Identification and localization of hookworm platelet inhibitor in Ancylostoma ceylanicum

Infection, Genetics and Evolution 77 (2020) 104102

Contents lists available at ScienceDirect

Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid

Research paper

Identification and localization of hookworm platelet inhibitor in Ancylostoma ceylanicum

T

Yue Huang, Asmaa M.I. Abuzeid, Yunqiu Liu, Long He, Qi Zhao, Xinxin Yan, Jianxiong Hang, ⁎ Rongkun Ran, Yongxiang Sun, Xiu Li, Jumei Liu, Guoqing Li Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Ancylostoma ceylanicum Hookworm platelet inhibitor Differential expression Immunolocalization Platelet aggregation

Ancylostoma ceylanicum is a zoonotic hookworm, which mainly causes iron deficiency anemia (IDA) in humans and animals. Hookworm platelet inhibitor (HPI) has been isolated from adult Ancylostoma caninum and linked to the pathogenesis of hookworm associated intestinal hemorrhage and IDA. However, there is no available data about HPI from A. ceylanicum. To study the molecular characteristics of A. ceylanicum HPI (Ace-HPI), its corresponding cDNA was amplified from adult A. ceylanicum mRNA using the primers designed based on the Ac-HPI gene sequence, and its sequence homology and phylogenetic relationship were analyzed. The differential expression of Ace-hpi mRNA in the adult and third larval (L3) stages was compared using the quantitative real-time PCR. Ace-HPI reactivity and tissue localization were studied by Western blot and immunofluorescence, respectively. Platelet aggregation activity was monitored in a 96-well microplate reader. The results showed that the Ace-HPI encoding gene was 603 bp in length. Ace-HPI showed 91% homology to Ac-HPI, was closely related to Ac-ASP3, and belonged to the CAP superfamily. Ace-hpi transcripts were most abundant in the adult stage, followed by serum-stimulated infective larvae (ssL3), and finally in L3 stage, with a significant difference. Escherichia coli-expressed recombinant protein had good reactivity with the positive serum of A. ceylanicuminfected dogs. Immunolocalization indicated that Ace-HPI was located in the esophagus and cephalic glands of the adult. As well as, recombinant Ace-HPI inhibited the platelet aggregation in-vitro. HPI overexpression, anatomical location in adults, antigenicity and its in-vitro activity indicate its possible role in adult worm bloodfeeding and as a valuable target for hookworm vaccine and drug development.

1. Introduction Hookworm infections cause a huge economic and public health problem in tropical regions of the world, especially in poor countries (Bartsch et al., 2016), by inducing iron deficiency anemia and malnutrition in their hosts (Keymer and Bundy, 1989; Wei et al., 2017). Hookworms infecting human mainly include Ancylostoma duodenale and Necator americanus, while Ancylostoma caninum, Ancylostoma braziliense, and Ancylostoma ceylanicum can infect both humans and animals. Among them, A. ceylanicum is the only animal-derived hookworm that can develop into adults in the human intestine (Traub, 2013). Currently, A. ceylanicum has been the second most common hookworm species infecting human in Asian countries, including Thailand (Traub et al., 2008), Japan (Kaya et al., 2016), Malaysia (Ngui et al., 2012), Laos (Conlan et al., 2012), and China (Chen et al., 2012). In endemic areas, hookworm control depends upon the mass treatment with benzimidazole anthelmintic drugs (Hotez, 2009). Because of the emerging

