Coxiella burnetii infection to the host cell needs Arf6 and its interacting protein EFA6 and PIP5K

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

Puerto Madryn

Congreso; XLVI Reunión Nacional de la Sociedad Argentina de Investigaciones Bioquímica y Biología Molecular (SAIB).; 2010

Sociedad Argentina de Investigaciones Bioquímica y Biología Molecular (SAIB).

It is known that remodeling of host actin cytoskeleton is needed for phagocytosis of Coxiella burnetii nevertheless the molecular mechanism is poorly characterized. The GTPases Arf6 is implicated in phagocytosis regulating actin cytoskeleton through PIP5 kinase and phospholipase D (PLD) activation. GTPases need to be loaded with GTP by a guanine-exchange factor (GEF) to become active. To determine if EFA6, an Arf6 GEF, is involved in C. burnetii phagocytosis, HeLa cells over-expressing EFA6 WT or its inactive mutant R386E were infected and processed for indirect immunofluorescence to quantify the internalized bacteria. We observed that the EFA6-R386E inhibited C. burnetii internalization. This result agrees with the previous one observed in cells over-expressing an Arf6 negative mutant. We study PIP5K activation during C. burnetii internalization determining the formation of PI(4,5)P2, the product of this kinase. To this end, cells over-expressing GFP-PH, a PLCalfa-PH domain which binds PI(4,5)P2, were infected for different times. We observed that after 15 min infection GFP-PH accumulated at the membrane in close contact with the bacteria. To evaluate PLD participation in C. burnetii infection we inhibited PLD with 2-butanol cell treatment. We observed no effect in the C. burnetii internalization. In summary our results suggest that host cell infection with C. burnetii is regulated by Arf6 and their interacting proteins EFA6 and PIP5K.