All animal experiments were approved by the Institutional Animal Care and Use Protocol Committees of the Pacific Immunology Corporation, CA, USA (protocol #11/11/19. Ref. SOP-1) and the University of Idaho, ID, USA (protocol #2010-54). The procedures were performed according to the U.S. National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and the ARRIVE guidelines and regulations.
Four New Zealand white rabbits (14481, 14482, 14483 and 14484) and five horses (H5, Te0018 (HO-168), HO-198, HO-209 and HO-183).
The T. equi Florida strain was used in the experiments described herein.
In silico analysis
The T. equi CLAMP amino acid sequence (accession number: BEWA_005470) was retrieved from GenBank. Phobius (http://phobius.sbc.su.se/) was used to predict the protein’s transmembrane topology and Protter15 was used for visualization and annotation of the transmembrane profile. B cell epitope mapping was performed using a proprietary suite of 20 separate predictive algorithms available at Pacific Immunology Corporation. This led to the identification of three immunogenic peptides i.e. peptide 1 (SERVNHIKTKGYLAIQNSAQNQKGNFNNLFVNEC), peptide 2 (DRFTHEMWKIAC) and peptide 3 (RQQMIPFPYLSYC) that map onto positions 103-136, 208-219 and 220-232 of the CLAMP amino acid sequence, respectively. The peptides were synthesized by Pacific Immunology Corporation.
Generation of T. equi-infected erythrocyte cultures and isolation of extracellular merozoites
T. equi-infected erythrocyte cultures were set up in five 25 cm2 Corning® cell culture flasks and maintained as previously described28 until a parasitemia of ~10% was attained. To isolate extracellular merozoites, the infected cell cultures were centrifuged at 650xg for 10 minutes to pellet erythrocytes. The supernatant was obtained and spun at 2800xg for 30 minutes at 10°C to pellet merozoites. The parasites were washed twice with 40 ml of 1X phosphate buffered saline (PBS) (pH7.2) and suspended in 10 ml of the same buffer.
Transcriptional analysis of the clamp gene in T. equi merozoites
RNA was extracted from T. equi merozoites using the Qiagen RNeasy® mini kit (Qiagen, Hilden, Germany) in compliance with the manufacturer’s instructions. Residual DNA was removed using the Invitrogen™ DNAse I kit (Thermo Fisher Scientific, Waltham, MA) and cDNA synthesis was performed using the Invitrogen™ SuperScript™ III first-strand synthesis system in accordance with the manufacturer’s instructions. Amplification of the transcript was performed using the Invitrogen™ Platinum™ SuperFi™ DNA Polymerase with specific primers (clamp fwd; 5’-GTA TAC ACA GAT AAG CCA TAA ATA ATC GTG-3’ and clamp rev; 5’-TCA AAA CTG GAA GTT ACG TGC C-3’). The PCR conditions used were as follows; initial denaturation at 98°C for 30 seconds, 40 cycles of denaturation at 98°C for 10 seconds, annealing at 57.6°C for 30 seconds and extension at 72°C for 90 seconds. Final extension was conducted at 72°C for 10 minutes and transcription of the clamp gene was visualized using a 1% agarose gel. The Qiagen QIAquick Gel Extraction Kit was used to clean-up the amplified transcript in accordance with the manufacturer’s instructions and Sanger sequencing was performed to confirm the integrity of the amplicon. Amplification of the equine merozoite antigen-1 (ema-1) transcript as previously described29 was used as a positive control, and amplification of RNA without addition of reverse transcriptase was used as a negative control.
Evaluation of expression of CLAMP in merozoites and intra-erythrocytic stages of T. equi development
Development of polyclonal antibody against synthetic CLAMP peptides
The polyclonal anti-CLAMP antibody was produced by Pacific Immunology Corporation (Pacific Immunology, Ramona, CA). Briefly, 5 ml of serum (pre-immunization serum) was obtained from each of the New Zealand white rabbits prior to immunization with 1 mg/ml of synthetic CLAMP peptides conjugated to the Keyhole limpet hemocyanin (KLH) carrier protein. The peptide conjugates were mixed with the Pacific Immunology AdjuLite™ Freund’s complete adjuvant at a 1:1 ratio. Rabbits 14481 and 14482 were immunized with peptide 1 and rabbits 14483 and 14484 were co-immunized with peptides 2 and 3. Three subsequent immunizations were performed at a 21-day interval, with the synthetic peptide conjugates mixed with the Pacific Immunology AdjuLite™ Freund’s incomplete adjuvant at a 1:1 ratio. 25 ml of serum was collected from the rabbits at 49- and 63-days post-initial immunization and at 7- and 21-days post-final boost to assess antibody responses. The rabbits were euthanized at 31 days post-final boost, and serum containing the polyclonal antibody was obtained from the individual rabbits and pooled for downstream analysis.
