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Molecular and functional characterization of vacuolar‐ ATPase from the A merican dog tick D ermacentor variabilis
Author(s) -
Petchampai N.,
Sunyakumthorn P.,
Guillotte M. L.,
Thepparit C.,
Kearney M. T.,
Mulenga A.,
Azad A. F.,
Macaluso K. R.
Publication year - 2014
Publication title -
insect molecular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.955
H-Index - 93
eISSN - 1365-2583
pISSN - 0962-1075
DOI - 10.1111/imb.12059
Subject(s) - dermacentor variabilis , biology , microbiology and biotechnology , tick , ixodes scapularis , amblyomma americanum , open reading frame , peptide sequence , virology , gene , biochemistry , ixodidae
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 D ermacentor variabilis chronically infected with R ickettsia 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– R ickettsia interactions, a full‐length 2887‐bp cDNA (2532‐bp open reading frame) clone corresponding to the transcript of the V 0 domain subunit a of D . variabilis V ‐ ATPase ( Dv VATPaseV 0 a ) 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 Dv VATPaseV 0 a showed the highest similarity to VATPaseV 0 a from I xodes scapularis . A potential N ‐glycosylation site and eight putative transmembrane segments were identified in the sequence. W estern blot analysis of tick tissues probed with polyclonal antibody raised against recombinant Dv VATPaseV 0 a revealed the expression of V ‐ ATPase in the tick ovary. Transcriptional profiles of Dv VATPaseV 0 a 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.