⁎

benzimidazole-resistance (Keiser and Utzinger, 2008) and the increasing number of pet owners, there is an urgent need to identify new target molecules for the development of vaccine and drugs against this parasitic infection. Platelets play a vital role in thrombosis and hemostasis. Platelet adhesion and aggregation are the first reactions in the formation of the hemostatic plug upon the injury of the blood vessels. At the site of injury, platelets encounter the subendothelial collagen and attach to it with several platelet receptors including GPIa/IIa. Following the activation of platelets, fibrinogen binds to the GPIIb/IIIa receptor forming bridges between platelets, thus creating platelet aggregates (Crab et al., 2002). Blood sucking parasites use different mechanisms to counteract the normal platelet hemostatic function in their host to facilitate their blood feeding. Mcmorran et al. (2009) demonstrated that platelets could participate in the innate immunity during the early stages of Plasmodium infection and kill Plasmodium parasites in red blood cells. Haemonchus contortus adult protein extract could inhibit the platelet

Corresponding author. E-mail address: [email protected] (G. Li).

https://doi.org/10.1016/j.meegid.2019.104102 Received 15 July 2019; Received in revised form 18 October 2019; Accepted 1 November 2019 Available online 02 November 2019 1567-1348/ © 2019 Elsevier B.V. All rights reserved.

Infection, Genetics and Evolution 77 (2020) 104102

Y. Huang, et al.

adhesion to collagen and fibrinogen and the platelet aggregation induced by collagen, thrombin, adenosine diphosphate (ADP), and ristocetin. Also, a small inhibitor of collagen-induced platelet aggregation was partially purified from H. contortus (Crab et al., 2002). Secreted proteins from Brugia malayi microfilariae inhibited platelet aggregation when incubated with human platelets (Liu and Weller, 1992). As well as, the recombinant antiplatelet protein from Anopheles mosquitoes inhibited the binding of platelets to immobilized fibrinogen and collagen (Hayashi et al., 2012). The whole hookworm protein extracts (HEX) and excretory/secretory (ES) products of hookworm inhibited platelets aggregation induced by ADP, thrombin, adrenaline, and collagen (Jr and Nossel, 1971; Carroll, 1984; Furmidge et al., 1996). Hookworm platelet inhibitor from A. caninum (Ac-HPI) was firstly cloned and expressed by Del Valle et al. (2003). After that, Ma et al. (2015) studied the structure of this protein and found that it belongs to the cysteine rich secretory/antigen 5/pathogenesis-related 1 (CAP) superfamily. The CAP superfamily are widely distributed in eukaryotes including hookworm. Members of this superfamily in hookworms include Ancylostoma-secreted proteins (ASPs) previously isolated from A. caninum (Hawdon et al., 1996), A. ceylanicum (Goud et al., 2004), and N. americanus (Goud et al., 2005). Although members of this superfamily are widely spread in hookworm secretome (Morante et al., 2017), their functions are generally unknown, except for neutrophil inhibitory factor (NIF), which can block neutrophil adhesion to endothelial cells (Moyle et al., 1994), and AcHPI, which can inhibit platelets function (Del Valle et al., 2003). Similarly, there are little reports on the molecular nature and function of HPI in A. ceylanicum. This study aims to clone, express, and characterize the HPI protein from adult A. ceylanicum (Ace-HPI). This research may aid in understanding the biological function of this protein and its role as a possible hookworm vaccine candidate.

