Ezrin and proteins of the RhoA pathway participate in Coxiella burnetii internalization

DISTEL JS; ORTIZ FLORES R; COLOMBO M; BERÓN W.

Santa Fé

Congreso; L Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Coxiella burnetii (Cb) is an obligate intracellular bacterium that causes Q fever in humans. We have shown that internalization of Cb by HeLa cells is regulated by actin and Rho GTPases. It is known that Rho proteins are regulated by GEF, GAP and RhoGDI. Moreover, RhoA activates ROCK, a kinase that phosphorylates Ezrin, a protein that acts as a linker between the plasma membrane and the cortical F-actin. Our goal was to study the role of Ezrin and some RhoA-interacting proteins in Cb internalization. HeLa cells were transfected with pMyc-ROCK (DN). -ROCK (CAT), pVSVG Ezrin-Nter, pEGFP-Ezrin WT, -Ezrin T567A, Ezrin T567D, pEGFP-RhoGDIα WT, -RhoGDIα (D45A) or -RhoGDIα (D185A), and then infected with Cb. After 6 h, cells were processed for indirect immunofluorescence and analyzed by confocal microscopy. We observed that overexpression of the truncated N-terminal ezrin inhibited Cb uptake. We also found that overexpression of a non-phosphorylatable (T567A) mutant decreased Cb internalization while the phosphomimetic (T567D) mutant increased it. An increased uptake was observed in cell overexpressing a catalytic domain (CAT) of ROCK. Opposite result was observed with the dominant negative mutant (DN). Finally, the overexpressión of RhoGDIα WT reduced Cb uptake. These results suggest that ezrin and proteins associated to RhoA pathway participate in Cb internalization into HeLa cells.