Fig 1.
Amino acid sequence analysis of IxsS17.
(A) Phylogenetic tree was constructed using the MEGA-X software, Maximum Likelihood method and Le_Gascuel_2008 model with Bootstrap set to 1,000 replications. Group A, B, C and D represent amino acid identity levels of IxsS17 to its homologs in percentage. (B) Multiple sequence alignment of IxsS17 reactive center loop (EEGSEAAAVTGFVIQLRTAAF) and its homologs as well as the antithrombin III outlier was done in MacVector using T-Coffee specifications. Amino acids in the grey box are identical.
Fig 2.
Expression and affinity purification of recombinant (r) IxsS17.
(A) Graphical illustration of three different rIxsS17 expression constructs that were custom synthesized: (1) C-terminal hexa-histidine tag, (2) N-terminal hexa-histidine tag and Tobacco Etch Virus (TEV) cutting site (ENLYFQG) included, and (3) hexa-histidine tag is cleaved off at the TEV cutting site in the non-tagged rIxsS17. Please note all three recombinant constructs contain full-length sequence of rIxsS17. (B) Western blotting analysis of daily expression of rIxsS17 in Pichia pastoris culture. Culture (1 mL) were precipitated by ammonium sulfate saturation and resolved on 10% SDS-PAGE. rIxsS17 were detected in western blot using the HRP-conjugated monoclonal antibody to the hexa-histidine tag. (C) Silver staining and (D) Western blotting analysis of affinity purified rIxsS17. The hexa-histidine tag was detected in the C-terminal (Lane 1) and N-terminal-His-rIxsS17 (Lane 2) but not in the non-tagged rIxsS17 (Lane 3).
Fig 3.
Inhibition profiling of rIxsS17 against 17 serine proteases related to host responses during tick feeding.
(A) Inhibition rates of C-terminal-Histidine tagged rIxsS17 (1 μM) against 17 serine proteases (with indicated concentrations) in the substrate hydrolysis assays. Substrate hydrolysis was monitored at A405nm every 11s for 30 min at 30°C. Inhibition rate was calculated using the formula: 100-Vi/V0 x 100, where Vi = activity in the present of, and V0 in the absence of rIxsS17. (B) Inhibition activity of C-terminal, N-terminal and non-Histidine tagged rIxsS17 against trypsin, factor Xa and human thrombin was determined using the substrate hydrolysis assay. Data represents mean ± SEM calculated from 3 biological replicates. The difference was analyzed using ANOVA in GraphPad Prism 9 and is statistically significant when P value ≤ 0.05.
Fig 4.
rIxsS17 is a moderate inhibitor of trypsin, rat trypsin IV and factor Xa.
Stoichiometry inhibition (SI) analysis calculated the amount of rIxsS17 needed to inhibit one molecule of bovine trypsin (A), rat trypsin IV (B), and factor Xa (C). Various molar ratios of rIxsS17 to proteases (0, 2.5, 5, 10, 20, 25, 50) were incubated for 15 min at 37°C with constant concentration of bovine trypsin (1.5 nM) or rat trypsin IV (2.0 nM) or factor Xa (13.9 nM). In the presence of appropriate substrates, residual enzymatic activity was measured and plotted against rIxsS17: protease molar ratio. The SI was determined by extrapolating to the rIxsS17: protease ratio where protease activity is zero (Y axis = 0). The inhibition rate (ka) of rIxsS17 was determined against bovine trypsin (D), rat trypsin IV (E) and factor Xa (F). Different concentrations of rIxsS17 (50, 100, 200, 400, 600 and 1000 nM) were incubated with constant amounts of bovine trypsin (14.6 nM), trypsin IV (12.5 nM) or factor Xa (13.9 nM) for different periods of time (0, 1, 2, 4, 6, 8, 10 and 15 min) at 37°C. The residual protease activity was measured and plotted against time to determine the pseudo-first order constant, kobs. Consequently, the second-rate constant (ka) was determined by the best fit line slope of the kobs values that were plotted against rIxsS17 concentration.
