receptor 4 (TLR4) selectively counteracted the increased cell invasion by Δ isp 2, but did not affect invasion by parental and add‐back lines. The same was observed following targeted gene silencing of PAR2, TLR4 or TMPRSS2 in host cells by siRNA. Furthermore, Δ isp 2 caused increased tissue edema in a BALB/c mouse footpad infection model after 3 h differently to that observed following infection with parental and add‐back lines. We propose that ISP2 contributes to protect T. cruzi from the anti‐microbial effects of human serum and to prevent triggering of PAR2 and TLR4 in host cells, resulting in the modulation of host cell invasion and contributing to decrease inflammation during acute infection.