Not I restriction sites, respectively. First strand cDNA was reverselytranscribed from total A. ceylanicum RNA according to the M-MLV first strand cDNA Synthesis Kit (OMEGA, Guangzhou, China).The detailed procedures were as follows: 1 μg RNA, 1 μL oligo (dT) primer (50 μmol/ L), 1 μL dNTP mix (10 mmol/L) were added in a 0.2 mL centrifuge tube, and the volume was adjusted to 18 μL using water treated with DEPC. The mix was placed at 75 °C for 5 min and in ice bath for 2 min, then 5 μL of 5 × RT buffer, 1 μL of M-MLV Reverse Transcriptase, 1 μL of RNase Inhibitor were added into the centrifuge tube. The mix was finally placed at 42 °C for 1 h, and at 85 °C for 5 min. PCR amplification was carried out using AceHPIF and AceHPIR primers with 2 × Pfu PCR Master Mix enzyme (TianGen, Beijing, China). PCR reaction (50 μL) contained cDNA (6 μL), forward and reverse primers (10 μmol/L), 2 × Pfu PCR MasterMix (25 μL), and ddH2O (15 μL). The PCR reaction conditions were: 94 °C for 3 min; 94 °C for 30 s, 63 °C for 30 s, 72 °C for 1 min, 30 cycles; 72 °C for another 5 min. PCR products were analyzed by ethidium bromide stained agarose gel electrophoresis, re-harvested using a DNA gel extraction Kit (Omega, Guangzhou, China). The purified PCR products were sub-cloned into the pMD19-T vector according to the kit's instructions (TaKaRa, Dalian, China). The obtained clone plasmids were sequenced by Shanghai Bio-Tech Company. Then, the purified PCR product and the plasmid pET-28a were digested with BamH I and Not I. The digested product was recovered, and then ligated using T4 ligase (TaKaRa, Dalian, China). After that, the recombinant plasmid pET-28a-Ace-HPI was transformed into E. coli DH5α competent cells (Sangon, Shanghai, China).

2.3. Expression and purification of recombinant Ace-HPI The recombinant plasmid pET-28a-Ace-HPI was extracted from DH5α and transformed into E. coli BL21 (DE3) competent cells (Sangon, Shanghai, China). Then the E. coli BL21 (DE3) containing the recombinant plasmid was cultured in Luria-Bertani (LB) medium with kanamycin (50 μg/mL) overnight, and transferred into 200 mL LB medium with kanamycin at the ratio of 1:20. After incubation at 37 °C for 3 h, 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture for induction of protein expression and the incubation was continued for 10 h at 28 °C with shaking at 165 r/min. The bacterial cells were collected by centrifugation and resuspended in 2.8 mL phosphate buffer saline (PBS). The mixture was ultra-sonicated on ice, and the obtained cell lysate was centrifuged at 12000g for 10 min. The supernatant was collected, and recombinant Ace-HIP was purified by His-tag Protein Purification Kit (Beyotime, Shanghai, China). Both the pET-28a-Ace-HPI/B21 (DE3) cell lysate and the purified recombinant Ace-HPI were separately analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue staining to determine its molecular mass. The protein concentration was determined by Bicinchoninic Acid (BCA) Protein Assay Kit (Sangon, Shanghai, China).

2. Materials and methods 2.1. Parasite samples Ancylostoma ceylanicum adult worms (n = 30) were collected at necropsy from the small intestines of a single dead naturally-infected dog obtained from the Surgery Laboratory of College of Veterinary Medicine, South China Agricultural University. Larvae L3 were obtained by fecal culture from A. ceylanicum-infected dogs with > 4000 EPG as previously described (Geiger et al., 2011). The parasite samples were identified according to the method previously described by Fu et al. (2018) and stored in the RNA sample preservation solution at −20 °C. 2.2. Cloning of Ace-HPI cDNA A. ceylanicum adult worms were grinded in liquid nitrogen and suspended in 1.0 mL TRIzol reagent (ThermoFisher Scientific, Massachusetts, USA) per each 50–100 mg of worm tissue. A 0.2 mL chloroform was added, then the mixture was centrifuged at 12,000g for 15 min at 4 °C, resulting in separation of the homogenate into a clear upper aqueous layer (containing RNA), an interphase, and a red lower organic layer (containing the DNA and proteins). The upper two-third of the clear aqueous phase was collected carefully to avoid contamination from the lower layer. Isopropanol (0.5 mL) was added to precipitate the RNA from the aqueous layer with centrifugation at 12,000g for 10 min at 4 °C. Precipitated RNA was washed with 75% alcohol and finally dissolved in 20 μL of RNase-free water (diethyl pyrocarbonate (DEPC)-treated water). Based on the sequence of HPI from A. caninum (GenBank: AF399709.1), primers AceHPIF (5´-CGGG ATCCGAAGGTGACTATTCGCTATGCCAGC-3′) and AceHPIR (5´-TGGC GGCCGCGTGACCAATGCAGAGCAAATTCT-3′) were designed to amplify the Ace-HPI cDNA. The underlined portions represent BamH I and