Fig 5.
Protein-to-protein interaction using differential precipitation of proteins (DiffPOP) analysis reveals novel IxsS17 functional insights.
25 μg of affinity purified rIxsS17 (A-C) or rIxsS4 (D-F) was incubated with human plasma in reaction buffer (20mM Tris-HCL and 150mM NaCl pH 7.4) overnight at 37°C. The reaction was stabilized using Phosphoprotein Kit- Buffer A and subjected to repeated precipitation (X10) using methanol and acetic solution (90% methanol to 1% acetic acid). Appropriately washed precipitates of each fraction were resolved on 10% SDS-PAGE and transferred onto PVDF membrane for western blot analysis using monospecific antibodies to rIxsS17. A and D = human plasma only, B and E = human plasma mixed with rIxsS17 or rIxsS4, and C and F = rIxsS17 or rIxsS4 alone. Ladder (L), Number (1–9) represents each fraction from differential precipitation. Please note that, fraction 10 for rIxsS17 is not shown in this figure; however, its LC-MS/MS data analysis is available in S1 File.
Table 1.
Fast fractionation and LC-MS/MS analyses identification of human plasma proteins that interacted with rIxsS17 and validation using in silico protein to protein interaction prediction PSOPIA software.
Fig 6.
IxsS17 binds heparin and the putative binding sites are located on the positive basic patch.
(A) Comparative modeling of IxsS17 secondary structure was predicted on Chimera X server and heparin binding predicted using Autodock Vina and Auto Dock Tools. Heparin ligand (in blue) was arranged accordingly to be flexible to rotate and to explore the most probable binding positions (in red) while the receptor was kept rigid. RCL = reactive center loop. (B) Two heparin binding sites at Lysine 188 and 210 are located on the positive basic patch (Electrostatic potential is color coded: positive is blue; neutral is white and negative is red). (C) Binding affinity of rIxsS17 to 4 different GAGs: heparin (black circle), heparan sulphate (red square), dermatan sulphate (green triangle) and chondroitin sulphate (yellow upside-down triangle). The rIxsS17 was added into 96-well microplates previously coated with different GAG at the concentration of 0, 1, 2, 5, 10 and 20 μg/mL. Binding was detected using HRP-conjugated antibody to the histidine tag and documented as A450nm. The data represent mean ± SEM from 3 biological replicates. (D) Silver staining of rIxsS17, rAAS19 (positive control), and r1E1 (negative control) eluted from heparin column. Recombinant proteins (~300 μg) were applied to the heparin column. After washing, the proteins were eluted using a gradient concentration of NaCl (0.25–0.5–1.0–2.0–3.0 M). Serpins = the proteins before applying to the column, FT = Flow through.
Fig 7.
Heparin binding enhances rIxsS17 anti-coagulant activity.
Anti-blood clotting effects of heparin binding on rIxsS17 was determined in the recalcification time assay. Universal coagulation reference human plasma was pre-incubated with (1) reaction buffer control, or (2) heparin only, or (3) rIxsS17 only, or (4) rIxsS17 and heparin pre-incubated together, or (5) rIxsS17 and heparin pre-incubated separately. CaCl2 was added to trigger blood clotting and the reaction was monitored at A650nm every 20s for 20 min. The A650nm data were then fitted in the Sigmoidal dose-response lines: blue (buffer control), red (heparin only), green (rIxsS17 only), black (rIxsS17 and heparin pre-incubated together), and brown (rIxsS17 and heparin pre-incubated separately). Clotting time was interpolated from the sigmoid line when A650nm increases by 10% with 95% confident interval. Drop vertical lines A, B, C, D, and E = clotting time for buffer only (circle), rIxsS17 only (triangle), heparin only (square), rIxsS17 and heparin pre-incubated with plasma separately (upside-down triangle), rIxsS17 and heparin pre-incubated with plasma together (diamond).
