Histidine acid phosphatase domain-containing protein from Haemonchus contortus is a stimulatory antigen for the Th1 immune response of goat PBMCs

Histidine acid phosphatase (HAP), a member of the histidine phosphatase superfamily, is widely found in parasites and is also a potential vaccine antigen or drug target. However, the biological function of HAP in Haemonchus contortus is still unclear. We cloned the HAP gene from H. contortus (Hc-HAP) and expressed the purified recombinant Hc-HAP (rHc-HAP) protein. The transcription of the Hc-HAP gene in the eggs, infective third-stage larvae (L3s), exsheathed third-stage larvae (xL3s) and adults (females/males) was analyzed by quantitative real-time-PCR (qPCR). An immunofluorescence assay was also used to detect the localization of Hc-HAP expression in adult worms. The effect of rHc-HAP on the function of peripheral blood mononuclear cells (PBMCs) was observed by co-culture of rHc-HAP protein with goat PBMCs. The qPCR results revealed that the Hc-HAP gene was transcribed at a higher level in the L3 and xL3 stages that there were gender differences in transcription at the adult stage, with females exhibiting higher transcription than males. Moreover, Hc-HAP was mainly expressed in adult intestinal microvilli. Additionally, western blot results revealed that rHc-HAP could be detected in goat sera artificially infected with H. contortus. In the experiments, rHc-HAP bound to goat PBMCs and released nitric oxide. The rHc-HAP also induced the expression of interferon gamma (IFN-γ) and the phosphorylated STAT 1 transcription factor, while inhibiting interleukin-4 expression. The results shows that rHc-HAP stimulated the IFN-γ/STAT1 signaling pathway and enabled polarization of PBMCs toward T-helper 1 immune responses.

lead to nematode resistance [3,4], and the emergence of multi-drug-resistant nematodes poses a potential threat to public health [5][6][7][8][9][10]. To develop an effective vaccine, it is imperative to understand the mechanisms of hostnematode interactions, particularly how nematode antigenic molecules modulate the host immune system.
The coexistence of H. contortus with the host relies on a delicate and complex immunomodulatory relationship between H. contortus and the host immune system. Gadahi et al. discovered that the excretory secretions of H. contortus inhibit the function of goat peripheral blood mononuclear cells (PBMCs) [11]. Moreover, we have recently demonstrated that trehalose-6-phosphate phosphatase (HcGOB) and trehalose phosphate synthase structural domain protein (Hc-TPS) in H. contortus inhibit PBMC function by activating the interleukin (IL)-10/signal transducer and activator of transcription (STAT) 3 (IL-10/STAT3) pathway [12,13].
Histidine acid phosphatase (HAP), a member of the histidine phosphatase superfamily, is characterized by a conserved catalytic core (histidine centric) in which histidine itself is phosphorylated [14]. A member of the HAP superfamily is found in a wide variety of parasites, so inhibiting its function has a potential therapeutic benefit [14]. For example, the lysosomal acid phosphatase of Giardia lamblia is required for its excystation [15]. RNA interference (RNAi) silencing of Caenorhabditis elegans pho-1 revealed its maternal effects; in that study, Tetsunari et al. demonstrated that HAP is essential for the development and growth of the worm and that its loss is lethal to the worm [16]. However, the biological function of HAP in H. contortus remains to be elucidated.
It has been shown that acid phosphatase (homolog of HAP) isolated from the external surface of the intracellular parasite Leishmania donovani can inhibit the production of oxidative metabolites in neutrophils [17]. However, almost nothing is known about the effect of HAP in nematodes on host immune function.
As part of the present study, we characterized the transcriptional features of the HAP gene from H. contortus (Hc-HAP) in the H. contortus egg, infective third-stage larvae (L3s), exsheathed third-stage larvae (xL3s) and adults, as well as its expression patterns in female and male adults. We also assessed the effects of the purified recombinant Hc-HAP (rHc-HAP) protein on immune cell function in goats, which may be helpful in developing new vaccine targets.