2.4. Reactivity of recombinant Ace-HPI using Western blotting The purified recombinant Ace-HPI protein (100 μg) was analyzed by SDS-PAGE, transferred to the nitrocellulose (NC) membrane, and then incubated with anti-6× His mouse IgG antibody (1:6000) (Sangon, Shanghai, China) or A. ceylanicum-positive dog serum (1:2000), followed by HRP-conjugated rabbit anti-mouse IgG antibody (1:20000) (Sangon, Shanghai, China) or HRP-conjugated rabbit anti-dog IgG antibody (1:10000) (Berseebio, Beijing, China), respectively. The reactivity of recombinant Ace-HPI with the positive dog serum and anti6 × His mouse IgG antibody was determined by 3, 3′-Diaminobenzidine substrate (DAB) Western blotting detection system (Solarbio, Beijing, China) according to the manufacturer's instructions.

2

Infection, Genetics and Evolution 77 (2020) 104102

Y. Huang, et al.

Bednar et al. (1995) with some modifications. The whole blood was collected from healthy dogs. The platelet-rich plasma was collected by centrifugation at 220 ×g for 10 min at 22 °C, then centrifuged at 850 ×g for 10 min at 22 °C, so that the resulted precipitate represented concentrated platelets. The platelet count was adjusted to 400,000/μL using Ca+-free Tyrode's buffer (137 mM NaCl, 3 mM KCl, 0.3 mM NaH2P04, 2 mM MgC12, 5 mM HEPES, 5 mM glucose, 12 mM NaHCO3, and 3.5 mg/mL bovine serum albumin at pH 7.4). Platelet suspension (100 μL) was divided into 4 groups and incubated with 10 μL Ace-HPI (experimental group A), 10 μL Ace-HPI with 1.8 mM CaCl2 (experimental group B), 10 μL normal saline (control group A), and 10 μL normal saline containing 1.8 mM CaCl2 (control group B) at room temperature for 15 min, and then added to the 96-well microtiter plate with a final volume of 0.1 mL per well. Adenosine diphosphate (ADP) (Leagene biotech, Beijing, China) was added to each well at a final concentration of 10 μmol/L and placed in a microplate reader. The results were examined at the wavelength of 405 and 630 nm as recommended by Bednar et al. (1995) and Chadderdon and Cappello (1999), respectively.

