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

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.

A repertoire of protease inhibitor families in Amblyomma americanum and other tick species: inter-species comparative analyses.

BACKGROUND: Protease inhibitors (PIs) are important regulators of physiology and represent anti-parasitic druggable and vaccine targets. We conducted bioinformatic analyses of genome and transcriptome data to determine the protease inhibitor (PI) repertoire in Amblyomma americanum and in 25 other ixodid tick species. For A. americanum, we compared the PI repertoires in fed and unfed, male and female A. americanum ticks. We also analyzed PI repertoires of female 48, 96 and 120 h-fed midgut (MG) and salivary gland (SG) tissues. RESULTS: We found 1,595 putative non-redundant PI sequences across 26 ixodid tick species. Ticks express PIs from at least 18 different families: I1, I2, I4, I8, I21, I25, I29, I31, I32, I35, I39, I43, I51, I53, I63, I68, I72 and I74 (MEROPS). The largest PI families were I2, I4 and I8 and lowest in I21, I31, I32, I35 and I68. The majority (75%) of tick PIs putatively inhibit serine proteases, with ~11 and 9% putatively regulating cysteine or metalloprotease-mediated pathways, respectively, and ~4% putatively regulating multiple/mixed protease types. In A. americanum, we found 370 PIs in female and 354 in male ticks. In A. americanum we found 231 and 442 in unfed and fed ticks, respectively. In females, we found 206 and 164 PIs in SG and MG, respectively. The majority of highly cross-tick species conserved PIs were in families I1, I2, I8, I21, I25, I29, I39 and I43. CONCLUSIONS: Ticks appear to express large and diverse repertoires of PIs that primarily target serine protease-mediated pathways. We speculate that PI families with the highest repertoires may contain functionally redundant members while those with the lowest repertoires are functionally non-redundant PIs. We found some highly conserved PIs in the latter category, which we propose as potential candidates for broad-spectrum anti-tick vaccine candidates or druggable targets in tick control.

Heparan sulfate/heparin glycosaminoglycan binding alters inhibitory profile and enhances anticoagulant function of conserved Amblyomma americanum tick saliva serpin 19.

Some serine protease inhibitor (serpin) regulators of essential life pathways bind glycosaminoglycans (GAGs) to enhance inhibitory functions and achieve physiologically relevant rates. This study demonstrates that highly conserved Amblyomma americanum tick saliva serpin 19 (AAS19), a broad-spectrum inhibitor of hemostasis and inflammation system proteases and anticoagulant, can bind heparan sulfate/heparin (HS)GAGs and that this interaction alters its function. Substrate hydrolysis and unpaired t-test analyses revealed that HSGAG binding caused rAAS19 inhibitory activity to: (i) significantly increase against blood clotting factors (f) IIa (thrombin) and fIXa, (ii) significantly reduce against fXa and fXIIa and (iii) moderate to no effect against trypsin, kallikrein, papain, and plasmin. Stoichiometry of inhibition (SI) analyses show that HSGAG binding improved the rAAS19 inhibitory efficiency against thrombin 2.7-4.3 fold as revealed by SI change from 13.19 in absence of HSGAGs to 4.83-3.04 in their presence. Our data show that HSGAG binding dramatically enhanced rAAS19 anticoagulant function. In the recalcification time assay, rAAS19 pre-incubated with HSGAGs prior to the assay, delayed plasma clotting by an additional 176-457 s above HSGAGs or rAAS19 alone. Our data suggest that formation of the HSGAGs and rAAS19 complex is important for the observed enhanced anticoagulant effect. Delay of plasma clotting was higher when HSGAGs and rAAS19 were co-incubated to allow complex formation prior to blood clotting assay as opposed to no co-incubation. We have discussed our finding with reference to tick feeding physiology and utility of the rAAS19 in blood clotting disorder therapy.

Identification of 24h Ixodes scapularis immunogenic tick saliva proteins.

