Abstract
This study evaluated the participation of host cell Rho-family GTPases and their effector proteins in the actin-dependent invasion by Trypanosoma cruzi extracellular amastigotes (EAs). We observed that all proteins were recruited and colocalized with actin at EA invasion sites in live or fixed cells. EA internalization was inhibited in cells depleted in Rac1, N-WASP, and WAVE2. Time-lapse experiments with Rac1, N-WASP and WAVE2 depleted cells revealed that EA internalization kinetics is delayed even though no differences were observed in the proportion of EA-induced actin recruitment in these groups. Overexpression of constitutively active constructs of Rac1 and RhoA altered the morphology of actin recruitments to EA invasion sites. Additionally, EA internalization was increased in cells overexpressing CA-Rac1 but inhibited in cells overexpressing CA-RhoA. WT-Cdc42 expression increased EA internalization, but curiously, CA-Cdc42 inhibited it. Altogether, these results corroborate the hypothesis of EA internalization in non-phagocytic cells by a phagocytosis-like mechanism and present Rac1 as the key Rho-family GTPase in this process.
Highlights
Trypanosoma cruzi is a protozoan parasite that causes Chagas’ disease and affects approximately 6–7 million people worldwide, mostly in Latin America (WHO., 2017)
Using cells depleted of or overexpressing these proteins and microcopy techniques, we found that Rac1 is the key Rho GTPase in this process, possibly acting together with WAVE2, whereas Cdc42 displays a minor role in parallel with N-WASP participation; RhoA had a negative role in the regulation of actin dynamics involved in extracellular amastigotes (EAs) entry
Considering the observations from our previous work, the present study aimed to evaluate host cell Rho-family GTPases and their effector proteins in actin dynamics during HeLa cell invasion by EAs, possibly providing new insights into this recently proposed mechanism for parasite entry
Summary
Trypanosoma cruzi is a protozoan parasite that causes Chagas’ disease and affects approximately 6–7 million people worldwide, mostly in Latin America (WHO., 2017). Unlike the metacyclic or bloodstream trypomastigote forms, host cell invasion by extracellular amastigotes (EAs) is highly dependent on the actin cytoskeleton of host cell (Mortara et al, 2005; Ferreira et al, 2012). Our group showed that EAs induce selective phosphorylation of cortactin by ERK, which is abolished if heat-killed parasites or non-infective epimastigote forms are used (Bonfim-Melo et al, 2015). These studies demonstrate the importance of the actin cytoskeleton and its regulatory proteins during EA invasion of non-phagocytic cells
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