Coxiella burnetii internalization is regulated by cholesterol and dynamins in epithelial cells

BERÓN, W; BERÓN, W; DISTEL, JS; DISTEL, JS; DIFRESCO, CP; DIFRESCO, CP

Mendoza

Congreso; Congreso: XXXVI Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2018

Sociedad de Biología de Cuyo

Cholesterol is an essential component of mammalian cell membranes. The level of cellular cholesterol results from the tight control of cholesterol neosynthesis and uptake. Dynamin proteins are a family of GTPases whose canonical function is the release of nascent endosomes from the plasma membrane, nevertheless, several works have also shown their participation in the metabolism and trafficking of cholesterol in different cell lines such as HeLa cells. A wide variety of bacteria preferentially interact with cholesterol-rich membrane microdomains, called "lipid rafts", to invade host cells. C. burnetii, a causal agent of Q Fiber, is an intracellular pathogen that enters into non-professional phagocytes through a mechanism poorly characterized. The goal of this work was to determine the role of cholesterol and dynamins in the internalization process of C. burnetii into non-professional phagocytic cells. To analyze the role of cholesterol in the internalization process, a cholesterol depleting agent Methyl-β-cliclodextrin (MβCD) and the inhibitor of cholesterol transport U18666A were used. HeLa cells seeded on coverslips in 24-well plates were infected for 6h with C. burnetii in the presence of MβCD or U18666A. Then, the cells were processed for indirect immunofluorescence and analyzed by confocal microscopy to quantify internalized bacteria. A significant decrease in the uptake of C. burnetii was observed in cells treated with MβCD. Internalization was lower in cells treated with U18666A than those treated with MβCD. To analyze the participation of dynamins in the bacterial entry into host cell, HeLa cells were infected in the presence of the inhibitor of dynamin Dynasore. The C. burnetii internalized by cells treated with the inhibitor decreased by 60% in comparison of untreated cells. To confirm the participation of dynamins in the internalization process, HeLa cells were transfected with pEGFP-dynamin I-WT (Wild Type), pEGFP-dynamin I-K44A (dynamin negative mutant defective in GTP binding and hydrolysis) or pEGFP-vector (control) and then infected with C. burnetii for 6h. While the overexpression of dynamin I WT showed a slight increase in the internalization, the overexpression of the mutant K44A produced a significant decrease. These results suggest that cholesterol and dynamins are involved in the internalization of C. burnetii by non-professional phagocytic cells.