Parasites, animals and PBMCs
Native goats (3-6 months old) were housed in the Laboratory Animal Center at the School of Animal Medicine and provided with sterile hay, corn feed and free access to water. Haemonchus contortus (Nanjing strain) were maintained by successive passages through worm-free local hybrid goats. Adults (females and males), L3s, eggs and xL3s were collected as described in previous studies [11,18]. Healthy goats were housed in individual cages to prevent accidental worm infections and provided with sterile hay, corn feed and free access to water.
Wistar rats weighing approximately 250 g were purchased from Qing Long Shan Animal Breeding Farm, housed in a barrier animal house at the Experimental Animal Center of Nanjing Agricultural University and provided with sterile food and water.
PBMCs were isolated from goat peripheral blood using the standard Ficoll-Hypaque (GE Healthcare, Little Chalfont, UK) gradient centrifugation method described previously [19]. The PBMCs were washed 3 times with sterile phosphate-buffered solution (PBS), and the cell density was adjusted to 1 × 10 6 cells/ml. Then, the cell viability of isolated PBMCs was detected with trypan blue dye [12].

Construction of recombinant expression plasmids and expression of the rHc-HAP protein
Haemonchus contortus adults were obtained from the fourth stomach of goats infected with H. contortus. As a first step, total RNA of adult worms was isolated by TRIzol reagent (Vazyme Biotech Co., Ltd., Nanjing, China). Then, 1 µg of RNA was synthesized into complementary DNA (cDNA) using the HiScript III First Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd.). Specific amplification primers (HAP-F and HAP-R; Additional file 1: Table S1) were synthesized based on the sequence of the coding region (CDS region) of the Hc-HAP gene (Gen-Bank: CDJ80664.1) published on the NCBI website. A 25-µl aliquot of the reaction system [1 µl of each primer, 12.5 µl of 2× Phanta Master Mix (Vazyme Biotech Co., Ltd.), 1 µl of cDNA and 9. 5 µl of ddH 2 O] was prepared in a PCR tube and amplified by following the PCR procedure described by Wen et al. [13]. The gene products were cleaned using a Gel Extraction Kit (Vazyme Biotech Co., Ltd.) and ligated to the pET32a(+) prokaryotic expression vector. The pET32a(+)/Hc-HAP plasmid was verified by enzymatic digestion reactions (BamHI/XhoI) and sequence alignment analysis with BLAST.

Preparation of polyclonal antibodies against rHc-HAP protein
The rat anti-Hc-HAP polyclonal antibody was prepared as described previously [13]. Briefly, rHc-HAP protein (300 μg) was mixed with adjuvant (Freund's complete adjuvant; Sigma-Aldrich, St. Louis, MO, USA) and immunized subcutaneously in Wistar rats. After 14 days, rHc-HAP protein (300 μg) was mixed with adjuvant (Freund's incomplete adjuvant; Sigma-Aldrich) and injected subcutaneously at multiple points in Wistar rats, followed by the injection of three more doses of rHc-HAP protein (300 μg) mixed with Freund's incomplete adjuvant at 7-day intervals. One week after the fifth immunization, antisera to rHc-HAP protein were collected and stored at − 80 °C.

Analysis of rHc-HAP protein by western blot assays
The relative protein expression of rHc-HAP was detected by western blot, as described in our previous studies [12,20]. Briefly, the proteins (30 µg) were transferred onto polyvinyl difluoride (PVDF) membranes [GE Healthcare Life Science (China) Co., Beijing, China] after 12% SDS-PAGE and the membranes incubated with 5% bovine serum albumin (BSA) at 37 °C for 1 h. After washing the membranes 5 times with TBST (Tris-buffered saline containing 0.05% Tween-20), the membranes were incubated overnight at 4 °C with goat serum artificially infected with H. contortus. The following day, the membranes were washed 5 times with TBST and incubated with rabbit anti-goat immunoglobulin G (H+L) horseradish peroxidase secondary antibody for 2 h at room temperature. Finally, the bound antibodies were detected following the instructions of the DAB Horseradish Peroxidase Chromogenic Kit (Beyotime, Shanghai, China).

