Disruption of the secretory pathway alters the development of the Coxiella replicative vacuole

CAMPOY, E.; ZOPPINO, F.C. M.; COLOMBO, M. I.

Tucumán, Argentina

Congreso; XLV Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

SAIB

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular bacterium, which replicates within large vacuoles with features of both, phagolysosomes and autolysosomes. We have previously demonstrated that the Coxiella replicative vacuole (CRV) interacts with the secretory pathway since Rab1b Q67L (GTPase defective mutant of Rab1b) was present in the vacuole membrane at later infection times (48 h). We also reported that overexpression of Rab1b altered the normal development of the CRV by changing its fusogenic capacity. In the present work we have analyzed the development of the CRV in conditions of disrupted secretory pathway. As a first strategy, pharmacological ERGolgi traffic disruption by Brefeldin A (BFA)-treatment was applied. BFA is an uncompetitive inhibitor of Arf1 activation. Cell treatment with BFA results in the specific inhibition of an Arf1 guanine-nucleotide exchange factor, and efficiently and reversibly blocks membrane traffic to the Golgi. As a second strategy, we blocked the secretory pathway at early steps by overexpression of GDP- and GTP-restricted mutants of Sar1 (Sar1 T39N and H79G) which strongly inhibits vesicle budding from the ER. The CRV diameter was dramatically altered under both conditions, indicating that a functional secretory pathway is essential for the normal C. burnetii intracellular development.