Molecular characterization and tissue‐specific gene expression of Dermacentor variabilis α‐catenin in response to rickettsial infection

Abstract

Alpha catenin is a cytoskeleton protein that acts as a regulator of actin rearrangement by forming an E-cadherin adhesion complex. In Dermacentor variabilis, a putative α-catenin (Dvα-catenin) was previously identified as differentially regulated in ovaries of ticks chronically infected with Rickettsia montanensis. To begin characterizing the role(s) of Dvα-catenin during rickettsial infection, the full-length Dvα-catenin cDNA was cloned and analyzed. Comparative sequence analysis demonstrates a 3069 basepair cDNA with a 2718 bp open reading frame with a sequence similar to Ixodes scapularis α-catenin. A portion of Dvα-catenin is homologous to the vinculin-conserved domain containing a putative actin binding region and β-catenin binding and dimerization regions. Quantitative RT-PCR analysis demonstrated that Dvα-catenin is predominantly expressed in tick ovaries and is responsive to tick feeding. The tissue-specific gene expression analysis of ticks exposed to Rickettsia demonstrates that Dvα-catenin expression was significantly downregulated at 12 hours post-exposure to R. montanensis, but not in R. amblyommii-exposed ovaries, compared to Rickettsia-unexposed ticks. Studying tick-derived molecules associated with rickettsial infection will provide a better understanding of the transmission dynamics of tick-borne rickettsial diseases.

Gene Expression of Tissue-Specific Molecules in Ex vivoDermacentor variabilis(Acari: Ixodidae) During Rickettsial Exposure

“…An ex vivo bioassay of D. variabilis tissues (backless tick explant) was modified from previously described protocols (Bell 1980, Sunyakumthorn et al 2012). Briefly, unfed female D. variabilis were cleaned with 70% ethanol and 10% benzalkonium chloride solution, and rinsed with sterile water three times.…”

“…Ten microliters of each reaction mixture were transferred into three wells of 384-well plates and amplification occurred in an ABI 7900HT unit (Applied Biosystems, Foster City, CA) using SDS v2.3 software. Actin gene was used as internal control gene as previously described (Sunyakumthorn et al 2012). Data for each sample were calculated as the difference in threshold cycle (C T ) value (ΔC T = C T dvactin − C T tick immune gene ).…”

Identification of Host Proteins Involved in Rickettsial Invasion of Tick Cells

“…Table S1 in the supplemental material). Although comprehensive analyses (11)(12)(13)(18)(19)(20)(21) confirmed gene sequences for some of the molecules, the partial transcript sequence data for several D. variabilis molecules suggest a level of identity between ticks and other invertebrates relative to cytoskeletal molecules. The specificity of the inhibitors on tick proteins and undefined downstream effects influencing rickettsial invasion cannot be confirmed without detailed molecular and functional characterization of each target molecule; however, the combined characterization of inhibitors in mammalian and Drosophila systems and corresponding identification of putative factors in the current study suggest the potential for specific activity in ticks.…”

“…As described by Sunyakumthorn et al (13), R. montanensis was propagated in Vero cells and maintained in a humidified 5% CO 2 incubator at 34°C. For each experiment, rickettsiae were purified using a modified protocol of Weiss et al (15) as previously described (11).…”

“…The Arp2/3 complex contributes to host cell invasion by Rickettsia montanensis in the natural arthropod vector, as determined using an ex vivo biochemical inhibition assay (12). Interestingly, downregulation of transcripts of ␣-catenin, a molecule associated with actin organization, in ticks upon rickettsial exposure suggests that ticks may actively respond to rickettsial infection by limiting the utilization of ␣-catenin (13). The presence of conserved molecules in multiple host backgrounds allows for further characterization in vector backgrounds.…”