The GTPase ARF6 regulates HeLa infection with Coxiella burnetii

CARMINATI S,; ROSALES E,; SALINAS R,; BERÓN W

Rosario, Santa Fe

Congreso; XLII Reunión Nacional de la Sociedad Argentina de Investigaciones Bioquímica y Biología Molecular (SAIB); 2006

Sociedad Argentina de Investigaciones Bioquímica y Biología Molecular (SAIB)

ARF6 is a GTPase involved in vesicular trafficking processes such as phagocytosis. Also, it regulates the actin dynamics through PIP5K and Rac proteins. It is known that actin cytoskeleton dynamics is critical for phagocytosis. After internalization, bacteria are transported to lysosomes for their degradation. The intracellular pathogen C. burnetii replicates in a parasitophorous vacuole (PV), a compartment with lysosomal characteristics. Our interest is to study the role of ARF6 and actin in the phagocytosis process of C. burnetii. HeLa cells were transfected with plasmids that encode ARF6WT (wild type), Q67L (constitutively active) or T27N (constitutively inactive) mutants fused to hemaglutinin (HA) or green fluorescent protein (GFP) tags. After 16 h infection, the cells were washed to remove extracellular bacteria and incubated for another 24h. Cells were processed by indirect immunofluorescence to detect C. burnetii or HA tag using specific antibodies and analyzed by fluorescence and confocal microscopy. We observed that infection was significantly inhibited in cell expressing the T27N mutant comparing with cells expressing the WT or Q67L mutant of ARF6. Interestingly, we observed that ARF6 WT and Q67L mutant were recruited to the PV together with actin. These results suggest that C. burnetii infection of host cells is regulated by ARF6 and actin.