Detection of the relative transcript levels of the Hc-HAP gene using quantitative real-time PCR
Detection of relative transcript levels of Hc-HAP genes in different life stages (adults, L3s, eggs and xL3s) was performed by quantitative real-time PCR (qPCR) using primers (Additional file 1: Table S2) as previously described [12,21]. Briefly, total RNA was isolated from adults (female and male), eggs, L3s and xL3s by the TRIzol method (Vazyme Biotech Co., Ltd.). Then, 1 µg of RNA was synthesized into cDNA using the HiScript III First Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd.). The β-tubulin gene of H. contortus was used as a reference gene. The data were analyzed according to raw cycle thresholds (Ct), which were obtained using ABI Prism 7500 software (Applied Biosystems, Foster City, CA, USA) using the comparative Ct (2 −ΔΔ Ct ) method.

Immunolocalization of Hc-HAP
The expression of native Hc-HAP protein in adult worms (females and males) was detected by immunofluorescence assay (IFA). Haemonchus contortus adults were obtained from the fourth stomach of goats and fixed in 4% tissue cell fixation solution for 12 h as previously described [12]. Samples were prepared into 4-μm paraffin sections, and antigens were repaired by microwave heating. Sections were placed in 5% BSA solution and incubated for 1 h at 37 °C, followed by incubation with rat anti-rHc-HAP serum and normal rat serum (negative control) overnight at 4 °C. The following day, after five washes with PBST wash solution, the sections were incubated with Cy3-labeled Goat Anti-Rat IgG (Beyotime, Shanghai, China) for 1 h at 37 °C, while being protected from light. After 5 washes with PBST, sections were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime), and fluorescence was observed with a laser confocal microscope (LSM 710; Carl Zeiss Spectroscopy GmbH, Jena, Germany).

Binding of rHc-HAP protein to goat PBMCs
The ability of the rHc-HAP protein to bind to goat PBMCs was examined following procedures described previously [12]. PBMCs were incubated with rHc-HAP protein (10 μg/ml) at 37 °C for 1 h in a 5% CO 2 cell culture incubator, and a control group was established (an equal volume of PBS was added). The PBMCs were washed with PBS and transferred to slides, following which the slides were fixed with 4% tissue cell fixation solution for 20 min. The specific IFA procedure was the same as that used for the immunolocalization assay of Hc-HAP.

Cell proliferation assays
Cell proliferation was detected using the Cell Counting Kit-8 (CCK-8; Beyotime) according to our previous report [13]. Briefly, freshly isolated PBMCs were spread on 96-well cell culture plates at a concentration of 1 × 10 6 /ml with 100 µl per well. Different concentrations (10, 20, 40 μg/ml) of rHc-HAP protein or pET-32a protein were added, respectively, and a control group was established (an equal volume of PBS was added). The cell culture plates were incubated in a 5% CO 2 cell incubator at 37 °C for 24 h. Following the addition of 10 μl of CCK-8 solution to each well, the cells were incubated for a further 4 h, at which time point the absorbance values at 450 nm were detected using a miniature flat panel reader.

Cell apoptosis assays
The group settings were the same as those used for the cell proliferation assays. The effect of rHc-HAP protein on the apoptosis of PBMCs was detected using flow cytometry, as previously described [22]. Cells were stained using the Annexin V-FITC Apoptosis Detection Kit (Beyotime) according to the manufacturer's instructions. The stained cells were analyzed by flow cytometry.

Nitric oxide production assays
The group settings were the same as those used for the cell proliferation assays. The cells were incubated in a 5% CO 2 cell incubator for 24 h at 37 °C, and the supernatant was collected. The secreted levels of nitric oxide (NO) in different groups were detected by the Total Nitric Oxide Assay Kit (Beyotime).

