Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages

Abstract

Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages.

Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth

“…Cytoskeletal requirements for CCV formation are poorly defined, but some information exists on the role of filamentous (F-) actin. Colonne and co-workers [ 25 ] described an actin ring circumscribing the mature CCV in THP-1 macrophages that labels with vasodilator-stimulated phosphoprotein (VASP), an actin regulatory protein. Depletion of VASP disrupts CCV formation and heterotypic fusion with latex bead-containing phagosomes.…”

“…Colonne at al . [ 25 ] propose that CCV-associated F-actin stabilizes the vacuole, and that actin rearrangements enable vacuole expansion. In HeLa cells, Aguilera and co-workers [ 26 ] observed CCV association with F-actin, either as an encompassing ring or in patches.…”

Type IV Secretion in Gram-Negative and Gram-Positive Bacteria

“…Computer modeling coupled with a validation approach also has facilitated the identification of T4SS secretion signals that may prove useful for discovering novel effector proteins in Legionella and Coxiella (Lifshitz et al, 2013). The endosomal nature of CCVs is reflected by the accumulation of late endosomal markers LAMP-1/-2, vasodilator-stimulated phosphoprotein (VASP), as well as the V-ATPase (Voth and Heinzen, 2007;Colonne et al, 2016). Genomewide gene silencing screens using siRNA identified additional host factors including the retromer complex (McDonough et al, 2013).…”

Transfer of HTLV-1 p8 and Gag to target T-cells depends on VASP, a novel interaction partner of p8

“…Contrary, during infections with the intracellular bacterial pathogens Shigella spp., which cause diarrheal disease in humans, VASP and the related protein Ena/VASP-like (EVL) limit bacterial spread [61]. Interestingly, VASP activity is also required for Coxiella burnetii growth in human macrophages [64]. Among viruses, Vaccinia uses actin-based motility for virion movement in host cells, and recruits the Ena/VASP family members N-WASP and VASP to the site of actin assembly [65,66].…”