The role of Rab27 in tick extracellular vesicle biogenesis and pathogen infection

Abstract

Background: The blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the US. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum. Nevertheless, it remains critically understudied compared to other arthropod vectors. Ixodes scapularis releases a variety of molecules that assist in the modulation of host responses. Recently, it was found that extracellular vesicles (EVs) carry several of these molecules and may impact microbial transmission to the mammalian host. EV biogenesis has been studied in mammalian systems and is relatively well understood, but the molecular players important for the formation and secretion of EVs in arthropods of public health relevance remain elusive. RabGTPases are among the major molecular players in mammalian EV biogenesis. They influence membrane identity and vesicle budding, uncoating, and motility.

Methods: Using BLAST, an in silico pathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking down rab27 to examine its role in tick EV biogenesis. Ixodes scapularis nymphs were injected with small interfering RNAs to knock down rab27 and then fed on naïve and A. phagocytophilum-infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition.

Results: Our BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencing rab27 in I. scapularis impacts tick fitness. Additionally, ticks acquire less A. phagocytophilum after rab27 silencing. Experiments in the tick ISE6 cell line show that silencing of rab27 causes a distinct range profile of tick EVs, indicating that Rab27 is needed to regulate EV biogenesis.

Conclusions: Rab27 is needed for successful tick feeding and may be important for acquiring A. phagocytophilum during a blood meal. Additionally, silencing rab27 in tick cells results in a shift of extracellular vesicle size. Overall, we have observed that Rab27 plays a key role in tick EV biogenesis and the tripartite interactions among the vector, the mammalian host, and a microbe it encounters.

Keywords: Extracellular vesicles; Rab27; Tick-borne diseases; Ticks.

Fig. 1

In silico analysis of EV biogenesis in the Ixodes scapularis genome. Sequences from proteins involved in mammalian EV biogenesis were input into the NCBI Basic Local Alignment Search Tool for proteins (BLASTp) against the I. scapularis genome and arranged based on mammalian literature to construct an in silico pathway in ticks. Accession numbers are listed in parentheses. Rab27 is highlighted

Fig. 2

rab27 silencing shifts the size of EVs being released by tick cells. A Experimental schematic for vesicle collection from tick cells. Tick ISE6 cells were nucleofected with small interfering (si-rab27) (green) or scrambled (sc-rab27) (gray) RNA for tick rab27. After 72 h, the cell culture media was replaced with vesicle free (VF) media. 24-h later, vesicles were collected through a series of tabletop and ultracentrifuge steps and measured using a nanosight machine. Graphs are representative of one two independent experiments. B Total vesicle amount released by tick cells does not change between the si-rab27 or sc-rab27 treatments. Statistical significance was evaluated by an unpaired, two-tailed t-test. Mean ± standard deviation (SD) is plotted. ns = not significant, P > 0.05. C Distribution of vesicles collected from sc-rab27 treated cells and compared to the si-rab27 treatment ranging from 0 to 600 nm

Fig. 3

rab27 silencing affects the fitness of Ixodes scapularis nymphs during hematophagy. A Ixodes scapularis nymphs were microinjected with small interfering (si-rab27)(green) or scrambled (sc-rab27)(gray) RNA, incubated for 24 h, and then placed on uninfected mice. B rab27 expression relative to actin after silencing. The graph represents three independent experiments combined. si-rab27 (n = 16). sc-rab27 (n = 25). Mean ± standard deviation (SD) is plotted. Statistical significance was evaluated by an unpaired, two-tailed t-test with Welch’s correction. *P < 0.05. C rab27 silencing effect on tick attachment. The graph represents three independent experiments combined. A Fisher’s exact test was performed to determine statistical differences. ns = not significant, P > 0.05. D Tick weight after rab27 silencing. The graph represents three independent experiments combined. si-rab27 (n = 16). sc-rab27 (n = 25). Mean ± SD is plotted. Statistical significance was evaluated by an unpaired, two-tailed t-test with Welch’s correction. *P < 0.05. Ticks recorded as a weight of 0 mg were below the limit of detection of our scale (1 mg)

Fig. 4

rab27 silencing impacts Anaplasma phagocytophilum acquisition during blood feeding of Ixodes scapularis nymphs. A C57BL/6 mice were intraperitoneally (i.p.) injected with 10⁷ A. phagocytophilum (Ap) 7 days prior to tick placement. Ticks were microinjected with small interfering (si-rab27) (green) or scrambled (sc-rab27)(gray) RNA, incubated for 24 h, and then placed on infected mice. Ticks were recovered daily from a water trap. Remaining ticks attached to the mouse at day 3 were removed by forceps. B rab27 silencing in I. scapularis ticks placed on Ap infected mice. The graph represents three independent experiments combined. si-rab27 (n = 9). sc-rab27 (n = 35). Mean ± standard deviation (SD) is plotted. Statistical significance was evaluated by an unpaired, two-tailed t-test with Welch’s correction. ***P < 0.0005. C Impact of rab27 silencing on tick attachment. The graph represents three independent experiments combined. Fisher’s exact test was performed to determine statistical differences. ****P < 0.0001. D Effect of rab27 silencing on tick weight. The graph represents three independent experiments combined. si-rab27 (n = 11). sc-rab27 (n = 37). Statistical significance was evaluated by an unpaired, two-tailed t-test with Welch’s correction. *P < 0.05. Ticks recorded as a weight of 0 mg were below the limit of detection of our scale (1 mg). E Impact of rab27 silencing on Ap acquisition. The graph represents three independent experiments combined. si-rab27 (n = 10). sc-rab27 (n = 32). Mean ± SD is plotted. Statistical significance was evaluated by an unpaired, two-tailed t-test with Welch’s correction. ****P < 0.0001