Assessment of the expression of CLAMP in extracellular T. equi merozoites
Immunoblotting was performed to evaluate expression of CLAMP in extracellular T. equi merozoites. The Protein Simple Wes™ kit (ProteinSimple, San Jose, CA) was used to conduct the analysis in accordance with the manufacturer’s instructions. Merozoite lysate was used as the antigen and the rabbit polyclonal anti-CLAMP antibody was used as a primary antibody at 1:100 dilution. The KPL affinity purified peroxidase labeled goat anti-Rabbit IgG (H+L) antibody (Seracare Life Sciences, Milford, MA) was used as a secondary antibody at 1: 500 dilution. In the case of negative and positive controls, merozoite lysates were used as the antigen and pre-immunization serum and the polyclonal anti-RAP-1a antibody were used as primary antibodies at 1:100 and 1:250 dilutions, respectively.
Live immunofluorescence antibody test was performed to confirm that CLAMP is expressed on the surface of T. equi as predicted by in silico analysis. Briefly, 6-CFDA staining was performed as previously described30 for viability assessment and quantification of the extracellular merozoites. 1 ml of 1X PBS containing live 2.5x108 merozoites was then pipetted into three 1.5 ml Eppendorf tubes®. The tubes were spun at 3000 xg for 5 minutes to pellet the parasites. The merozoites were obtained and incubated with 10% bovine serum albumin (BSA) in 1X PBS (blocking buffer) at room temperature for 15 minutes. The parasites were pelleted and washed twice using 500 µl of 1XPBS prior to being incubated with the polyclonal anti-CLAMP antibody diluted in the blocking buffer at 1:100 (test sample). Monoclonal anti-EMA-1 antibody and the pre-immunization serum were used as positive and negative controls at 1:200 and 1:100 dilutions, respectively. Incubation was performed at 37°C for 30 minutes and the parasites were spun and washed thrice prior to incubation with fluorescent-labeled secondary antibodies. The test sample and negative control were incubated with the Invitrogen™ goat anti-Rabbit IgG (H+L) Alexa Fluor™ Plus 647 and the positive control was incubated with Invitrogen™ Alexa Fluor™ 594 goat anti-mouse IgG (H+L) antibody. Both antibodies were diluted in the blocking buffer at 1:200 and the Sigma Aldrich® DAPI was added to the secondary antibody solutions at 0.1 µg/ml. The parasites were incubated at 37°C for 30 minutes prior to being pelleted once more and washed thrice. The merozoites were suspended in 50 µl of 1X PBS and 10 µl of the suspensions from each of the Eppendorf tubes® were transferred onto respective wells of a 12-well teflon printed diagnostic microscopic slide. The slide was air-dried and fixed using ice cold acetone and methanol as previously described31. It was then air-dried, and mounting was performed using 5% glycerol diluted in 1X PBS. A coverslip was used to cover the slide and confocal microscopy was performed using the Leica microsystems SP8-X white light pulsed laser point scanner with lightning confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL) and the data was collected using the Leica Application Suite X (LAS X) software.
Evaluation of the localization and expression of CLAMP in T. equi’s intra-erythrocytic developmental stages
The fixed immunofluorescence antibody test was performed to assess expression of CLAMP in the intra-erythrocytic stages of T. equi development. Briefly, infected erythrocyte smears were permeabilized and fixed using ice cold acetone and methanol at equal ratios as previously described31. The slides were air dried and blocked with 200 µl of the blocking buffer for 15 minutes at 37°C in a wet chamber prior to being washed twice with 1X PBS. 200 µl of the polyclonal anti-CLAMP antibody diluted in the blocking buffer at 1:10 dilution was pipetted onto one of the slides (test slide). 1:10 and 1:50 dilutions of the pre-immunization serum and monoclonal anti-EMA-1 antibody were used as negative and positive controls, respectively. The slides were incubated at 37° C for 30 minutes prior to being washed twice with 1X PBS. The test and negative control slides were then incubated with 200 µl of the Invitrogen™ goat anti-Rabbit IgG (H+L) Alexa Fluor™ Plus 647 diluted in the blocking buffer at 1:200. The positive control was incubated with 200 µl of the Invitrogen™ Alexa Fluor™ 594 goat anti-mouse IgG (H+L) antibody diluted at 1:200. DAPI was added to the secondary antibody suspensions at 0.1 µg/ml. The three slides were incubated at 37°C for 30 minutes prior to being washed twice using 1X PBS. They were then air-dried, and 5% glycerol diluted in 1X PBS was used for mounting. Evaluation of the localization and expression of CLAMP was performed using the Leica microsystems SP8-X white light pulsed laser point scanner with lightning confocal microscope and LAS X software was used for data acquisition.
