Deorphanization and target validation of cross-tick species conserved novel Amblyomma americanum tick saliva protein

Abstract

We previously identified a cross-tick species conserved tick feeding stimuli responsive Amblyomma americanum (Aam) AV422 gene. This study demonstrates that AamAV422 belongs to a novel group of arthropod proteins that is characterized by 14 cysteine amino acid residues: C23-X7/9-C33-X23/24-C58-C8-C67X7-X75-X23-C99-X15-C115-X10-C126X24/25/33-C150C151-X7-C159-X8-X168-X23/24-C192-X9/10-C202 predicted to form seven disulfide bonds. We show that AamAV422 protein is a ubiquitously expressed protein that is injected into the host within the first 24 h of the tick attaching onto the host as revealed by western blotting analyses of recombinant (r)AamAV422, tick saliva and dissected tick organ protein extracts using antibodies to 24 h and 48 h tick saliva proteins (TSPs). Native AamAV422 is apparently involved with mediating tick anti-hemostasis and anti-complement functions in that rAamAV422 delayed plasma clotting time in a dose responsive manner by up to ~160 s, prevented platelet aggregation by up to ~16% and caused ~24% reduction in production of terminal complement complexes. Target validation analysis revealed that rAamAV422 is a potential candidate for a cocktail or multivalent tick vaccine preparation in that RNA interference (RNAi)-mediated silencing of AamAV422 mRNA caused a statistically significant (~44%) reduction in tick engorgement weights, which is proxy for amounts of ingested blood. We speculate that AamAV422 is a potential target antigen for development of the highly desired universal tick vaccine in that consistent with high conservation among ticks, antibodies to 24 h Ixodes scapularis TSPs specifically bound rAamAV422. We discuss data in this study in the context of advancing the biology of tick feeding physiology and discovery of potential target antigens for tick vaccine development.

Dual silencing of long and short Amblyomma americanum acidic chitinase forms weakens the tick cement cone stability

“…Tick organs SG, MG and CA were pooled in 1 ml Trizol and stored at −80°C until total RNA extraction. Within the first hour of detachment, ticks were prepared for dissections as described by Mulenga et al (Mulenga et al, 2013). Prior to dissecting, tick mouthparts were inspected to remove any remaining rabbit tissue and washed in RNase inhibitor diethylpyrocarbonate (DEPC)-treated water.…”

“…Western blotting analysis of rAamAch-L using antibodies to 48 h (Chalaire et al, 2011) and replete fed (Mulenga et al, 2013) A. americanum tick saliva proteins was carried out to investigate the possibility of native AamAch-L being injected into the host during tick feeding. The expectation is that if native AamAch-L was immunogenic and injected into the host during tick feeding, rAamAch-L would specifically bind antibodies to 48 h and/or replete fed A. americanum tick saliva proteins.…”

“…The expression plasmid was constructed by subcloning the mature AamAch-L coding domain into pPICZαA KpnI and NotI sites, using forward (5′-GGTACCATGCCCCAGCAAGACGGGGATG-3′) and reverse (5′-GCGGCCGCAGCGGGTGATGCGGTAATCTCG-3′) primers with added restriction enzyme sites in bold. The pPICZαA-AamAch-L expression plasmid was linearized with PmeI and used to transform P. pastoris X-33 strain (Life Technologies) by electroporation as described previously (Mulenga et al, 2013). Likewise, induction, validation and affinity purification of recombinant protein expression were done as described previously (Mulenga et al, 2013).…”

Ixodes scapularistick saliva proteins sequentially secreted every 24h during blood feeding

“…Additionally one protein was identified in I. scapularis nymphs as an immunogenic protein that bound to human serum from exposure to tick bites [198]. The remaining proteins were found in saliva proteomes of R. microplus (n = 28, [50]), H. longicornis (n = 22, [52]), D. andersoni (n = 2, [51]), O. moubata (n = 5, [53], sequencing of I. scapularis tick saliva by Edman degradation (n = 4) [48], and others were verified as secreted in western blotting studies [42,43,[199][200][201][202][203][204].…”

“…al., [40] and Lewis et. al., [41] used antibodies to 24-48 h tick saliva proteins [42,43] to immunoscreen phage display cDNA expression libraries to identify 24-48 h Amblyomma americanum and 24 h I. scapularis immunogenic tick saliva proteins. Similar immunoscreening approaches were used to identify immunodominant I. scapularis tick saliva proteins [44][45][46][47].…”