Cytokine transcript abundance detected by qPCR
The group settings were the same as those used for the cell proliferation assays. The relative transcript abundance of IL-4, IL-9, IL-17, and interferon gamma (IFNγ) was analyzed by qPCR. The reaction system contained 5 μl 2× ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.), 3.6 μl ddH 2 O, 0.2 μl forward and reverse primers and 1 μl cDNA. Primer sequences are shown in Additional file 1: Table S2 [23,24]. To calculate raw cycle thresholds (Ct), the relative Ct (2 −ΔΔCt ) method was used with ABI Prism 7500 software (Applied Biosystems).

The expression levels of inducible nitric oxide synthase, IL-17, IL-4, IFN-γ, IL-9 and p701-STAT1 were detected by western blot assays
The group settings were the same as those used for the cell proliferation assays. The specific procedure was performed as described previously for western blot assays in section Analysis of rHc-HAP protein by western blot assays. The expression of the target proteins was detected by chemiluminescence. Image J software was used to analyze the relative protein expression levels. The source antibodies were: inducible nitric oxide synthase (iNOS), IL-4, IFN-γ, IL-17, STAT1 and p701-STAT1 (1:1000; Affinity Biosciences, China); IL-9 (prepared by our research team); and β-actin (ABclonal, Wuhan, China).

Data analysis
Student's t-test and one-way analysis of variance (ANOVA) analyses were used to compare two groups and different groups, respectively. Data are presented as the mean ± standard error of the mean and were considered significant at *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. All experiments were repeated a minimum of three times.

Cloning, expression and western blot analysis of Hc-HAP
No signal peptide and transmembrane structure were found in the Hc-HAP protein sequence (Additional file 2: Figures S1, S2). The cDNA of adult H. contortus worms was used as a template to amplify the Hc-HAP gene using primers HAP-F and HAP-R, and the size of the amplified target gene was 990 bp (Fig. 1a). The Hc-HAP gene was successfully inserted into the pET32a vector and verified by enzymatic digestion and online BLAST analysis (Fig. 1b). The recombinant plasmid (pET32a/ Hc-HAP) was induced by IPTG to be expressed in E. coli BL21 (DE3). After separation by SDS-PAGE, staining with Coomassie Brilliant Blue revealed that the size of the fusion protein rHc-HAP was approximately 52 kDa (Fig. 1c). The rHc-HAP protein was expressed in the form of inclusion bodies (Fig. 1d) and purified by passage through the HisTrap TM FF Column. SDS-PAGE showed a single band for the purified rHc-HAP, indicating good purification (Fig. 1e).
As shown in Fig. 1g, rHc-HAP was recognized by sera from goats infected with H. contortus, whereas normal goat sera did not recognize rHc-HAP. This result suggests that Hc-HAP is exposed to the host immune system during H. contortus infection and has the potential to be a vaccine candidate antigen.

Relative transcript abundance of Hc-HAP gene in different life stages of H. contortus
The relative transcript levels of the Hc-HAP gene in different developmental stages of the H. contortus were examined using qPCR. As shown in Fig. 3, Hc-HAP transcript levels were significantly upregulated at

Immunolocalization of native Hc-HAP protein in adult H. contortus
Localization of Hc-HAP protein in adult nematodes was examined by IFA. Figure 4 shows the longitudinal sections of female and male worms, respectively, where the red fluorescent signal represents the Hc-HAP protein.
Expression of rHc-HAP was observed in the intestinal microvilli (the main expression sites of Hc-HAP protein), gonads and body wall of H. contortus. Control sections exhibited no red fluorescent signal.

Binding of rHc-HAP protein to goat PBMCs
Goat PBMCs were incubated with rHc-HAP protein, and the ability of the PBMCs to bind to rHc-HAP protein was  The results presented here are representative of three independent experiments. Asterisks indicate significant differences at *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Abbreviations: L3, Infective third-state larva; mRNA, messenger RNA; ns, no significant difference; SEM, standard error of the mean; xL3s, exsheathed L3s determined by IFA. The IFA results showed that red fluorescence was observed on the surface of PBMCs treated with rHc-HAP protein, while no red fluorescence was detected in the control group (Fig. 5). These results suggest that rHc-HAP protein can bind to goat PBMCs.