Assessment of antibody responses to CLAMP in T. equi infected horses
Indirect ELISA was conducted to determine whether CLAMP elicits antibody responses in T. equi infected horses. Horse sera obtained from previous studies were used in this analysis. This included sera from horses H532, Te0018 (HO-168)33 and HO-198, HO-209 & HO-18334 that were collected prior to T. equi infection and at 34, 14, 11, 1.5 and 18 months post-infection respectively. Briefly, two 96-well Thermo Scientific™ Nunc™, Immulon™ plates (Thermo Fisher Scientific, Waltham, MA) were labeled as pre- and post-infection plates. The wells were then coated with 100 µl/ well of the combined CLAMP synthetic peptides diluted in 0.05 M carbonate-bicarbonate (Na2CO3-NaHCO3) buffer (pH 9.6) to final concentrations of 0.05 mg/ml each. Coating was performed in triplicates and the plates were incubated overnight at 4°C. They were then washed once using 0.05% tween 20 diluted in 1X PBS (wash buffer) and blocked with 200 µl of 20% skimmed milk diluted in the wash buffer at room temperature for 1 hour. The blocking buffer was discarded and 75 µl of the pre- and post-infection sera diluted in 0.3% BSA in 1X PBS (dilution buffer) were pipetted into wells of the respective plates at 1:100 dilution. The plates were incubated at room temperature for one hour prior to being washed four times. 100 µl of the KPL affinity purified peroxidase labeled goat anti-Rabbit IgG (H+L) antibody diluted in the dilution buffer at 1:10,000 was pipetted into the wells, and the plates were incubated at room temperature for one hour. The wells were then washed four times and 100 µl of the Thermo Scientific™ 1-Step™ Ultra TMB-ELISA substrate solution was added to each well. Incubation was performed at room temperature for 15 minutes prior to addition of 100 µl of 2M sulfuric acid to stop the reaction. The SpectraMax® 190 microplate reader (Molecular Devices, San Jose, CA) was used to measure the absorbance of each well at 450 nm. Cut-off was calculated using the formula: mean (of the pre-infection serum) + 3*SD (of the pre-infection serum)35. Any value above the cut-off was considered to be a true positive. Two-way ANOVA available in GraphPad Prism 8.4.3 (GraphPad Software, San Diego, CA) was used to perform the statistical analysis at a significance level (α) of 0.05.
Evaluation of the role of T. equi CLAMP in the invasion of equine erythrocytes
The in vitro neutralization assay was performed to determine whether T. equi utilizes CLAMP to invade host cells. Briefly, heat inactivated post-immunization serum (polyclonal anti-CLAMP antibody) was diluted in 162 µl of the T. equi growth medium 28 at 1:10, 1:20 and 1:40 dilutions prior to being added to the wells of a Corning® Costar® 96-well flat-bottom tissue culture-treated plate (Millipore Sigma, Burlington, MA) in triplicate. Heat inactivated pre-immunization serum was used as a negative control at dilutions identical to the polyclonal anti-CLAMP antibody. 18 µl of T. equi infected erythrocytes at 0.2% parasitemia were added to the wells and the plate was incubated at 37°C and 5% CO2 for 96 hours. The antibody containing medium was replaced after every 24 hours.
5µl of the infected cells were harvested from the bottom of the wells after every 24 hours and centrifuged at 500xg for 5 minutes at 4°C. The pelleted cells were obtained and washed twice with 200 µl of 1X PBS (pH 7.2) prior to being suspended in 100 µl of the same buffer containing 25 µg/µl of the Invitrogen™ dihydroethidium to stain the parasites’ nuclei. The cell suspension was incubated at 5% CO2 and 37°C for 30 minutes in the dark prior to being washed twice with 200µl of 1X PBS. They erythrocytes were then suspended in 200 µl of the same buffer.
