Novel Identification of Dermacentor variabilis Arp2/3 Complex and Its Role in Rickettsial Infection of the Arthropod Vector

Abstract

Tick-borne spotted fever group (SFG) Rickettsia species must be able to infect both vertebrate and arthropod host cells. The host actin-related protein 2/3 (Arp2/3) complex is important in the invasion process and actin-based motility for several intracellular bacteria, including SFG Rickettsia in Drosophila and mammalian cells. To investigate the role of the tick Arp2/3 complex in tick-Rickettsia interactions, open reading frames of all subunits of the protein including Arp2, Arp3, ARPC1, ARPC2, ARPC3, ARPC4, and ARPC5 were identified from Dermacentor variabilis. Amino acid sequence analysis showed variation (ranging from 25–88%) in percent identity compared to the corresponding subunits of the complex from Drosophila melanogaster, Mus musculus, Homo sapiens, and Saccharomyces cerevisiae. Potential ATP binding sites were identified in D. variabilis (Dv) Arp2 and Arp3 subunits as well as five putative WD (Trp-Asp) motifs which were observed in DvARPC1. Transcriptional profiles of all subunits of the DvArp2/3 complex revealed greater mRNA expression in both Rickettsia-infected and -uninfected ovary compared to midgut and salivary glands. In response to R. montanensis infection of the tick ovary, the mRNA level of only DvARPC4 was significantly upregulated compared to uninfected tissues. Arp2/3 complex inhibition bioassays resulted in a decrease in the ability of R. montanensis to invade tick tissues with a significant difference in the tick ovary, indicating a role for the Arp2/3 complex in rickettsial invasion of tick cells. Characterization of tick-derived molecules associated with rickettsial infection is imperative in order to better comprehend the ecology of tick-borne rickettsial diseases.

Identification of Host Proteins Involved in Rickettsial Invasion of Tick Cells

“…Utilization of the natural host cell background may illuminate unique pathways or identify novel rickettsial mechanisms for cell invasion. Consistently with the studies in mammalian and Drosophila cell lines and whole tick tissues (6,9,12), the Arp2/3 complex and actin are the central molecules activated during the entry DVE1 cells were treated with C. difficile toxin B (ToxB), a general inhibitor for Rho GTPases (A), or with the Rac1 inhibitor (Rac) (B), and the mean (Ϯ standard error of the mean) percent relative rickettsial invasion of each treatment was compared to that of the untreated (vehicle only) control. Each concentration of inhibitor was assessed in quadruplicate, and means represent a combination of results from two independent experiments.…”

“…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.…”

“…The experiments were performed in triplicate for each group, and the results are a combination of the results from two independent experiments. ****, P Ͻ 0.0001. tein capable of nucleating actin filaments (36, 37); a homologous complex was recently identified in D. variabilis (12). For several bacterial pathogens, the Arp2/3 complex plays a role in invasion of host cells (38)(39)(40)(41)(42)(43)(44)(45).…”

“…For several bacterial pathogens, the Arp2/3 complex plays a role in invasion of host cells (38)(39)(40)(41)(42)(43)(44)(45). Previous in vitro studies revealed the importance of the Arp2/3 complex in rickettsial internalization in Drosophila and mammalian cells (6,9), as well as in whole tick organs ex vivo (12). To examine whether the molecule is essential for the entry of R. montanensis into tick cells, DVE1 cells were treated with CK-666, an Arp2/3 complex inhibitor, at 500, 50, and 5 M for 2 h before being infected with R. montanensis for 1 h. Compared to the controls (no inhibitor, vehicle only), inhibition of the Arp2/3 complex significantly reduced (P Ͻ 0.0001) the percent relative invasion to 8% at the highest concentration of the inhibitor used (Fig.…”

“…Similarly, a recent molecular and functional characterization of the D. variabilis Arp2/3 complex identified a relatively conserved molecule that is involved in rickettsial entry. 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).…”