Figure 1.
Tick histamine release factor (tHRF) was up-regulated in B. burgdorferi- infected ticks.
(A) The expression of tHRF in B. burgdorferi-infected or uninfected ticks during I. scapularis feeding. Results are expressed as the mean ± the SEM (*p<0.05; **p<0.01). (B) tHRF-antiserum recognizes recombinant tHRF generated in Escherichia coli (tHRF E.coli) or Drosophila S2 cells (tHRF DES). (C–D) The up-regulation of tHRF protein in B. burgdorferi-infected ticks. (*p<0.05) (E) The detection of tHRF in adult tick saliva (Saliva), nymphal tick salivary glands (S.G.), midgut (M.G.) and whole ticks (Tick).
Figure 2.
Silencing tHRF impairs tick feeding and B. burgdorferi transmission.
(A) tHRF and SSRB (another tick gene, as a control) were silenced efficiently and specifically in nympal tick salivary gland by micro-injection of dsRNA. Results are expressed as the mean ± the SEM. (B) The weight of B. burgdorferi-infected ticks after feeding on naive mice. (C) The B. burgdorferi burden in whole ticks at 72 h post tick-engorgement. Spirochete burden in (D) murine skin at day 7, (E) joints and (F) heart at day 21 post-infection. (G) The weight of uninfected ticks after feeding on naive mice. Horizontal lines and bars represent the mean values ± the SEM. * p<0.05 and ** p<0.01 when compared with MOCK controls. Results are pooled from 3 independent experiments.
Figure 3.
tHRF antiserum interferes with tick feeding and B. burgdorferi transmission.
(A) Weights of B. burgdorferi-infected tick after feeding on tHRF antiserum-immunized, or control, mice. (NRS: normal rabbit serum; rabbit anti-Salp25D antiserum, both used as controls) (B) Spirochete burden in whole I. scapularis nymphs after feeding on tHRF antiserum-immunized, or control, mice. (C) B. burgdorferi burden in murine skin at day 7 post-tick feeding. Spirochete burden in the (D) joints and (E) heart at day 21 post-tick feeding. (F) Weights of uninfected ticks after feeding on tHRF antiserum-immunized, or control, mice. Horizontal lines and bars represent the mean values ± the SEM. * p<0.05 and ** p<0.01, when compared with the NRS controls. Results are a composite of independent experiments.
Figure 4.
Tick feeding and B. burgdorferi transmission was significantly impaired in tHRF immunized mice.
(A) tHRF probed with sera from mice or rabbits actively immunized with tHRF. (B) Weights of B. burgdorferi uninfected or (C) infected tick after feeding on tHRF-immunized, or control, mice. (D) Spirochete burden in whole I. scapularis after feeding on tHRF-immunized, or control, mice. (E) B. burgdorferi burden in murine skin at day 7 post-tick feeding. Spirochete burden in the (F) joints and (G) heart at day 21 post-tick feeding. Horizontal lines and bars represent the mean values ± the SEM. * p<0.05 and ** p<0.01, when compared with the control group. Results are pooled from 3 independent experiments.
Figure 5.
tHRF binds to basophils and stimulates histamine release.
(A) Flow cytometric analysis of recombinant tHRF binding to basophils, – as defined by the shift in the FL1 (green) channel when compared to the controls. (B) Confocal imaging of recombinant tHRF binding to the surface of basophils (tHRF+ Alexa 488 labeled tHRF Ab). and Negative control (PBS+Alexa 488 labeled tHRF Ab). Nuclei were counterstained with TOPRO3. (C) Recombinant tHRF (generated from Drosophila (DES) or E. coli) and extract from nymphal tick salivary gland (T. SGE) stimulate histamine release from basophils. Recombinant tHRF (DES) induces histamine release in a dose-dependent manner. Recombinant GST used as a negative control; Substance C48/80, a calcium ionophore shown to induce histamine release in basophils [22] served as a positive control. Results are expressed as the mean ± the SEM. Representative results from at least 3 independent experiments were shown.
Figure 6.
tHRF is required for tick rapid feeding.
Quantitative RT-PCR assessment of the expression pattern of: (A) tick histamine binding protein 1 (HBP1), (B) HBP2, (C) HBP3 and (D) tick histamine release factor (tHRF). (E) Weights of ticks at 72 h post tick attachment on mice treated with histamine (10mM) or PBS (control). (F) Weights of ticks at 72 h post tick attachment on mice treated with recombinant tHRF (10µg) or GST (control). (G) B. burgdorferi burden in ticks fed on mice treated with recombinant tHRF (10µg) or GST (control). Horizontal lines and bars represent the mean values ± the SEM. * p<0.05 and ** p<0.01. Representative results from at least 3 independent experiments were shown.