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Lyme disease (LD) caused by Borrelia burgdorferi is among the most important human vector borne diseases for which there is no effective prevention method. Identification of tick saliva transmission factors of the LD agent is needed before the highly advocated tick antigen-based vaccine could be developed. We previously reported the highly conserved Ixodes scapularis (Ixs) tick saliva serpin (S) 17 (IxsS17) was highly secreted by B. burgdorferi infected nymphs. Here, we show that IxsS17 promote tick feeding and enhances B. burgdorferi colonization of the host. We show that IxsS17 is not part of a redundant system, and its functional domain reactive center loop (RCL) is 100% conserved in all tick species. Yeast expressed recombinant (r) IxsS17 inhibits effector proteases of inflammation, blood clotting, and complement innate immune systems. Interestingly, differential precipitation analysis revealed novel functional insights that IxsS17 interacts with both effector proteases and regulatory protease inhibitors. For instance, rIxsS17 interacted with blood clotting proteases, fXII, fX, fXII, plasmin, and plasma kallikrein alongside blood clotting regulatory serpins (antithrombin III and heparin cofactor II). Similarly, rIxsS17 interacted with both complement system serine proteases, C1s, C2, and factor I and the regulatory serpin, plasma protease C1 inhibitor. Consistently, we validated that rIxsS17 dose dependently blocked deposition of the complement membrane attack complex via the lectin complement pathway and protected complement sensitive B. burgdorferi from complement-mediated killing. Likewise, co-inoculating C3H/HeN mice with rIxsS17 and B. burgdorferi significantly enhanced colonization of mouse heart and skin organs in a reverse dose dependent manner. Taken together, our data suggests an important role for IxsS17 in tick feeding and B. burgdorferi colonization of the host., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Tick serine protease inhibitors (serpins) play crucial roles in tick feeding and pathogen transmission. We demonstrate that Ixodes scapularis (Ixs) nymph tick saliva serpin (S) 41 (IxsS41), secreted by Borrelia burgdorferi (Bb)-infected ticks at high abundance, is involved in regulating tick evasion of host innate immunity and promoting host colonization by Bb. Recombinant (r) proteins were expressed in Pichia pastoris, and substrate hydrolysis assays were used to determine. Ex vivo (complement and hemostasis function related) and in vivo (paw edema and effect on Bb colonization of C3H/HeN mice organs) assays were conducted to validate function. We demonstrate that rIxsS41 inhibits chymase and cathepsin G, pro-inflammatory proteases that are released by mast cells and neutrophils, the first immune cells at the tick feeding site. Importantly, stoichiometry of inhibition analysis revealed that 2.2 and 2.8 molecules of rIxsS41 are needed to 100% inhibit 1 molecule of chymase and cathepsin G, respectively, suggesting that findings here are likely events at the tick feeding site. Furthermore, chymase-mediated paw edema, induced by the mast cell degranulator, compound 48/80 (C48/80), was blocked by rIxsS41. Likewise, rIxsS41 reduced membrane attack complex (MAC) deposition via the alternative and lectin complement activation pathways and dose-dependently protected Bb from complement killing. Additionally, co-inoculating C3H/HeN mice with Bb together with rIxsS41 or with a mixture (rIxsS41 and C48/80). Findings in this study suggest that IxsS41 markedly contributes to tick feeding and host colonization by Bb. Therefore, we conclude that IxsS41 is a potential candidate for an anti-tick vaccine to prevent transmission of the Lyme disease agent., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bencosme-Cuevas, Kim, Nguyen, Berry, Li, Adams, Smith, Batool, Swale, Kaufmann, Jones-Hall and Mulenga.)

Background: Lyme disease (LD) caused by Borrelia burgdorferi is the most prevalent tick-borne disease. There is evidence that vaccines based on tick proteins that promote tick transmission of B. burgdorferi could prevent LD. As Ixodes scapularis nymph tick bites are responsible for most LD cases, this study sought to identify nymph tick saliva proteins associated with B. burgdorferi transmission using LC-MS/MS. Tick saliva was collected using a non-invasive method of stimulating ticks (uninfected and infected: unfed, and every 12 h during feeding through 72 h, and fully-fed) to salivate into 2% pilocarpine-PBS for protein identification using LC-MS/MS., Results: We identified a combined 747 tick saliva proteins of uninfected and B. burgdorferi infected ticks that were classified into 25 functional categories: housekeeping-like (48%), unknown function (18%), protease inhibitors (9%), immune-related (6%), proteases (8%), extracellular matrix (7%), and small categories that account for <5% each. Notably, B. burgdorferi infected ticks secreted high number of saliva proteins (n=645) than uninfected ticks (n=376). Counter-intuitively, antimicrobial peptides, which function to block bacterial infection at tick feeding site were suppressed 23-85 folds in B. burgdorferi infected ticks. Similar to glycolysis enzymes being enhanced in mammalian cells exposed to B. burgdorferi : eight of the 10-glycolysis pathway enzymes were secreted at high abundance by B. burgdorferi infected ticks. Of significance, rabbits exposed to B. burgdorferi infected ticks acquired potent immunity that caused 40-60% mortality of B. burgdorferi infected ticks during the second infestation compared to 15-28% for the uninfected. This might be explained by ELISA data that show that high expression levels of immunogenic proteins in B. burgdorferi infected ticks., Conclusion: Data here suggest that B. burgdorferi infection modified protein content in tick saliva to promote its survival at the tick feeding site. For instance, enzymes; copper/zinc superoxide dismutase that led to production of H 2 O 2 that is toxic to B. burgdorferi were suppressed, while, catalase and thioredoxin that neutralize H 2 O 2 , and pyruvate kinase which yields pyruvate that protects Bb from H 2 O 2 killing were enhanced. We conclude data here is an important resource for discovery of effective antigens for a vaccine to prevent LD.