A Rap1b activation and ERK phosphorylation during cAMP/Epac-mediated invasion by Trypanosoma cruzi G

MARTIN EDREIRA; GABRIEL FERRI; DANIEL MUSIKANT

Congreso; Molecular Parasitology Meeting XXXI; 2020

Cyclic AMP has been shown to play critical roles during host cell invasion by T. cruzi. Ca2+ release from cellular compartments, such as theendoplasmic reticulum, is accompanied by an elevation of intracellular cAMP levels and it has been shown that cAMP is able to potentiate the Ca2+-dependentexocytosis of lysosomes during host cell invasion. We previously demonstrated that Epac1-mediated signaling represents the main mechanism for cAMPmediated host cell invasion. Furthermore, Epac1 has been involved in PI3K/Akt and MEK/ERK pathways, and members of these pathways, including Rap1, werelocalized at late endosomes/lysosomes. In this work, we investigated the involvement of two downstream effectors, Rap1b and ERK, in Epac-mediated invasion.Active GTP-bound Rap1 was detected in lysates from infected cells by pull-down experiments through specific protein interaction with a GST-RalGDS Rap1-binding domain. In line with these results, invasion significantly increased in cells transfected with the constitutively active form of Rap1b (G12V) when comparedwith the DMSO control and a dominant negative mutant of Rap1b (N17). Regarding ERK participation, we first showed that trypomastigotes induced ERKphosphorylation. In addition, treatment with PD98059, an inhibitor of MEK1/2, suppressed ERK phosphorylation and induced a significant decrease in T. cruziinvasion. Interestingly, co-inhibition of Epac1 and ERK phosphorylation showed no additive or synergistic effects, suggesting that ERK is a downstream effector ofcAMP/Epac-mediated host cell invasion.