-containing vacuoles interact with host recycling endosomal proteins Rab11a and Rab35 for vacuolar expansion and bacterial growth.

Introduction: is a gram-negative obligate intracellular bacterium and a zoonotic pathogen that causes human Q fever. The lack of effective antibiotics and a licensed vaccine for in the U.S. warrants further research into pathogenesis. Within the host cells, replicates in an acidic phagolysosome-like vacuole termed -containing vacuole (CCV). Previously, we have shown that the CCV pH is critical for survival and that the Type 4B secretion system regulates CCV pH by inhibiting the host endosomal maturation pathway. However, the trafficking pattern of the 'immature' endosomes in - infected cells remained unclear.

Methods: We transfected HeLa cells with GFP-tagged Rab proteins and subsequently infected them with mCherry- to visualize Rab protein localization. Infected cells were immunostained with anti-Rab antibodies to confirm the Rab localization to the CCV, to quantitate Rab11a and Rab35- positive CCVs, and to quantitate total recycling endosome content of infected cells. A dual-hit siRNA mediated knockdown combined with either immunofluorescent assay or an agarose-based colony-forming unit assay were used to measure the effects of Rab11a and Rab35 knockdown on CCV area and intracellular growth.

Results: The CCV localization screen with host Rab proteins revealed that recycling endosome-associated proteins Rab11a and Rab35 localize to the CCV during infection, suggesting that CCV interacts with host recycling endosomes during maturation. Interestingly, only a subset of CCVs were Rab11a or Rab35-positive at any given time point. Quantitation of Rab11a/Rab35-positive CCVs revealed that while Rab11a interacts with the CCV more at 3 dpi, Rab35 is significantly more prevalent at CCVs at 6 dpi, suggesting that the CCV preferentially interacts with Rab11a and Rab35 depending on the stage of infection. Furthermore, we observed a significant increase in Rab11a and Rab35 fluorescent intensity in -infected cells compared to mock, suggesting that increases the recycling endosome content in infected cells. Finally, siRNA-mediated knockdown of Rab11a and Rab35 resulted in significantly smaller CCVs and reduced intracellular growth, suggesting that recycling endosomal Rab proteins are essential for CCV expansion and bacterial multiplication.

Discussion: Our data, for the first time, show that the CCV dynamically interacts with host recycling endosomes for intracellular survival and potentially uncovers novel host cell factors essential for pathogenesis.