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

CAMPOY, EMANUEL MARTIN; ZOPPINO, FELIPE CARLOS; COLOMBO MARIA ISABEL

Tucuman

Congreso; Reunion SAIB XLV; 2009

SAIB

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellularγ-proteobacterium, which replicates within large vacuoles with features ofboth, phagolysosomes and autolisosomes. We have previouslydemonstrated that the Coxiella replicative vacuole (CRV) interacts with thesecretory pathway since Rab1b Q67L (GTPase defective mutant of Rab1b)was present in the vacuole membrane at later infection times (48 h). Wealso reported that overexpression of Rab1b altered the normal developmentof the CRV by changing its fusogenic capacity. In the present work we haveanalyzed the development of the CRV in conditions of disrupted secretorypathway. As a first strategy, pharmacological ER–Golgi traffic disruption byBrefeldin A (BFA)-treatment was applied. BFA is an uncompetitive inhibitorof Arf1 activation. Cell treatment with BFA results in the specific inhibition ofan Arf1 guanine-nucleotide exchange factor, and efficiently and reversiblyblocks membrane traffic to the Golgi. As a second strategy, we blocked thesecretory pathway at early steps by overexpression of GDP- and GTPrestrictedmutants of Sar1 (Sar1 T39N and H79G) which strongly inhibitsvesicle budding from the ER. The CRV diameter was dramatically alteredunder both conditions, indicating that a functional secretory pathway isessential for the normal C. burnetii intracellular development.