Ixodes scapularis is arguably the most medically important tick species in the United States. This tick transmits 5 of the 14 human tick-borne disease (TBD) agents in the USA: Borrelia burgdorferi, Anaplasma phagocytophilum, B. miyamotoi, Babesia microti, and Powassan virus disease. Except for the Powassan virus disease, I. scapularis-vectored TBD agents require more than 24h post attachment to be transmitted. This study describes identification of 24h immunogenic I. scapularis tick saliva proteins, which could provide opportunities to develop strategies to stop tick feeding before transmission of the majority of pathogens. A 24h fed female I. scapularis phage display cDNA expression library was biopanned using rabbit antibodies to 24h fed I. scapularis female tick saliva proteins, subjected to next generation sequencing, de novo assembly, and bioinformatic analyses. A total of 182 contigs were assembled, of which ∼19% (35/182) are novel and did not show identity to any known proteins in GenBank. The remaining ∼81% (147/182) of contigs were provisionally identified based on matches in GenBank including ∼18% (27/147) that matched protein sequences previously annotated as hypothetical and putative tick saliva proteins. Others include proteases and protease inhibitors (∼3%, 5/147), transporters and/or ligand binding proteins (∼6%, 9/147), immunogenic tick saliva housekeeping enzyme-like (17%, 25/147), ribosomal protein-like (∼31%, 46/147), and those classified as miscellaneous (∼24%, 35/147). Notable among the miscellaneous class include antimicrobial peptides (microplusin and ricinusin), myosin-like proteins that have been previously found in tick saliva, and heat shock tick saliva protein. Data in this study provides the foundation for in-depth analysis of I. scapularis feeding during the first 24h, before the majority of TBD agents can be transmitted.

A 24-48 h fed Amblyomma americanum tick saliva immuno-proteome.

BACKGROUND: Multiple tick saliva proteins, the majority of which are unknown, confer tick resistance in repeatedly infested animals. The objective of this study was to identify the 24-48 h fed Amblyomma americanum tick saliva immuno-proteome. The 24-48 h tick-feeding phase is critical to tick parasitism as it precedes important events in tick biology, blood meal feeding and disease agent transmission. Fed male, 24 and 96 h fed female phage display cDNA expression libraries were biopanned using rabbit antibodies to 24 and 48 h fed A. americanum female tick saliva proteins. Biopanned immuno-cDNA libraries were subjected to next generation sequencing, de novo assembly, and bioinformatic analysis. RESULTS: More than 800 transcripts that code for 24-48 h fed A. americanum immuno-proteins are described. Of the 895 immuno-proteins, 52% (464/895) were provisionally identified based on matches in GenBank. Of these, ~19% (86/464) show high level of identity to other tick hypothetical proteins, and the rest include putative proteases (serine, cysteine, leukotriene A-4 hydrolase, carboxypeptidases, and metalloproteases), protease inhibitors (serine and cysteine protease inhibitors, tick carboxypeptidase inhibitor), and transporters and/or ligand binding proteins (histamine binding/lipocalin, fatty acid binding, calreticulin, hemelipoprotein, IgG binding protein, ferritin, insulin-like growth factor binding proteins, and evasin). Others include enzymes (glutathione transferase, cytochrome oxidase, protein disulfide isomerase), ribosomal proteins, and those of miscellaneous functions (histamine release factor, selenoproteins, tetraspanin, defensin, heat shock proteins). CONCLUSIONS: Data here demonstrate that A. americanum secretes a complex cocktail of immunogenic tick saliva proteins during the first 24-48 h of feeding. Of significance, previously validated immunogenic tick saliva proteins including AV422 protein, calreticulin, histamine release factor, histamine binding/lipocalins, selenoproteins, and paramyosin were identified in this screen, supporting the specificity of the approach in this study. While descriptive, this study opens opportunities for in-depth tick feeding physiology studies.