Figure 1. Tick salivary protein IsC1ql3 is induced by B. burgdorferi, and IsC1ql3 antibodies are elicited in animals by natural tick bites.
(A) IsC1ql3 encodes a signal peptide (SP) and a gC1q domain. Simplified tree diagram showing the relationships between C1qDCs and IsC1ql3 (highlighted in red). The tree was adapted from Ye et al.18
(B) RT-qPCR assessment of IsC1ql3 transcript levels in the nymphal tick salivary glands (SGs) and midgut (MG). Each dot represents one biological replicate. Statistical significance was assessed using a non-parametric Mann-Whitney test (****p < 0.0001).
(C) Saliva collection from I. scapularis adults.
(D) SDS-PAGE and western blots of IsC1ql3 in tick saliva. SDS-PAGE gels were run under non-reducing conditions except where specified. Western blots were probed with polyclonal mouse anti-IsC1ql3 or mouse anti-ovalbumin (OVA) sera as a control. The 50 mM reducing agent dithiothreitol (DTT) was applied to reduce IsC1ql3 disulfide-linked multimers or undefined complexes.
(E) Western blots of IsC1ql3 in unfed and fed tick SGs and MG under non-reducing conditions.
(F) Western blots of IsC1ql3 in clean (B. burgdorferi-free) and infected tick saliva. The SDS-PAGE gel was run under reducing conditions. Data are represented as mean ± SD from three replicate experiments. Statistical significance was assessed using an unpaired t test (****p < 0.0001).
(G) IsC1ql3 antibodies are elicited by natural tick bites. Western blots of recombinant IsC1ql3 (rIsC1ql3) by probing with naive serum and serum from mice, guinea pigs, and humans bitten by clean (B. burgdorferi-free) and B. burgdorferi-infected ticks. The asterisks indicate rIsC1ql3.
(H) ELISA analysis of rIsC1ql3 by probing with naive serum and serum from mice and guinea pigs bitten by clean (B. burgdorferi-free) and B. burgdorferi-infected ticks. Humans exposed to tick bites were also examined. Data are represented as mean ± SD from three replicate experiments.