ROLE OF ORP1L AND NPC1 PROTEINS IN THE BIOGENESIS OF THE Coxiella burnetii PARASITOPHOROUS VACUOLE

ORTIZ RM; DISTEL JS; ALBANES M; AGUILERA MO; BERÓN W

Mar del Plata

Congreso; LI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2015

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L, forming the tripartite motor complex. ORP1L sense cholesterol levels in LEs. Under high cholesterol conditions, LEs accumulate at the microtubule minus end as the result of dynein motor activity. Coxiella burnetii (Cb) is an intracellular obligate pathogen, which transits through the phagocytic pathway of host cell to form a parasitophorous vacuole (PV). To determine the role of ORP1L in the PV biogenesis, HeLa cells were transfected with pGFP or pRFP-ORP1L, pEGFP-p150Glued, pEGFP-RILP and the negative mutants DsRed-ORP1LΔORD and then infected with Cb. In the other hand, HeLa cells were infected with Cb followed by the incubation with the U188666A inhibitor. For Indirect Immunofluorescence cells were processed and following parameters were determined: number of infected cells, Cb intracellular multiplication, number and size of PVs, and recruitment of the studied proteins on PVs. The results showed that wild type forms of overexpressed proteins recruited on the PV membrane without significantly affecting its size and number. The size of VPs decreased and their number increased when the mutated proteins were overexpressed, similarly effects we saw with inhibitor. In conclusion, our results show an important role of cholesterol in the biogenesis and integrity of the PV.