Flow cytometry was performed to assess the neutralization capacity of the polyclonal anti-CLAMP antibody as previously described36. Briefly, the suspended cells were analyzed by the Guava® easyCyte flow cytometer (Luminex Corporation, Austin, Tx) at a proportion of 800-1,000 cells/µl with 20,000 events acquired. Normal, uninfected equine erythrocytes (nRBC) and infected erythrocytes cultured in the absence of antibodies (iRBC) were used as negative and positive controls respectively in the flow cytometric analysis. The results were analyzed using the De Novo™ FCS Express v6 software (De Novo Software, Pasadena, CA) and the output was presented as a percentage of parasitized erythrocytes (PPE). Statistical analysis was performed using two-way ANOVA at a significance level of 0.05.
Given the fact that optimum neutralization activity of the polyclonal anti-CLAMP antibody was observed at 72 hours post infection, percentage inhibition of merozoites was calculated at this time point. The formula: 100 - [(Test – nRBC / iRBC - nRBC) x 100]37 was used to determine the percentage of merozoites that were inhibited at the three antibody dilutions, and the results were compared to percentage inhibition in the presence of pre-immunization serum.
Isolation and identification of equine erythrocyte proteins that interact with CLAMP
crosslinking and co-immunoprecipitation of interacting proteins
To crosslink erythrocyte surface proteins to CLAMP, 5 ml of uninfected equine erythrocytes suspended in 10 ml of 1X PBS were incubated with 5 x 108 T. equi merozoites for 1 hour at 4°C. The Thermo Scientific™ DTSSP crosslinker was added to the suspension to a final concentration of 2 mM, and further incubation was performed on ice for 2 hours. Tris was then added to the solution to a final concentration of 20 mM, and incubation was performed at room temperature for 15 minutes to quench the reaction.
To crosslink intramembrane and intracellular erythrocyte proteins to CLAMP, 5 ml of T. equi infected erythrocytes at ~11% PPE were washed twice in 1X PBS to remove T. equi growth media prior to being suspended in 10 ml of 1X PBS. The Thermo Scientific™ DSP crosslinker was then dissolved in DMSO to a final concentration of 25 mM prior to being added to the suspended erythrocytes to a final concentration of 2 mM. Incubation was performed on ice for 2 hours and tris was added to a final concentration of 20mM to quench the reaction.
The crosslinked proteins were isolated using the Thermo Scientific™ Pierce™ co-immunoprecipitation kit in accordance with the manufacturer’s instructions, and agarose resins supplied by the manufacturer were used as negative controls. SDS-PAGE analysis was performed using the Invitrogen™ NuPAGE™ 4-12% Bis-Tris protein gel for detection of CLAMP’s interacting partners, and immunoblot analysis was conducted as described herein to confirm that CLAMP was isolated alongside its interacting partners.
Mass spectrometry analysis
The protein gel was stained overnight with the Thermo Scientific™ Pierce coomassie brilliant blue R-250 prior to being washed twice with double distilled water at 15 minutes each. Protein bands on the gel were cut-out and transferred into sterile 2 ml Eppendorf tubes®, and trypsin digestion was performed as previously described38. Nano LC-MS/MS was performed to analyze the digested peptides using the Thermo Scientific™ Easy-nLC 1000 ultra-high-pressure liquid chromatography connected to the Thermo Scientific™ Orbitrap Fusion Tribrid mass spectrometer. The Waters NanoAcquity HSS T3 column with a Thermo Scientific™ trap column was used to separate the peptides as previously described39. Full MS scans (MS1) were acquired from peptides eluted from the orbitrap’s electrospray source and MS2 scans were conducted on 3s timed scans that were data-dependent and were detected in the ion trap as previously described39.
The Thermo Scientific™ Proteome Discoverer software (version 2.2) was used to search the LC-MS/MS raw data against the Equus caballus proteome sequences available in Uniprot (www.uniport.org) to identify equine erythrocyte proteins that interact with T. equi CLAMP. The static peptide and dynamic modification analyses, and a decoy database search to control for false discovery rate (FDR) were performed at previously described settings39.