Identification and characterization of proteins in the Amblyomma americanum tick cement cone

Abstract

The adaptation of hard ticks to feed for long periods is facilitated by the cement cone, which securely anchors the tick mouthparts onto host skin and protects the tick from being groomed off by the host. Thus, preventing tick cement deposition is an attractive target for the development of innovative tick control. We used LC-MS/MS sequencing to identify 160 Amblyomma americanum tick cement proteins that include glycine-rich proteins (GRP, 19%), protease inhibitors (12%), proteins of unknown function (11%), mucin (4%), detoxification, storage, and lipocalin at 1% each, and housekeeping proteins (50%). Spatiotemporal transcription analysis showing mRNA expression in multiple tick organs and transcript abundance increasing with feeding suggest that selected GRPs (n = 13) regulate multiple tick feeding functions, being classified as constitutively expressed (CE), feeding induced (FI), and up-regulated with feeding (UR). We show that transcription of CE GRPs is likely under the control of tick appetence associated factors in that mRNA abundance increased several thousand fold in 1 week old adult ticks, the time period that coincides with tick attainment of appetence. Given the high number of targets, we synthesized and injected unfed ticks with combinatorial (co) double stranded (ds)RNA and disrupted GRP mRNA in clusters according to similar transcription patterns: CE (n = 3), FI, (n = 4), and UR (n = 6) to streamline the work. Our data suggest that CE and FI GRPs are important for maintenance of the tick feeding site in that reddening and subsequent bleeding were observed around the mouthparts of CE and FI GRP co-dsRNA injected ticks during feeding. Furthermore, although not significantly different, indices for blood meal size and fecundity were apparently reduced in FI and UR ticks. We discuss our data with reference to A. americanum tick feeding physiology.

Time-resolved proteomic profile ofAmblyomma americanumtick saliva during feeding

“…Tick cement deposition is completed during the first 96 h of tick feeding (125), and thus it is conceivable that some of the glycine-rich proteins in this study might be involved with tick cement formation. It is interesting to note that some of the glycine-rich proteins that were identified from tick cement in our lab (131) and others (130) were also found in this study (Supplemental table 3). Some of the glycine-rich proteins were secreted from the 144-h time point, long after tick cement formation; these might regulate other tick feeding functions.…”

Tick and Host Derived Compounds Detected in the Cement Complex Substance

“…However, probably due to cement composition with previously unknown components, it was not fully dissolved in SDS-containing buffer. Therefore, to improve protein extraction for cementome analysis, a urea-containing buffer previously applied to tick cement analysis [16,30] was used in addition to SDS. This approach resulted in a higher protein identification in the tick cementome (654 proteins) when compared to SDS-based (388 proteins) or urea-based (266 proteins) extraction protocols alone (Figure 2A).…”

“…Ixodid ticks feed on blood for a relatively long period and an evolutionary adaptation to overcome host inflammatory and immune responses using molecules produced in tick salivary glands or recycled from the host and inoculated with saliva into the feeding site [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. Cattle tick Rhipicephalus microplus (Canestrini, 1888) is an Ixodid one-host tick species (all developmental stages remain on the same host) with an impact on cattle industry in tropical and subtropical regions of the world [3,9,10].…”

“…The cement has not only adhesive properties but has been proposed to have a possible role in antimicrobial properties, seals the lesion during feeding, facilitates feeding and pathogen transmission, and protects ticks from host immune and inflammatory responses [22,29]. Tick cement has been studied using different methodological approaches to characterize its structure, protein and chemical composition, and functional implications such as decrease in the width of cement cone as it goes deeper into the host skin or after Glycine-rich protein coding gene knockdown in Amblyomma americanum [16,20,29,30]. However, the biochemical properties of tick cement during different ectoparasite feeding stages have not been fully characterized.…”

Tick-Borne Encephalitis Virus Infection Alters the Sialome of Ixodes ricinus Ticks During the Earliest Stages of Feeding

“…This cement is immunogenic, and though largely structural it has an impact on modulating host responses. Glycine-rich proteins are one of the cement's characteristic constituents (Hollmann et al, 2018).…”