2.5. Phylogenetic trees, homology analysis and tertiary structure prediction The Ace-HPI nucleotide sequence was used to search GenBank using BLAST to detect its relative sequences. Ace-HPI were aligned with the related protein-encoding genes and members of the CAP superfamily using CLUSTAL (http://www.ebi.ac.uk/clustalw/#). The phylogenetic tree was established using the neighbor-joining method by MEGA5 software. The amino acid sequence of Ace-HPI was predicted based on the nucleotide sequence of Ace-HPI gene. The homology between AcHPI and Ace-HPI amino acid sequence was determined by DANMAN software, and its structural characteristics were analyzed according to the methods of Gibbs et al. (2008) and Ma et al. (2015). Molecular modelling of Ace-HPI was performed using 3D-PSSM fold recognition server (http://www.sbg.bio.ic.ac.uk/~3dpssm/). 2.6. Determination of Ace-HPI gene expression by real time PCR Following the method previously described by Wiśniewski et al. (2016), the expression levels of Ace-hpi mRNA in the different developmental stages of A. ceylanicum were analyzed. Briefly, adults and third stage larvae (L3) were firstly washed with PBS. About 5000 larvae were cultured for 2 h in RPMI1640 (37 °C, 5% CO2) supplemented with 50% fetal bovine serum to prepare serum-stimulated L3 (ssL3). RNAs were extracted from L3, ssL3, and adults using TRIzol reagent according to the methods described above. RNA concentration and purity were determined by spectrophotometry, and only pure samples (OD260/ OD280 ratio of ~2.0) were used for cDNA synthesis. Then, the first strand cDNA was synthesized from 1 μg RNA samples by reverse transcription using the M-MLV first strand cDNA Synthesis Kit (OMEGA, Guangzhou, China) as described above. The real-time PCR was performed in 96-well plates using a LightCycler® 96 System (Roche, Basel, Switzerland). Gene-specific primers QAceHF (5´-GGAGCTGCACAATG GCTACAGG-3′) and QAceHR (5´-ATTAAGAGTAGTTGC GCCGTC CTC-3′) were used to analyze the Ace-hpi mRNAs expression levels in L3, ssL3 and adult. The real time PCR reaction (20 μL) contained cDNA (1 μL), forward and reverse primers (10 μmol/L), 10 μL 2× qPCR Master Mix (Novogene, Beijing, China), ddH2O (complete the volume to 20 μL). The cycling conditions were as follows: 10 min at 95 °C, 40× (15 s at 95 °C, 30 s at 60 °C). Ace-HPI cDNA were cloned into the PMD19-T cloning vectors according to the manufacturer's instructions (TaKaRa, Dalian, China). Ace-hpi mRNA copy number of each sample was determined by using plasmid pMD-19 T-Ace-HPI with a known concentration run in parallel and statistically analyzed by the student ttest (STATISTICA 10).

3. Results 3.1. Ace-HPI cloning, expression, and purification The cDNA encoding HPI was isolated from adult A. ceylanicum using RT-PCR. The full length Ace-HPI cDNA was 603 bp in length and encoded an open reading frame (ORF) of 200 amino acids (aa). The translated cDNA predicted a mature protein of 183 amino acids in addition to a signal sequence of 17 amino acids at its N-terminal. The nucleotide and translated amino acid sequence named Ace-HPI gene was deposited in GenBank (Accession No. MK087839). The recombinant Ace-HPI protein was expressed in E. coli and then purified at the concentration of approximately 1.1 mg/mL. The pET28a-Ace-HPI/B21 (DE3) expression cell lysate and the purified recombinant Ace-HPI were analyzed by SDS-PAGE (Fig. 1). The results showed that both the expression mixture and purified protein produced a clear band at about 26 kDa. Western blot analysis showed that the purified recombinant protein could be recognized by the anti-6 × His mouse IgG antibody and serum of A. ceylanicum-infected dog, with a visible protein band at approximately 26 kDa, similar to the predicted molecular weight (Fig. 1).

2.7. Polyclonal antibody preparation and immunolocalization A polyclonal antiserum was prepared by immunizing a six-week-old Kunming mouse intraperitoneally with 100 μg of recombinant Ace-HPI emulsified in complete Freund's adjuvant. The mouse was subsequently boosted twice at 2-week intervals using 50 μg of protein in incomplete Freund's adjuvant. A week after the last immunization, the mouse was euthanized, and the serum was collected. The antibody titer in mouse serum was tested by ELISA. The individual worms embedded in paraffin were cut longitudinally or transversely on the head and continuous sagittal sections were fixed to glass slides. Each section was incubated with 1:250 diluted antiserum (test group) or pre-immune mouse serum (control group) overnight at 4 °C. After that, these sections were incubated with anti-mouse fluorescein isothiocyanate (FITC)-conjugated IgG (Sangon, Shanghai, China) for 2 h at room temperature and observed with an Olympus BX-60 fluorescent microscope using a 488 nm excitation filter block and emission at 525 nm.

