title: 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.
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
authors.forename: Punsiri M
authors.initials: PM
authors.lastname: Colonne
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
authors.forename: Caylin G
authors.initials: CG
authors.lastname: Winchell
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Graham
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
authors.forename: Frances I
authors.initials: FI
authors.lastname: Onyilagha
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: MacDonald
authors.affiliation: Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, United States of America.
authors.forename: Heike R
authors.initials: HR
authors.lastname: Doeppler
authors.affiliation: Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, United States of America.
authors.forename: Peter
authors.initials: P
authors.lastname: Storz
authors.affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.|Arkansas Children's Hospital Research Institute, Little Rock, Arkansas, United States of America.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Kurten
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Beare
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Robert A
authors.identifier: http://orcid.org/0000-0001-6870-4619
authors.initials: RA
authors.lastname: Heinzen
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Voth
mesh_terms: D015815:Cell Adhesion Molecules / Q000378:metabolism*; D016997:Coxiella burnetii; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D055785:Gene Knockdown Techniques; D054884:Host-Pathogen Interactions / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D016676:Macrophages, Alveolar / Q000378:metabolism* / Q000382:microbiology*; D008840:Microfilament Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D010750:Phosphoproteins / Q000378:metabolism*; D011778:Q Fever / Q000378:metabolism*; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D017868:Cyclic AMP-Dependent Protein Kinases