Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development

Abstract

The Q fever bacterium Coxiella burnetii replicates inside host cells within a large Coxiella-containing vacuole (CCV) whose biogenesis relies on the Dot/Icm-dependent secretion of bacterial effectors. Several membrane trafficking pathways contribute membranes, proteins, and lipids for CCV biogenesis. These include the endocytic and autophagy pathways, which are characterized by phosphatidylinositol 3-phosphate [PI(3)P]-positive membranes. Here we show that the C. burnetii secreted effector Coxiella vacuolar protein B (CvpB) binds PI(3)P and phosphatidylserine (PS) on CCVs and early endosomal compartments and perturbs the activity of the phosphatidylinositol 5-kinase PIKfyve to manipulate PI(3)P metabolism. CvpB association to early endosome triggers vacuolation and clustering, leading to the channeling of large PI(3)P-positive membranes to CCVs for vacuole expansion. At CCVs, CvpB binding to early endosome-and autophagy-derived PI(3)P and the concomitant inhibition of PIKfyve favor the association of the autophagosomal machinery to CCVs for optimal homotypic fusion of the Coxiella-containing compartments. The importance of manipulating PI(3)P metabolism is highlighted by mutations in cvpB resulting in a multivacuolar phenotype, rescuable by gene complementation, indicative of a defect in CCV biogenesis. Using the insect model Galleria mellonella, we demonstrate the in vivo relevance of defective CCV biogenesis by highlighting an attenuated virulence phenotype associated with cvpB mutations.Coxiella burnetii | host-pathogen interactions | phosphoinositides

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

“…Despite no obvious replication defect, the altered, multi-vacuole CCV state appears to significantly affect the virulence of Coxiella . In Galleria mellonella , injection of WT Coxiella results in efficient killing of larvae and this is attenuated in the absence of cig2 [15,34]. Similarly, cig2 ::Tn bacteria were recovered from the spleen and lung of severe combined immunodeficiency (SCID) mice at significantly lower levels than WT Coxiella [35].…”

“…Recent research into the pathogenesis of Coxiella has identified several important Dot/Icm effectors including Cig2, which binds phosphatidylinositol-3-phosphate and phosphatidylserine [34] and is necessary for LC3B delivery to the CCV, and Cig57, which interacts with FCHO2 and is necessary for recruitment of CLTC to the CCV [18]. Given that we did not see LC3B-I conversion to LC3B-II during infection with cig57 ::Tn, there may be a role for Cig57 in linking CLTC to autophagosomes.…”

Right on Q: Genetics begin to Unravel Coxiella Burnetii host cell Interactions

“…Collectively, these data suggest CvpA targets clathrin-mediated vesicular trafficking that c ontributes to CCV biogenesis. Three groups have demonstrated that mutation of the effector gene cbu0021 (cvpB, cig2) results in aberrant CCV formation and/or Coxiella growth [50,60,107,131]. Vacuoles harboring cbu0021 mutants do not homotypically fuse, resulting in a multivacuole phenotype [60,107,131].…”

“…Three groups have demonstrated that mutation of the effector gene cbu0021 (cvpB, cig2) results in aberrant CCV formation and/or Coxiella growth [50,60,107,131]. Vacuoles harboring cbu0021 mutants do not homotypically fuse, resulting in a multivacuole phenotype [60,107,131]. In two investigations, cbu0021 mutants also display deficient replication [50,107].…”

“…Depletion of the autophagy regulators syntaxin 7, ATG5 or ATG7 during infection with wild-type bacteria results in a multiple vacuole phenotype similar to that associated the cbu0021 mutant, suggesting a functional link between the Coxiella effector, autophagy and CCV fusogenic properties [60]. Indeed, CBU0021 binds phosphoinositides on host cell membranes and perturbs the activity of the phosphatidylinositol 3-kinase PIKfyve to enrich phosphatidylinositol 3-phosphate (PI[3] P) on CCVs [131]. This favors association of autophagosomal machinery with CCVs and their homotypic fusion [131].…”