The putative role of Rhipicephalus microplus salivary serpins in the tick-host relationship

Abstract

Inflammation and hemostasis are part of the host's first line of defense to tick feeding. These systems are in part serine protease mediated and are tightly controlled by their endogenous inhibitors, in the serpin superfamily (serine protease inhibitors). From this perspective ticks are thought to use serpins to evade host defenses during feeding. The cattle tick Rhipicephalus microplus encodes at least 24 serpins, of which RmS-3, RmS-6, and RmS-17 were previously identified in saliva of this tick. In this study, we screened inhibitor functions of these three saliva serpins against a panel of 16 proteases across the mammalian defense pathway. Our data confirm that Pichia pastoris-expressed rRmS-3, rRmS-6, and rRmS-17 are likely inhibitors of pro-inflammatory and pro-coagulant proteases. We show that rRmS-3 inhibited chymotrypsin and cathepsin G with stoichiometry of inhibition (SI) indices of 1.8 and 2.0, and pancreatic elastase with SI higher than 10. Likewise, rRmS-6 inhibited trypsin with SI of 2.6, chymotrypsin, factor Xa, factor XIa, and plasmin with SI higher than 10, while rRmS-17 inhibited trypsin, cathepsin G, chymotrypsin, plasmin, and factor XIa with SI of 1.6, 2.6, 2.7, 3.4, and 9.0, respectively. Additionally, we observed the formation of irreversible complexes between rRmS-3 and chymotrypsin, rRmS-6/rRmS-17 and trypsin, and rRmS-3/rRmS-17 and cathepsin G, which is consistent with typical mechanism of inhibitory serpins. In blood clotting assays, rRmS-17 delayed plasma clotting by 60 s in recalcification time assay, while rRmS-3 and rRmS-6 did not have any effect. Consistent with inhibitor function profiling data, 2.0 μM rRmS-3 and rRmS-17 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner by up to 96% and 95% respectively. Of significant interest, polyclonal antibodies blocked inhibitory functions of the three serpins. Also notable, antibodies to Amblyomma americanum, Ixodes scapularis, and Rhipicephalus sanguineus tick saliva proteins cross-reacted with the three R. microplus saliva serpins, suggesting the potential of these proteins as candidates for universal anti-tick vaccines.

Keywords: Cathepsin G; Immune response; Platelet aggregation inhibitor; Tick saliva.

Figure 1. Recombinant expression of R. microplus salivary serpins in Pichia pastoris

(A) Daily expression levels of rRmS-3, rRmS-6, and rRmS-17 throughout five days (1–5). Recombinant proteins in spent culture media were precipitated by ammonium sulfate and verification of protein expression was performed using an antibody to the C-terminus hexa histidine tag. (B) Total expression (1) and affinity purified-(2) rRmS-3, rRmS-6, and rRmS-17 were resolved on a 12.5 % SDS-PAGE following Coomassie brilliant blue staining. (C) Recombinant serpins treated with deglycosylation enzyme mix (D+) or without treatment (D−) were resolved on a 12.5 % SDS-PAGE following Coomassie brilliant blue staining and western blotting using anti-C-terminus hexa histidine tag antibody.

Figure 2. Inter-species cross-reactivity assay

Purified rRmS-3, rRmS-6 and rRmS-17 treated with deglycosylation enzyme mix (D+) or without treatment (D−) were resolved on a 12.5 % SDS-PAGE following western blotting analysis with: cattle serum generated by R. microplus tick infestation (dilution 1:50), rabbit serum generated by adult R. sanguineus infestations (dilution 1:50), A. americanum infestation (dilution 1:50), and adult I. scapularis infestation (dilution 1:25). Anti-C-terminus hexa histidine tag antibodies (dilution 1:5000) and rabbit pre-immune serum (dilution 1:50) were used as control.

Figure 3. Transcription and expression profile of salivary serpins

(A) Comparison of the feeding stage of ticks used for serpin expression profile.. (B) Total RNA was extracted from ticks and subjected to RT-PCR to amplify RmS-3, RmS-6, and RmS-17 fragments. Tick ribosomal protein S3a was used as reference. (C) Total protein extracts of SG from eight groups of ticks were subjected to western blotting analyses using antirRmS-3, anti-rRmS-6, rRmS-17 sera as well as pre-immune serum. Arrows indicate the position of the native protein.