Fig. 1. SDS-PAGE (lane 1 and 2) and Western blot (lane 3, 4 and 5) analysis of the expression product of pET-28a-Ace-HPI. M: Protein molecular weight marker; lane 1: Total cell lysate of pET-28a-Ace-HPI/BL21(DE3) with induction; lane 2: Purified recombinant Ace-HPI proteins; lane 3: Recombinant proteins detected by anti-6× His mouse IgG antibody; lane 4: Recombinant proteins detected by serum of A. ceylanicum-infected dogs; lane 5: Recombinant proteins detected by serum of healthy dog.

2.8. Platelet aggregation assay The experiment was performed following the method described by 3

Infection, Genetics and Evolution 77 (2020) 104102

Y. Huang, et al.

Fig. 2. Alignment of deduced amino acid sequences of Ace-HPI with Ac-HPI. The amino acid sequence homology between the Ace-HPI and Ac-HPI proteins was 91%. Both proteins had 10 conserved cysteine sites and the incompletely conserved CAP1~CAP4 domains.

37.05% (405 nm) and 44.16% (630 nm). After adding exogenous calcium ions, the OD value of the control group B decreased much more than that of the experimental group B and the percentage reduction in platelet aggregation by Ace-HPI was 63.73% (405 nm) and 65.49% (630 nm) (Fig. 7). This result demonstrates that Ace-HPI can inhibit platelets aggregation.

3.2. Bioinformatic analyses The amino acid sequence homology between the Ace-HPI (200 aa) and Ac-HPI proteins (198 aa) was 91%, with a 16-amino acid difference. However, Ace-HPI lacked a potential disintegrin-like motif (KGD) previously detected in Ac-HPI. Both proteins had 10 conserved cysteine sites and the incompletely conserved CAP1~CAP4 structure (Fig. 2). Phylogenetic analysis showed that Ace-HPI was genetically related to Ac-HPI (GenBank: AAK81732.1) and Ac-ASP-3 (GenBank: AAO63575. 1) from A. caninum, which are members of the CAP superfamily (Fig. 3). The protein had six β-sheets and six alpha-helices as predicted by the tertiary structure, and its N-terminus and C-terminus were exposed on the outside (Fig. 4).

4. Discussion Human hookworms, which currently infect nearly 500 million people in tropical regions of the world (Pullan et al., 2014), are a major cause of iron-deficiency anemia in developing countries. Iron-deficiency anemia can be related to the high blood loss caused by the adult worm sucking blood from the host intestine. Hookworm secrete different antithrombotic factors, including anticoagulants and hookworm platelet inhibitor (HPI), to maintain its ability to feed on blood (Jones and Cappello, 2004). The study of HPI began in the canine hookworm, A. caninum. Adult A. caninum HEX and ES products showed an activity that could inhibit the binding of platelets to immobilized fibrinogen and collagen, via the blockage of their corresponding integrin receptors, GPIIb/IIIa and GPIa/IIa (Calvete, 1995; Sixma et al., 1995; Du and Ginsberg, 1997; Moroi and Jung, 1997; Chadderdon and Cappello, 1999). Later, Del Valle et al. (2003) isolated HPI with the above inhibitory activity from adult A. caninum and amplified for the first time the Ac-HPI by RACE-PCR. Presently, A. ceylanicum has become the second largest hookworm infecting human beings (Traub et al., 2008; Jiraanankul et al., 2011; Conlan et al., 2012; Ngui et al., 2012), which can cause abdominal pain, bloat, diarrhea, occult blood in the feces, and anemia (Traub, 2013). These hookworm-associated symptoms result from the blood-feeding behavior of adult worms in the small intestine. HPI facilitate the bloodsucking by adults, whereas there is no available data about the characterization of this protein from A. ceylanicum. Thus, it is important to understand the molecular characteristics of Ace-HPI, which might provide a target for reducing the pathogenesis or controlling A. ceylanicum hookworm disease. Here, we described for the first time the isolation of HPI cDNA from adult A. ceylanicum RNA using primers designed based on Ac-HPI sequence. Even though, A. ceylanicum genome is available, we did not find a sequence named or identified as platelet inhibitor in GenBank so that we used the sequence of A. caninum HPI for designing our primers.

