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

Abstract

The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system–mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases 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 protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro. This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella-containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein–protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication.

Targeting Endosomal Recycling Pathways by Bacterial and Viral Pathogens

“…More recently, the C. burnetii effector Cstk1 was shown to regulate the biogenesis of the pathogen vacuole ( Martinez et al, 2020 ). Cstk1 is a Ser/Thr/Tyr kinase and interacts with TBC1D5.…”

“…Remarkably, recent studies have also uncovered that many intracellular bacteria, including Chlamydia trachomatis ( Mirrashidi et al, 2015 ), Legionella pneumophila ( Finsel et al, 2013 ), Salmonella Typhimurium ( D’Costa et al, 2019 ), Burkholderia cenocepacia ( Walpole et al, 2020 ), and Coxiella burnetii ( Martinez et al, 2020 ), target host endosomal recycling pathways for their survival and replication ( Table 1 ). These bacteria pathogens utilize different types of secretion systems and a plethora of effector proteins to create a unique vacuolar compartment known as pathogen vacuoles or inclusion, in which they can replicate.…”

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

“…For example, when ectopically expressed the effector CpeB (CBUA0013) colocalizes with LC3B, and CpeL (CBUDA0024) partially colocalized with autophagosomes (Voth et al, 2011;Maturana et al, 2013). Additionally, the protein kinase CstK (CBU0175) was found to localize at vesicles and the CCV and interacts with a homologue of mammalian TBC1D5 in an amoeba model (Martinez et al, 2020). TBC1D5 is a GTPase activating protein for Rab7, which is involved in lysosomal biogenesis, positioning and function, as well as fusion with autophagosomes (Guerra and Bucci, 2016).…”

Ticks convert pathogenicCoxiellainto endosymbionts

“…Among them are genes for T4BSS and for effectors secreted through T4BSS ( Fig. 2; Supplemental Table S2) that enable C. burnetii to generate its intracellular replicative niche, termed Coxiella-containing vacuole (CCV) (Chen et al 2010;Beare et al 2011;Martinez et al 2014Martinez et al , 2020Newton et al 2014). In addition, genes for PmrAB and EirA that control T4BSS activity, and for eight T4BSS effectors present on C. burnetii's QpH1 plasmid have been inactivated in CLEOA (Maturana et al 2013;Beare et al 2014;Kuba et al 2020).…”