Effect of rHc-HAP protein on the proliferation of goat PBMCs
The effect of rHc-HAP protein on PBMC proliferation was examined using a CCK-8 kit. The results showed that rHc-HAP protein did not affect the proliferation of goat PBMCs  (Fig. 6).

Effect of rHc-HAP protein on NO secretion from goat PBMCs
As shown in Fig. 8, rHc-HAP protein increased nitrate levels in the cell supernatant in a dose dependent manner, indicating that rHc-HAP protein upregulates NO production in goat PBMCs   (Fig. 8b).

Discussion
The nematode H. contortus, which is considered to be one of the most common parasites infesting ruminant grazers, represents a major threat to the health of livestock herds [25,26]. Currently, Barbervax ® (Wormvax Australia Pty Ltd., Albany, WA, Australia) is the only vaccine that is commercially available in Australia. This vaccine uses natural H11 and H-gal-GP proteins of adult worms as antigens [27][28][29]. However, Barbervax ® vaccine antigen extraction is relatively expensive, so there is a need to explore new vaccine targets. As nematode antigens are complex and immune-modulatory, vaccine development is challenging, and a comprehensive study of how the host immune system is influenced by H. contortus antigens is warranted. A transcript level of Hc-HAP has been found in the egg, L3, xL3 and adult stages of H. contortus, with the highest level of transcription in the L3 stage, suggesting that Hc-HAP may play an important role in the development of L3. However, the role of Hc-HAP in L3 development and environmental resistance remains to be demonstrated by subsequent studies. Interestingly, an aspartyl protease inhibitor protein previously identified in our laboratory from H. contortus has a similar transcriptional pattern to that of Hc-HAP and also affects the function of PBMCs [30]. In addition, the immunofluorescence results revealed that Hc-HAP was mainly expressed on the microvilli of H. contortus, which showed similar immunolocalization patterns to H11 and H-gal-GP antigens [31]. Theoretically, Hc-HAP can induce circulating antibodies, and these antibodies enter the parasite's intestine via the blood-sucking route and attack native Hc-HAP in the intestinal microvilli, thus potentially affecting the development and reproduction of the H. contortus. Interestingly, Caenorhabditis elegans pho-1 (a homolog of HAP, necessary for the development of the adult intestine) was also found mainly in the microvilli, consistent with immunolocalization data of Hc-HAP [16]. Based on these results, Hc-HAP may be a candidate for vaccine or drug development. Fig. 8 Effect of rHc-HAP on NO secretion from goat PBMCs. Cells were incubated with serial concentrations of rHc-HAP and pET-32a for 24 h at 37 °C and 5% CO 2 . a The effect of rHc-HAP on NO release from PBMCs was examined using a commercial kit. b The effect of rHc-HAP on the expression level of iNOS in PBMCs was analyzed using western blot assays. Data are presented as the mean ± SEM from three independent experiments. Asterisks indicate significant difference at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs the control group. Abbreviations: iNOS, Iducible nitric oxide synthase; NO nitric oxide A major function of PBMCs is to present antigens to the host and assist with the host's innate defense against foreign pathogens [32,33]. In previous studies, our group has shown that many antigenic molecules of H. contortus bind to PBMCs and affect their function [34][35][36][37][38]. The IFA results showed that rHc-HAP could bind to goat PBMCs, which may be a mechanism by which rHc-HAP could regulate the function of PBMCs. In addition, rHc-HAP could be recognized by sera from goats infected with H. contortus, suggesting that rHc-HAP may also serve as a diagnostic antigen for haemonchosis.
As a result of immune cells producing NO, the host is more likely to resist infection by parasites, bacteria and other pathogens [39][40][41][42]. Our study showed that rHc-HAP promoted the release of NO from PBMCs in a dosedependent manner, which may be one of the defense mechanisms of the host to prevent parasite invasion.