Figure 4. rRmS-3 stoichiometry inhibition (SI) assay

Residual protease activity in the presence and absence of rRmS-3 was evaluated pre-incubating serpin for 1 h at 37 °C with (A) cathepsin G (200 nM), (B) chymotrypsin (10 nM), and (C) pancreatic elastase (50 nM), resulting in molar ratios (serpin:protease) ranging from 0 to 10. Protease activity was measured using specific colorimetric substrate for each protease as described in Materials and Methods. The data were plotted as residual protease activity (V_i/V₀) versus molar ratio (serpin:protease).

Figure 5. rRmS-6 stoichiometry inhibition (SI) assay

Residual protease activity in the presence and absence of rRmS-6 was evaluated pre-incubating serpin for 1 h at 37 °C with (A) trypsin (10 nM), (B) chymotrypsin (10 nM), (C) factor Xa (5 nM), (D) factor XIa (5 nM), and (E) plasmin (50 nM), resulting in a molar ratio (serpin:protease) ranging from 0 to 10 for trypsin, chymotrypsin, and plasmin, and from 0 to 20 for factor Xa and factor XIa. Protease activity was measured using specific colorimetric substrate for each protease as described in Materials and Methods. The data were plotted as residual protease activity (V_i/V₀) versus molar ratio (serpin:protease).

Figure 6. rRmS-17 stoichiometry inhibition (SI) assay

Residual protease activity in the presence and absence of rRmS-17 was evaluated pre-incubating serpin for 1 h at 37 °C with (A) trypsin (10 nM), (B) chymotrypsin (10 nM), (C) cathepsin G (200 nM), (D) factor XIa (5 nM), and (E) plasmin (50 nM), resulting in a molar ratio (serpin:protease) ranging 0 to 10 (for factor XIa molar ratio ranges between 0 to 20). Protease activity was measured using specific colorimetric substrate for each protease as described in Materials and Methods. The data were plotted as residual protease activity (V_i/V₀) versus molar ratio (serpin:protease).

Figure 7. Heat and SDS-stable complex formation assay

Increasing amounts of recombinant serpins were pre-incubated for 1 h at 37 °C with a constant concentration of (A) chymotrypsin and cathepsin G for rRmS-3, (B) trypsin and cathepsin G for rRmS-17, and (C) trypsin for rRmS-6, resulting in molar ratios varying from 0.6:1 to 10:1 (serpin:protease). Samples were resolved on 12.5% SDS–PAGE and Coomassie blue-stained to identify SDS-stable complexes (indicated by arrows).

Figure 8. rRmS-3 and rRmS-17 effect upon cathepsin G-induced platelet aggregation

Platelet aggregation function induced by cathepsin G assay was done using bovine platelet rich plasma (PRP) as described in Materials and Methods. (A) Tyrode solution with varying amounts of rRmS-3 or rRmS-17 (3 µM, 2.5 µM, 2 µM, 1.5 µM, 1 µM, and 0.5 µM) were pre-incubated with cathepsin G (0.7 µM) in a 50-µL reaction for 15 min at 37 °C. Platelet aggregation was initiated with addition of 100 µL pre-warmed PRP and monitored at 20-s intervals over 30 min at OD_650nm. (B) Percent of reduction of cathepsin G-induced platelet aggregation inhibited by rRmS-3 and rRmS-17.

Figure 9. Effect of R. microplus serpins upon plasma recalcification time

Human reference plasma (50 µL) was incubated with rRmS-3 (10 µM), rRmS-6 (10 µM), and rRmS-17 (10 µM) in 90 µL of Tris–HCl reaction buffer for 15 min at 37 °C followed by the addition of 150 mM CaCl₂ (10 µL). Clotting was measured every 20 s for 30 min.

Figure 10. Reactivity of anti-rRmS antibodies with RmS-3, RmS-6 and RmS-17 from tick saliva

Pilocarpine-induced tick saliva were harvested as described in Material and Methods. Saliva total protein treated with deglycosylation enzyme mix (D+) or non-treated (D−) were resolved on a 12 % SDS-PAGE following western blotting using polyclonal sera (A) anti-rRmS-3, (B) anti-rRmS-6, and (C) anti-rRmS-17.

Figure 11. Effect of antibodies upon serpin inhibitory activity

Purified IgG from (A) rabbit polyclonal serum or (B) from mice MAbs (anti-rRmS-3, anti-rRmS-6, and rRmS-17) were incubated with rRmS-3 (55 nM), rRmS-6 (55 nM) or rRmS-17 (88 nM) at 37 °C for 30 min before the addition of chymotrypsin (4.9 nM) for rRmS-3, or trypsin (1.6 nM) for rRmS-6 and rRmS-17, following a new 15-min incubation. Protease kinetics was monitored for 15 min every 11 s.