Cantet, F. | bioRxiv Science

bioRxiv2024

The multifunction Coxiella effector Vice stimulates macropinocytosis and interferes with the ESCRT machinery

Intracellular bacterial pathogens divert multiple cellular pathways to establish their niche and persist inside their host. Coxiella burnetii, the causative agent of Q fever, secretes bacterial effector proteins via its Type 4 secretion system to generate a Coxiella-containing vacuole (CCV). Manipulation of lipid and protein trafficking by these effectors is essential for bacterial replication and virulence. Here, we have characterized the lipid composition of CCVs and discovered that the effector Vice interacts with phosphoinositides and membranes enriched in phosphatidylserine (PS) and lysobisphosphatidic acid (LBPA). Remarkably, eukaryotic cells ectopically expressing Vice present compartments that resemble early CCVs in both morphology and composition. We discovered that the biogenesis of these compartments relies on the double function of Vice. The effector protein initially localizes at the plasma membrane of eukaryotic cells where it triggers the internalization of large vacuoles by macropinocytosis. Then, Vice stabilizes these compartments by perturbing the ESCRT machinery and inhibiting the formation of intraluminal vesicles (ILVs). Collectively, our results reveal that Vice is an essential C. burnetii effector protein capable of hijacking two major cellular pathways to shape the bacterial replicative niche. Significance statementCoxiella burnetii is a unique bacterial pathogen that secretes more than a hundred effector proteins to manipulate cellular processes and establish a replicative niche, the Coxiella-containing vacuole (CCV). Our study identified host cell lipids that are actively recruited by the bacterium to the CCV. Using a library of effector mutants, we identified the protein Vice (for Vacuole-inducing Coxiella effector) as the first bacterial effector capable of interacting with lysobisphosphatydic acid-enriched membranes and accumulating this lipid to CCVs. We show that Vice is also capable of stimulating macropinocytosis and inhibiting the ESCRT machinery. Together, our data show how a single bacterial effector can manipulate different cellular processes to favor the biogenesis of a bacterial pathogens niche.

microbiology↗

bioRxiv2019

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

Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to the pathogenesis of this intracellular pathogen is the secretion of bacterial effectors into host cells by a Type 4b Secretion System (T4SS), to subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole allowing intracellular replication. The characterization of prokaryotic Serine/Threonine Protein Kinases (STPKs) in bacterial pathogens is emerging as an important strategy to better understand host-pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a bacterial protein kinase identified in C. burnetii by in silico analysis. Here, we demonstrated that this putative protein kinase undergoes autophosphorylation on Ser, Thr, and Tyr residues, and phosphorylates a classical eukaryotic protein kinase substrate in vitro. This dual Ser/Thr and Tyr kinase activity is similarly observed for eukaryotic dual specificity Tyr phosphorylation-regulated kinase class. CstK is translocated during infections and localizes at Coxiella-containing vacuoles (CCVs). Moreover, a C. burnetii mutant strain overexpressing CstK displays a severe CCVs development phenotype, suggesting a finely tuned regulation by the bacterial kinase during infection. Protein-protein interaction studies identified the Rab7-GTPase activating protein (GAP) TBC1D5 as a candidate CstK-specific host target, suggesting a role for this eukaryotic GAP in Coxiella infections. Indeed, CstK colocalizes with TBC1D5 in non-infected cells, and TBC1D5 is recruited at CCVs during infection. Accordingly, depletion of TBC1D5 from infected cells significantly affects CCVs development. Our results indicate that CstK has a critical role during infection as a bacterial effector protein that interacts with host proteins to facilitate vacuole biogenesis and intracellular replication.

microbiology↗