title: Coxiella burnetii effector proteins that localize to the parasitophorous vacuole membrane promote intracellular replication.
abstract: The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supports C. burnetii replication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promote C. burnetii intracellular growth and PV expansion. We predict additional C. burnetii effectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predicted C. burnetii T4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion by C. burnetii during infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termed Coxiella vacuolar protein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins. C. burnetii ΔcvpB, ΔcvpC, ΔcvpD, and ΔcvpE mutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ΔcvpD and ΔcvpE mutants rescued intracellular growth and PV generation, whereas the growth of C. burnetii ΔcvpB and ΔcvpC was rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicate C. burnetii encodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages.
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Charles L
authors.initials: CL
authors.lastname: Larson
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Beare
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Voth
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Dale
authors.initials: D
authors.lastname: Howe
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Diane C
authors.initials: DC
authors.lastname: Cockrell
authors.affiliation: Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University, Durham, North Carolina, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Bastidas
authors.affiliation: Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University, Durham, North Carolina, USA.
authors.forename: Raphael H
authors.initials: RH
authors.lastname: Valdivia
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA rheinzen@niaid.nih.gov.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Heinzen
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems / Q000502:physiology; D045744:Cell Line, Tumor; D002462:Cell Membrane; D016997:Coxiella burnetii / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D017353:Gene Deletion; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D008264:Macrophages / Q000382:microbiology*; D021381:Protein Transport / Q000235:genetics*; D011778:Q Fever / Q000382:microbiology / Q000473:pathology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D011993:Recombinant Fusion Proteins