3.3. Ace-HPI differential transcription We used the real-time PCR analysis to compare the transcription of Ace-hpi mRNA in adults, L3, and ssL3. Results showed that the expression level of Ace-hpi mRNA in adults was the highest among the analyzed stages (p < .001). Additionally, Ace-hpi expression in ssL3 was significantly higher than that in L3 (p < .05) (Fig. 5). 3.4. Ace-HPI immunolocalization in adult A. ceylanicum The localization of Ace-HPI in the adult tissues of A. ceylanicum was detected by probing of adult worm longitudinal and transverse sections with the anti-Ace-HPI mouse serum. Immunofluorescent microscopy showed that Ace-HPI protein was localized to the esophagus and cephalic glands of the adult, while no significant staining was observed in the sections of adult worms probed with pre-immune mouse serum (the control group) (Fig. 6). 3.5. In-vitro platelet aggregation inhibitory activity of Ace-HPI When platelets are placed in a 96-well plate, they aggregate, and the absorbance and the optical density (OD) value detected by the microplate reader decrease. Therefore, platelets aggregation can be detected based on the change in the OD value. In this experiment, after ADPinduced platelet aggregation, the OD value in group A decreased and the percentage reduction in platelet aggregation by Ace-HPI was 4

Infection, Genetics and Evolution 77 (2020) 104102

Y. Huang, et al.

Fig. 3. Phylogenetic analysis of hookworm CAP superfamily members inferred by neighbor-joining method. ASPs: Ancylostoma-secreted proteins, HPI: hookworm platelet inhibitor, NIF: neutrophil inhibitory factor, MTP: metalloprotease, APRs: aspartic proteins, GSTs: Glutathione-S-Transferase.

Fig. 4. Molecular modelling using the 3D-PSSM fold recognition server generated a single significant three-dimensional model of Ace-HPI. β-pleated sheets, α-helices and β-bends are represented in blue, red and green, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Analysis of Ace-hpi expression in the different developmental stages (L3, ssL3 larvae and adults) of A. ceylanicum by real time PCR. The statistical significance between groups was marked with “*(p < 0.05)” and “** (p < 0.001)”, respectively.

Ace-HPI cDNA was a 603 bp fragment with an ORF of 200 aa. Ac-HPI is a hydrophobic protein and its recombinant protein was expressed in the form of insoluble inclusion body without a biological activity (Del Valle et al., 2003). To induce the soluble expression of Ace-HPI in E. coli, we 5

Infection, Genetics and Evolution 77 (2020) 104102

Y. Huang, et al.

Fig. 6. Immunolocalization of Ace-HPI in adult A. ceylanicum. A1 and B1 show a bright field of longitudinal and cross section of adult A. ceylanicum, respectively. A2 and B2 show longitudinal and cross section of adult A. ceylanicum probed with mouse anti-Ace-HPI serum followed by FITC conjugated anti-mouse IgG, respectively. Es: esophagus, cg: cephalic gland. No significant staining was observed with pre-immune mouse serum (A3 and B3).

