Figure 4.

Functional analysis by RNAi supports a role for tick and human Rab14 in A. phagocytophilum infection of host cells. (A) A 75–83% knockdown by RNAi of tick rab14 (B7QHS7) in tick cells resulted in a 40% decrease in A. phagocytophilum infection levels, suggesting that A. phagocytophilum increases the levels of Rab14 to facilitate infection. Tick ISE6 cells were treated with rab14 dsRNA and control cells were treated with the unrelated Rs86 dsRNA. DNA samples from infected cells were analyzed by real-time PCR using the A. phagocytophilum major surface protein 4 (msp4) gene-specific primers. Normalized Ct values were compared between groups by Student's t-test with unequal variance (p = 0.02; n = 6 biological replicates). (B) Tick rab14 knockdown did not affect cell viability. The percent of apoptotic tick ISE6 cells was determined after RNAi with rab14 test and Rs86 control dsRNAs by flow cytometry using the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit. The percentage of apoptotic cells was compared between both test and control dsRNA treated UtC and ItC by Student's t-test with unequal variance (p > 0.05; n = 6 biological replicates). (C) Representative images of immunofluorescence analysis of UhC and IhC incubated with either ON-TARGETplus SMARTpool Human rab14 siRNA or control ON-TARGETplus Non-targeting Control Pool siRNA. Cells were stained with rabbit anti-A. phagocytophilum msp4 antibodies, labeled with FITC (green, arrows) and DAPI (blue). To confirm the uptake of siRNA, cells were treated with Accell Red Non-targeting Control siRNA (red, arrows) and labeled with DAPI (blue). (D) Human rab14 was up-regulated at the mRNA level in response to infection. The RNA levels of human rab14 (P61106) were determined by real-time RT-PCR in UhC and IhC. Normalized Ct values were compared between groups by Student's t-test with unequal variance (p = 0.03; n = 4 biological replicates). (E) A 31–52% knockdown by RNAi of rab14 in human HL60 cells did not affect A. phagocytophilum infection levels, suggesting that Rab14 protein levels decrease post-transcriptionally in human neutrophils to control A. phagocytophilum infection. Human HL60 cells were treated with rab14 siRNA or control ON-TARGETplus Non-targeting Control Pool siRNA. DNA samples from infected cells were analyzed by real-time PCR using the A. phagocytophilum major surface protein 4 (msp4) gene-specific primers. Normalized Ct values were compared between groups by Student's t-test with unequal variance (non-significant, p > 0.05; n = 4 biological replicates). (F) Proposed model of ras-related protein function in A. phagocytophilum-infected tick and human cells. In tick cells, A. phagocytophilum (Ap) increases the levels of active ras-related proteins Rab14 in phagosomal membranes to prevent the transfer of bacteria from phagosomes to lysosomes and hijacks Rab10 and other endoplasmic reticulum membrane proteins to its vacuole to complete the infection cycle and favor pathogen survival and facilitate infection. In human neutrophils, the decrease in Rab10 levels appears as a post-transcriptional mechanism to control A. phagocytophilum infection.