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: C(23)-X7/9-C(33)-X23/24-C(58)-X8-C(67)-X7-C(75)-X23-C(99)-X15-C(115)-X10-C(126)-X24/25/33-C(150)C(151)-X7-C(159)-X8-C(168)-X23/24-C(192)-X9/10-C(202) 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 24h 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 and 48 h tick saliva proteins. 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 24h Ixodes scapularis tick saliva proteins 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.

Fig. 1

Predicted disulfide bonds and zinc binding sites in Amblyomma americanum (Aam)AV422 predicted protein sequence. The translated AamAV422 protein was scanned on the DiAminoacid Neural Network Application (DiANNA 1.1 web server, http://clavius.bc.edu/~clotelab/DiANNA/). Numbering of cysteine residues forming disulfide bonds starts from the first methionine of AamAV422 pre-protein. Predicted disulfide bond pairings, C²³-C¹¹⁵, C³³-C⁵⁸, C⁶⁷-C¹⁵⁰, C⁷⁵-C¹⁵¹, C⁹⁹-C¹²⁶, C¹⁵⁹-C¹⁹², C¹⁶⁸-C²⁰², are marked with solid lines.

Fig. 2

Multiple sequence alignment of Amblyomma americanum (Aam)AV422 amino acid sequence with homologs in other ticks (A) and non-tick arthropods (B). Amino acid sequences were aligned using the CustalWsequence alignment tool in MacVector DNA analysis software. Conserved amino acid residues are shaded gray. Stars (★) denotes conserved cysteine amino acid residues. Sequences are denoted by an abbreviation of their species name followed by their GenBank accession number. AamAV422, A. americanum (KC222016); Rhimic, Rhipicephalus microplus (CK189031); Rhapp, Rhipicephalus appendiculatus (CD782378); Amac, Amblyomma maculatum (AE033794, AE035804, AE036190); Avar, Amblyomma variegatum (DAA34157 and BM293119); Ixsc, Ixodes scapularis (Y66605, EEC1287, EEC13894, EEC18200, EEC18201, EEC18204, EEC18638, EEC18639 and EEC19641); Angamb, Anopheles gambiae (EAA00770); Meu, Mesobuthus eupeus (ABR21053); Hoju, Hottentotta judaica (ADY39539); Bter, Bombus terrestris (XP_003396818); Dyak, Drosophila yakuba (EDW92254).

Fig. 2

Multiple sequence alignment of Amblyomma americanum (Aam)AV422 amino acid sequence with homologs in other ticks (A) and non-tick arthropods (B). Amino acid sequences were aligned using the CustalWsequence alignment tool in MacVector DNA analysis software. Conserved amino acid residues are shaded gray. Stars (★) denotes conserved cysteine amino acid residues. Sequences are denoted by an abbreviation of their species name followed by their GenBank accession number. AamAV422, A. americanum (KC222016); Rhimic, Rhipicephalus microplus (CK189031); Rhapp, Rhipicephalus appendiculatus (CD782378); Amac, Amblyomma maculatum (AE033794, AE035804, AE036190); Avar, Amblyomma variegatum (DAA34157 and BM293119); Ixsc, Ixodes scapularis (Y66605, EEC1287, EEC13894, EEC18200, EEC18201, EEC18204, EEC18638, EEC18639 and EEC19641); Angamb, Anopheles gambiae (EAA00770); Meu, Mesobuthus eupeus (ABR21053); Hoju, Hottentotta judaica (ADY39539); Bter, Bombus terrestris (XP_003396818); Dyak, Drosophila yakuba (EDW92254).

Fig. 3

Neighbor joining guide phylogenetic tree showing the relationship of Amblyomma americanum AamAv422 putative protein to AamAV422-like proteins from ticks (n = 15) and non-tick arthropods (n = 6). The predicted AamAV422 protein sequence was aligned with AamAV422-like sequences: Rhimic, Rhipicephalus microplus (CK189031); Rhapp, Rhipicephalus appendiculatus (CD782378); Amac, Amblyomma maculatum (AE033794, AE035804, AE036190); Avar, Amblyomma variegatum (DAA34157 and BM293119); Ixsc, Ixodes scapularis (Y66605, EEC1287, EEC13894, EEC18200, EEC18201, EEC18204, EEC18638, EEC18639 and EEC19641); Angamb, Anopheles gambiae (EAA00770); Meu, Mesobuthus eupeus (ABR21053); Hoju, Hottentotta judaica (ADY39539); Bter, Bombus terrestris (XP_003396818) and Dyak, Drosophila yakuba (EDW92254), using ClustalW. The guide phylogenetic tree was developed using the MacVector Neighbor joining method default parameters as described in Section 2.3. Groups 1 and 2, and subgroups BA and BB are indicated. Numbers on each branch represent bootstrap values that signify the level of confidence in the branch.

