title: Anaplasma phagocytophilum APH_0032 is expressed late during infection and localizes to the pathogen-occupied vacuolar membrane.
abstract: Anaplasma phagocytophilum infects neutrophils and myeloid, endothelial, and tick cell lines to reside within a host cell-derived vacuole that is indispensible for its survival. Here, we identify APH_0032 as an Anaplasma-derived protein that associates with the A. phagocytophilum-occupied vacuolar membrane (AVM). APH_0032 is a 66.1 kDa acidic protein that electrophoretically migrates with an apparent molecular weight of 130 kDa. It contains a predicted transmembrane domain and tandemly arranged direct repeats that comprise 46% of the protein. APH_0032 is undetectable on Anaplasma organisms bound to the surfaces of HL-60 cells, but is detected on the AVM and surfaces of intravacuolar bacteria beginning 24 h post-infection. APH_0032 localizes to the AVM in HL-60, THP-1, HMEC-1, and ISE6 cells. APH_0032, along with APH_1387, which encodes a confirmed AVM protein, is transcribed during A. phagocytophilum infection of tick salivary glands and murine neutrophils. APH_0032 localizes to the AVM in neutrophils recovered from infected mice. The Legionella pneumophila Dot/IcM type IV secretion system (T4SS) can heterologously secrete a CyaA-tagged version of the A. phagocytophilum VirB/D T4SS effector, AnkA, but fails to secrete CyaA-tagged APH_0032 or APH_1387. These data confirm APH_0032 as an Anaplasma-derived AVM protein and hint that neither it nor APH_1387 are T4SS effectors.
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298-0678, USA.
authors.forename: Bernice
authors.initials: B
authors.lastname: Huang
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Troese
authors.forename: Dale
authors.initials: D
authors.lastname: Howe
authors.forename: Shaojing
authors.initials: S
authors.lastname: Ye
authors.forename: Jonathan T
authors.initials: JT
authors.lastname: Sims
authors.forename: Robert A
authors.initials: RA
authors.lastname: Heinzen
authors.forename: Dori L
authors.initials: DL
authors.lastname: Borjesson
authors.forename: Jason A
authors.initials: JA
authors.lastname: Carlyon
mesh_terms: D000595:Amino Acid Sequence; D041081:Anaplasma phagocytophilum / Q000472:pathogenicity*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D054884:Host-Pathogen Interactions; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry*; D016952:Legionella pneumophila / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009504:Neutrophils / Q000382:microbiology; D017434:Protein Structure, Tertiary; D020080:Tandem Repeat Sequences; D013987:Ticks; D014158:Transcription, Genetic; D014617:Vacuoles / Q000737:chemistry* / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins