Rac1/WAVE2 and Cdc42/N-WASP Participation in Actin-Dependent Host Cell Invasion by Extracellular Amastigotes of Trypanosoma cruzi

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.

Autophagy: A necessary process during the Trypanosoma cruzi life-cycle

“…The infective forms of T. cruzi are metacyclic trypomastigotes and bloodstream trypomastigotes, derived from epimastigotes and amastigotes, respectively, both with the capacity to invade a large number of different cell types in a process independent of host cell actin polymerization [3]. The so-called extracellular amastigotes, originated from the premature rupture of infected cells or transformed from swimming trypomastigotes, are also infective forms that, in contrast to trypomastigotes, require functional intact microfilaments to infect non-phagocytic host cells [4,5].…”

Amastigote Synapse: The Tricks of Trypanosoma cruzi Extracellular Amastigotes

“…We previously observed that Rac1 is important for the EA invasion of transfected MDCK (Madin-Darby canine kidney) cells ( Fernandes and Mortara, 2004 ). Re-examining the effects of Rho GTPases by depletion (inhibition) or overexpression of constitutively active (CA, enhancer) or dominant negative (DN, inhibitor) forms, we confirmed that among these proteins, Rac1 has a major role in cup formation and parasite invasion in HeLa cells ( Bonfim-Melo et al, 2018 ). Cells overexpressing Rac1-CA had increased EA internalization, whereas a decrease was observed in cells overexpressing Rac1-DN ( Bonfim-Melo et al, 2018 ).…”

“…In 2013, we showed that EAs mobilize host phosphoinosites (light and dark gray) along with actin in a phagocytosis-like mechanism ( Fernandes et al, 2013 ). In 2015 Bonfim-Melo et al (2018) described the activation (upward blue arrows) of PKD1 and ERK but not SFKs upon EA-HeLa interaction with cortactin and PKD1 phosphorylation (orange P) and participation in EA invasion. In 2016 Ferreira et al (2016) described that TcMVK (yellow) is secreted by EAs promoting their invasion and the activation of diverse actin related host proteins (orange).…”

“…Additionally, in 2017, we described that ezrin and radixin (cyan), but not moesin (light gray) are involved in actin regulation and EA invasion independent of their phosphorylation (light gray P) activation mechanism ( Ferreira et al, 2017 ). Recently, we observed the participation of Rho GTPases (Rac1 and Cdc42) and their effector proteins regulating actin polymerization signaling and promoting EA invasion, although RhoA inhibited these events (green) ( Bonfim-Melo et al, 2018 ). The surface protein Ssp-4 is differentially expressed among T. cruzi strains and can modulate EA invasion through its 1D9 carbohydrate epitope, ligand of host galectin-3 (brown) ( Florentino et al, 2018 ).…”

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

“…Exogenous Gal-3 is able to activate Rac-1 in corneal epithelial cells ( Saravanan et al, 2009 ). Interestingly, our group has previously revealed the participation of proteins associated with the Rho-GTPase family, such as Rac-1, in EA internalization ( Fernandes and Mortara, 2004 ; Bonfim-Melo et al, 2018 ). Members of these families are responsible for mediating the recruitment of the actin cytoskeleton with cell surface receptors.…”