title: Molecular and functional characterization of vacuolar-ATPase from the American dog tick Dermacentor variabilis.
abstract: Vacuolar (V)-ATPase is a proton-translocating enzyme that acidifies cellular compartments for various functions such as receptor-mediated endocytosis, intracellular trafficking and protein degradation. Previous studies in Dermacentor variabilis chronically infected with Rickettsia montanensis have identified V-ATPase as one of the tick-derived molecules transcribed in response to rickettsial infection. To examine the role of the tick V-ATPase in tick-Rickettsia interactions, a full-length 2887-bp cDNA (2532-bp open reading frame) clone corresponding to the transcript of the V0 domain subunit a of D. variabilis V-ATPase (DvVATPaseV0a) gene encoding an 843 amino acid protein with an estimated molecular weight of ~96 kDa was isolated from D. variabilis. Amino acid sequence analysis of DvVATPaseV0a showed the highest similarity to VATPaseV0a from Ixodes scapularis. A potential N-glycosylation site and eight putative transmembrane segments were identified in the sequence. Western blot analysis of tick tissues probed with polyclonal antibody raised against recombinant DvVATPaseV0a revealed the expression of V-ATPase in the tick ovary. Transcriptional profiles of DvVATPaseV0a demonstrated a greater mRNA expression in the tick ovary, compared with the midgut and salivary glands; however, the mRNA level in each of these tick tissues remained unchanged after infection with R. montanensis for 1 h. V-ATPase inhibition bioassays resulted in a significant decrease in the ability of R. montanensis to invade tick cells in vitro, suggesting a role of V-ATPase in rickettsial infection of tick cells. Characterization of tick-derived molecules involved in rickettsial infection is essential for a thorough understanding of rickettsial transmission within tick populations and the ecology of tick-borne rickettsial diseases.
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.
authors.forename: N
authors.initials: N
authors.lastname: Petchampai
authors.forename: P
authors.initials: P
authors.lastname: Sunyakumthorn
authors.forename: M L
authors.initials: ML
authors.lastname: Guillotte
authors.forename: C
authors.initials: C
authors.lastname: Thepparit
authors.forename: M T
authors.initials: MT
authors.lastname: Kearney
authors.forename: A
authors.initials: A
authors.lastname: Mulenga
authors.forename: A F
authors.initials: AF
authors.lastname: Azad
authors.forename: K R
authors.initials: KR
authors.lastname: Macaluso
mesh_terms: D000818:Animals; D003870:Dermacentor / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D020869:Gene Expression Profiling; D012333:RNA, Messenger / Q000096:biosynthesis; D012281:Rickettsia / Q000235:genetics / Q000472:pathogenicity*; D012282:Rickettsia Infections / Q000235:genetics* / Q000635:transmission; D012469:Salivary Glands; D014481:United States; D025262:Vacuolar Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D025262:Vacuolar Proton-Translocating ATPases
keywords: Dermacentor variabilis; Rickettsia montanensis; vacuolar-ATPase