Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence

Abstract

2020): Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence, Autophagy, ABSTRACT Coxiella burnetii, the etiological agent of the zoonosis Q fever, replicates inside host cells within a large vacuole displaying autolysosomal characteristics. The development of this compartment is mediated by bacterial effectors, which interfere with a number of host membrane trafficking pathways. By screening a Coxiella transposon mutant library, we observed that transposon insertions in cbu0626 led to intracellular replication and vacuole biogenesis defects. Here, we demonstrate that CBU0626 is a novel member of the Coxiella vacuolar protein (Cvp) family of effector proteins, which is translocated by the Dot/Icm secretion system and localizes to vesicles with autolysosomal features as well as Coxiellacontaining vacuoles (CCVs). We thus renamed this effector CvpF for Coxiella vacuolar protein F. CvpF specifically interacts with the host small GTPase RAB26, leading to the recruitment of the autophagosomal marker MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) to CCVs. Importantly, cvpF::Tn mutants were highly attenuated compared to wild-type bacteria in the SCID mouse model of infection, highlighting the importance of CvpF for Coxiella virulence. These results suggest that CvpF manipulates endosomal trafficking and macroautophagy/autophagy induction for optimal C. burnetii vacuole biogenesis.

Interfering with Autophagy: The Opposing Strategies Deployed by Legionella pneumophila and Coxiella burnetii Effector Proteins

“…During infection of U2OS osteosarcoma cells, both WT and CvpF transposon mutant C. burnetii strains developed acidified CCVs decorated with LAMP1, however CCVs produced by CvpF mutants were deficient in LC3B. Further, CvpF transposon mutants did not induce an increase in lipidated LC3B as seen in WT infections (Siadous et al, 2020). However, unlike Cig57, SQSTM-1 was still increased during infection with CvpF transposon mutants and reduced by starvation-induced autophagy.…”

“…A yeast two-hybrid screen identified the GTPase Rab26 as a binding partner of CvpF, with fluorescence microscopy confirming their colocalization and also observing that CvpF increases the membrane targeting of Rab26 (Siadous et al, 2020). CvpF was found to promote the accumulation of Rab26 at the CCV, although low level accumulation was still observed in CvpF mutants.…”

“…More recently, an additional C. burnetii vacuolar protein, CvpF (CBU0626) was identified as important for intracellular replication of C. burnetii and CCV formation while being dispensable for replication in axenic medium (Siadous et al, 2020). During infection of U2OS osteosarcoma cells, both WT and CvpF transposon mutant C. burnetii strains developed acidified CCVs decorated with LAMP1, however CCVs produced by CvpF mutants were deficient in LC3B.…”

The secreted protein kinase CstK from Coxiella burnetii influences vacuole development and interacts with the GTPase-activating host protein TBC1D5

“…Among these, the Rab GAP/TBC domain-containing protein, DDB_G0280253 (UniProtKB -Q54VM3), is a 136.4 kDa protein homologous to mammalian TBC1D5 (http://dictybase.org), a GTPaseactivating protein (GAP) for Rab7a and Rab7b (44)(45)(46) that acts as a molecular switch between the endosomal and the autophagy pathway (47). Given the recently reported implication of TBC1D5 in the biogenesis of L. pneumophila-containing vacuoles (48) and the role of autophagy in the biogenesis of CCVs (25,31,49), we aimed at validating the interaction between human TBC1D5 (Hs-TBC1D5) and CstK in HEK-293T cells. Cells coexpressing Hs-TBC1D5-GFP and HA-CstK or CstK mutants were used for immunoprecipitation using anti-HA beads.…”

“…Some of these effectors modulate important signaling pathways of infected cells, including apoptosis and inflammasome activation (27)(28)(29), whereas others are essential for the development of the intracellular replicative niche. Among these CvpB and CvpF have been recently implicated in the manipulation of autophagy for optimal vacuole development (30)(31)(32)(33). C. burnetii genome analysis revealed a close homology to the facultative intracellular pathogen Legionella pneumophila, in particular at the level of Dot/Icm core genes (34).…”