Role of host cell Dynein/Dynactin in the biogenesis of the parasitophorous vacuole where Coxiella burnetii resides and multiplies

ORTIZ FLORES, RM; DISTEL, JS; COLOMBO, MI; AGUILERA, MO; BERÓN, W

Puerto Natales

Workshop; Workshop Current advances in membrane trafficking: Implications for polarity and diseases; 2014

EMBO Courses & Workshops

Dynein/dynactin motor complex plays an important role in membrane trafficking in mammalian cells. This is a multisubunit molecular motor that drives centripetal transport of membranous cargoes along microtubules (Mts). Dynactin consists of distinct subunits such as p150Glued and dynamitinp50. Small GTPases of Rab family interact with motor proteins. Rab7, through its effector RILP, associates dynein/dynactin motor to late endosomal compartments which are transported to perinuclear region. Coxiella burnetii (Cb) is an intracellular obligate pathogen, causal agent of fever Q, that transits through the phagocytic pathway of host cell to form a compartment with autophagolysosomal features called parasitophorous vacuole (PV). Rab7 is recruited to PV and regulates PV formation. Little evidence exists about the interrelation of intracellular trafficking of Cb with the microtubule-motor system. To determine the role of Mts/dynein/dynactin and Rab7/RILP in the PV biogenesis, HeLa cells were transfected with pEGFP-p150Glued, pEGFP-Dynamitina-p50, pEGFP-RILP, pEGFP-Rab7 or negative mutants (pRFP-p150Glued CC1, pRFP-RILPΔN, pEGFP-Rab7T22N) and then infected with Cb. After 48 h, cells were processed for Indirect Immunofluorescence and analyzed by confocal microscopy. The following parameters were determined: number of infected cells, Cb intracellular multiplication (FFU assay), number and size of PVs, and recruitment of the studied proteins on PVs. The results showed that wild type forms of overexpressed proteins were recruited on the PV membrane without significantly affecting its size and number compared with the EGFP. The size of VPs decreased and their number increased when the mutated proteins were overexpressed. Interestingly these mutants were recruited onto PV membrane. The Cb intracellular multiplication was diminished in cell overexpressing the mutated proteins regarding to those overexpressing the wild type proteins. In conclusion, our results suggest the transport of early C. burnetii-containing phagosomes would use the Mts/dynein/dynactin system to be transported from the cell periphery to the perinuclear region to promote PV formation.