Fig. 4

Expression and affinity purification of recombinant (r) Amblyomma americanum AamAV422 expression in Pichia pastoris. (A) Cumulative daily expression levels of rAamAV422 through 5 days of culture. Construction of expression plasmid, induction and validation of expression levels using the antibody to the C-terminus hexahistidine tag and affinity purification of rAamAV422 were performed as described in Section 2.4. (B) Validation of affinity purification of rAamAV422 using Coommassie blue staining was accomplished by a combination of ammonium sulfate precipitation and affinity purification on NiCl²⁺ charged columns as described in Section 2.4. (C) Validation of N-glycosylation posttranslational modification of rAamAV422: 20 μg of affinity-purified rAamAV422 was treated with the PNGase F enzyme as described in Section 2.4. De-glycosylated rAamAV422 was detected by western blotting analysis using antibodies to the C-terminus hexahistidine tag. Lane A, Non-treated rAamAV422; lane B, rAamAV422 treated with PNGase F buffer only, lane C, deglycosylated rAamAV422.

Fig. 5

Western blotting analyses. Validation of Amblyomma americanum AamAV422 protein secretion into the host, cross-reactivity of recombinant (r)AamAV422 with antibodies to other ticks, temporal and spatial expression analyses of AamAV422 were done by routine western blotting analyses as described in section 2.6. rAamAV422 western blots were screened with rabbit pre-immune serum (A), antibodies to 24 h (B) and 48 h (C) A. americanum tick saliva proteins (TSPs) and antibodies to Ixodes scapularis (D) TSPs. Mono-specific antibodies were eluted as described and used to screen western blots of tick saliva (TS) proteins, tick hemolymph (TH) and protein extracts of whole ticks (WT) (E), and dissected tick organs, salivary glands (SG), Midgut (MG) and carcass (CA) (F). TS lanes 1, 2 and 3 are TSPs harvested from 48, 96 and 120 h fed ticks, respectively. TH, WT, SG, MG and CA lanes 1, 2 and 3 are hemolymph and protein extracts of 24, 72 and 120 h fed ticks. PC, rAamAV422 used as positive control; *, a potentially glycosylated native AamAV422 form.

Fig. 6

Anti-platelet aggregation function assay. The effect of recombinant (r) Amblyomma americanum AamAV422 protein (rAamAV422) on platelet aggregation was done using the whole blood approach as described in Section 2.8. Five hundred μL of cattle whole blood mixed 9:1 with sodium citrate diluted with equal volume normal saline (0.9% sodium chloride) was pre-incubated with indicated various amounts of rAamAV422 for 10 min at 37°C. Adding 20 μM ADP triggered platelet aggregation, which was subsequently monitored as a function of electrical resistance using the Chrono-Log aggregometer.

Fig. 7

The effect of recombinant (r) Amblyomma americanum AamAV422 protein (rAamAV422) against plasma clotting in the recalcification time assay. Twenty μL of citrated human plasma diluted up to 90 μL with 10 mM HEPES buffer plus 150 mM NaCl, pH 7.4, was pre-incubated at 37°C for 5 and 10 min with indicated amounts of affinity purified rAamAV422. In the microplate, clotting time was triggered by adding 10 μL of 25 mM CaCl₂₊ and then monitored as described in Section 2.8.

Fig. 8

The effect of recombinant (r) Amblyomma americanum AamAV422 protein (rAamAV422) against classical complement activation pathway. Indicated amounts of rAamAV422 protein were pre-incubated with serum for 10 min at 37°C and subjected to the classical complement activation assay as described in Section 2.8.

Fig. 9

Validating the significance of Amblyomma americanum AamAV422 protein in tick feeding success. (A) Validating the disruption of AamAV422 mRNA in AamAV422-double stranded (ds) RNA injected ticks. Approximately 25-30 ticks were microinjected with 0.5-1 μL (2-3 μg/μL) of AamAV422 or GFP (control) dsRNA in Tris-EDTA buffer. At 48 h post-attachment, three ticks per treatment, non-injected and GFP-dsRNA injected control ticks, together with the AamAV422-dsRNA injected ticks, were manually detached. Ticks were individually processed for total RNA extraction, DNase treated to eliminate genomic DNA contamination and then subjected to two-step reverse transcriptase (RT)-PCR using AamAV422 PCR primers. Equivalent amounts, ~15 μL of PCR products, were resolved on 2% agarose gels containing 1 μg/ml of ethidium bromide. (B) Physical phenotype of AamAV422-dsRNA injected ticks. Ticks were photographed after spontaneous detachment. (C) Effect of RNA interference (RNAi)-mediated silencing of the AamAV422 mRNA disruption on Amblyomma americanum female tick feeding success. After spontaneously detaching from the host, ticks were individually weighed to determine engorgement weights (EWs) as indices for amounts of blood imbibed by ticks. EWs were subjected to one-way ANOVA as described in Section 2.9 to determine statistical significance. Mean (M) EWs (MEW ± S.D.) were plotted as bar graphs. *, significant differences in MEWs between AamAV422-dsRNA injected ticks and both control groups. P < 0.0001.