Fig 8.
rIxsS17 dose dependently inhibited membrane attack complex deposition of the Mannose-Binding Lectin pathway.
A Blank, Negative control, Positive control or Positive control incubated with 2-fold-serial dilution of rIxsS17starting from 4 μM were added to the Mannan binding lectin (MBL) pathway Kit and incubated at 37°C for 60 min. After the washing step, conjugate and substrate were subsequently added following the instructions of the manufacturer. Mac deposition rate was calculated using the formular: (Sample-NC)/(PC-NC) x100%, where NC is negative control and PC (or 0.0 μM of rIxsS17) is positive control. Data is presented as percent inhibition of MAC deposition mean ± SEM calculated from 3 biological replicates.
Fig 9.
rIxsS17 impaired complement mediated killing of Borrelia burgdorferi.
Normal human serum (NHS) was pre-incubated with serial dilutions of rIxsS17 (0.25, 0.5, 0.75, and 1.0 μM) or heat-inactivated rIxsS17 (1.0 μM) or Phosphate buffered saline (PBS) at 37°C for 30 min prior to addition of 85 μl of 106 cells/mL of B. burgdorferi B314/pBBE22luc (complement sensitive strain) and incubated in a bio-shaker at 32°C, 100 rpm. NHS incubated with B. burgdorferi B314/pPCD100 (complement resistant strain) were used as positive control. Survival rates of B. burgdorferi were assessed at 1.5 h (A), 2 h (B), 2.5 h (C) and 3 h (D) post incubation. Data represents mean ± SEM of 3 biological replicates. Statistical significance was evaluated using t test in GraphPad Prism 9 (ns: no significance, *:P value ≤ 0.05, **: P value ≤ 0.01, ***: P value ≤ 0.001). Negative control: black circle, PBS: red diamond, HI-rIxsS17: green cross, 0.25 μM rIxsS17: maroon square, 0.5 μM rIxsS17: green triangle, 0.75μM rIxsS17: purple upside-down triangle, 1μM rIxsS17: blue hexagon, Positive control: orange star.
Fig 10.
Mouse groups co-inoculated with low dose of rIxsS17 have higher B. burgdorferi load in organs than high dose injected mice.
Four mice/group were inoculated with B. burgdorferi only (104 cells) mixed with or without various amounts of rIxsS17 (0.060, 0.125, 0.250, 0.500 μM). At 21 days post inoculation, B. burgdorferi burden in mouse heart (A), joint (B), ear (C) and bladder tissues (D) was quantified by real-time qPCR method. The data were presented as fold change of the rIxsS17 treated groups in comparison with B. burgdorferi group (2 -ΔΔCt = [(Ct Flab—Ct β-Actin) B. burgdorferi-rIxsS17 co-injected group—(Ct Flab—Ct β-Actin) Bb only group]). Blue: B. burgdorferi only group, Red: B. burgdorferi-rIxsS17 co-injected group.
Fig 11.
Mouse groups co-inoculated with low dose of rIxsS17 have higher IgM titers compared to high dose injected mice.
ELISA plates previously coated with B. burgdorferi crude antigen (200 ng/well) were tested with mouse sera that diluted at 1:250 (A) and 1:500 (B). IgM titers were determined using anti-mouse IgM monospecific antibody conjugated with HRP and absorbance were read at 450 nm. The data were presented as mean ± SEM; each dot is individual mouse. NC = negative control; Bb = B. burgdorferi. Statistical significance was evaluated using t test in GraphPad Prism 9 (***:P value ≤ 0.001, ****: P value ≤ 0.0001). (C). Western blot analysis of Bb lysate (2 μg) incubated with antisera from mice (diluted to 1:200) and anti-mouse IgM antibody-HRP conjugate (diluted to 1:5000). Images were taken at 18 seconds of exposure. Asterisks indicate extra and intense bands detected in mice challenged with Bb plus 0.06 and 0.125 μM rIxsS17.