title: Anaplasma phagocytophilumAPH0032 Is Exposed on the Cytosolic Face of the Pathogen-Occupied Vacuole and Co-opts Host Cell SUMOylation.
abstract: Anaplasma phagocytophilum, a member of the familyAnaplasmataceaeand the obligate intracellular bacterium that causes granulocytic anaplasmosis, resides in a host cell-derived vacuole. Bacterial proteins that localize to theA. phagocytophilum-occupied vacuole membrane (AVM) are critical host-pathogen interfaces. Of the few bacterial AVM proteins that have been identified, the domains responsible for AVM localization and the host cell pathways that they co-opt are poorly defined. APH0032 is an effector that is expressed and localizes to the AVM late during the infection cycle. Herein, the APH0032 domain that is essential for associating with host cell membranes was mapped. Immunofluorescent labeling of infected cells that had been differentially permeabilized confirmed that APH0032 is exposed on the AVM's cytosolic face, signifying its potential to interface with host cell processes. SUMOylation is the covalent attachment of a member of the small ubiquitin-like modifier (SUMO) family of proteins to lysines in target substrates. Previous work from our laboratory determined that SUMOylation is important forA. phagocytophilumsurvival and that SUMOylated proteins decorate the AVM. Algorithmic prediction analyses identified APH0032 as a candidate for SUMOylation. Endogenous APH0032 was precipitated from infected cells using a SUMO affinity matrix, confirming that the effector co-opts SUMOylation during infection. APH0032 pronouncedly colocalized with SUMO1, but not SUMO2/3 moieties on the AVM. Ectopic expression of APH0032 inA. phagocytophiluminfected host cells significantly boosted the bacterial load. This study delineates the first domain of anyAnaplasmataceaeprotein that is essential for associating with the pathogen-occupied vacuole membrane, demonstrates the importance of APH0032 to infection, and identifies it as the secondA. phagocytophilumeffector that co-opts SUMOylation, thus underscoring the relevance of this post-translational modification to infection.
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine Richmond, VA, USA.
authors.forename: Aminat T
authors.initials: AT
authors.lastname: Oki
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine Richmond, VA, USA.
authors.forename: Bernice
authors.initials: B
authors.lastname: Huang
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine Richmond, VA, USA.
authors.forename: Andrea R
authors.initials: AR
authors.lastname: Beyer
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine Richmond, VA, USA.
authors.forename: Levi J
authors.initials: LJ
authors.lastname: May
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine Richmond, VA, USA.
authors.forename: Hilary K
authors.initials: HK
authors.lastname: Truchan
authors.affiliation: Department of Pathology, Microbiology, and Immunology, University of California School of Veterinary Medicine Davis, CA, USA.
authors.forename: Naomi J
authors.initials: NJ
authors.lastname: Walker
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine Richmond, VA, USA.
authors.forename: Nathan L
authors.initials: NL
authors.lastname: Galloway
authors.affiliation: Department of Pathology, Microbiology, and Immunology, University of California School of Veterinary Medicine Davis, CA, USA.
authors.forename: Dori L
authors.initials: DL
authors.lastname: Borjesson
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine Richmond, VA, USA.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Carlyon
mesh_terms: D041081:Anaplasma phagocytophilum / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity*; D000712:Anaplasmosis / Q000382:microbiology*; D000818:Animals; D058491:Bacterial Load; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003600:Cytosol / Q000382:microbiology*; D004269:DNA, Bacterial; D015870:Gene Expression; D005798:Genes, Bacterial; D057809:HEK293 Cells; D018922:HL-60 Cells; D054884:Host-Pathogen Interactions / Q000502:physiology*; D006801:Humans; D050296:Microbial Viability; D018613:Microscopy, Confocal; D011499:Protein Processing, Post-Translational; D058207:Sumoylation / Q000502:physiology*; D014617:Vacuoles / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial
keywords: Anaplasmataceae; Rickettsia; SUMOylation; inclusion membrane protein; intracellular bacteria