qualitatively, it could effectively inhibit the platelet aggregation whether there was exogenous calcium ion in platelet suspension (can promote platelet aggregation) or not. Thus, KGD sequence may not mediates the inhibition of platelet function by HPI as Ma et al. (2015) reported that KGD sequence is positioned at the apex of a tight turn in tertiary structure of Ac-HPI, making it unlikely interacts with the integrin. Prediction of Ace-HPI tertiary structure in our study demonstrated that the protein had six β-sheets and six α-helix helices, and its N-terminus and C-terminus were exposed on the outside. On the contrary, Ac-HPI had only three β-sheets and four α-helix (Ma et al., 2015). This structural difference between both recombinant proteins may explain the difference in their in-vitro platelet inhibitory activity and may support the hypothesis of Ma et al. (2015) that Ac-HPI might require post-translational modification or have a different biological function. Moreover, further studies are required to perform a deeper analysis and find the functional fragments of Ace-HPI gene, as well as to test their binding to the platelet glycoproteins GPIa/IIa and GPIIb/IIIa in order to gain a deeper understanding of Ace-HPI. Multiple alignment of Ace-HPI to members of the CAP family and phylogenetic analysis showed that Ace-HPI and Ac-HPI clustered together and were relatively close to Ac-ASP3, but far from Ac-ASP4, AcASP5 and Ac-ASP6. CAP superfamily proteins are widely distributed in eukaryotes, but most of them have not been functionally characterized.

adopted strategies of low temperature, low speed, low IPTG concentration and extended induction time. Our results showed that AceHPI could be expressed in a soluble form under 0.1 mmol/L of IPTG induction for 10 h at 165 r/min (28 °C). On SDS-PAGE, both the expression mixture lysates and the purified recombinant protein produced clear bands at the same size (26 kDa). In this study, the purified recombinant Ace-HPI protein reacted with the dog positive serum forming a band with the predicted size at 26 kDa on Western blotting. These findings indicate that this recombinant protein has a good immunogenicity and could provide candidate antigen for hookworm serological diagnosis and vaccine development. Although we did not test the presence of Ace-HPI in A. ceylanicum ES products, our protein had a hydrophobic signal peptide and lacked a transmembrane domain in its deduced amino acid sequence, suggesting that this molecule is a secreted protein (Briggs and Gierasch, 1986). The translated amino acid sequence of Ace-HPI showed 91% homology to that of Ac-HPI, with sharing the same incomplete conserved CAP1~CAP4 domain and 10 conserved cysteine sites, which indicates that Ace-HPI belongs to the CAP superfamily. Nevertheless, Ace-HPI didn't contain the tripeptide sequence Lys-Gly-Asp (KGD), which is a possible recognition site for the integrin receptor GPIIb/IIIa and was previously detected in Ac-HPI (Del Valle et al., 2003). When we tested the biological function of the purified recombinant Ace-HPI

6

Infection, Genetics and Evolution 77 (2020) 104102

Y. Huang, et al.

Fig. 7. Inhibitory effect of Ace-HPI on platelet aggregation. In the absence of exogenous calcium ions, the OD values of both the experimental group and the control group decreased after the addition of ADP, but the control group decreased a little more (A); In the presence of exogenous calcium ions, the OD value of the control group decreased much more than that of the experimental group (B). These results indicate Ace-HPI functions to inhibit platelets aggregation whether exogenous calcium ions exist or not. The platelet aggregation was tested every 6 min in a 96-well microplate reader. Parallel experiments were represented with three consecutive symbols.

worm cephalic gland, a main secretory structure that directly empties into the buccal capsule of the worm, as well as, AcAPc3 localized to the esophagus to facilitate the blood feeding (Mieszczanek et al., 2004). Thus, we believe that Ace-HPI can facilitate the worm blood-feeding not only by inhibition of coagulation at the site of attachment (by its secretion from cephalic glands) but also by binding platelets entering the esophagus and preventing their aggregation. Interestingly, Ac-ASP3 protein, which was closely related to Ace-HPI in the phylogenetic tree, had also a higher expression level in the adult stage and localized to the esophagus (Zhan et al., 2003). These findings indicate a possible functional similarity between these two proteins. As the Ace-HPI does not present the desintegrin-like motif, that is present in A. caninum and besides that, the immunolocalization is also different from the one in A. caninum, we consider that this protein may be another member of the family. In conclusion, we successfully cloned and expressed Ace-HPI recombinant protein in E. coli, which was closely related to Ac-HPI and Ac-ASP3 belonging to the CAP superfamily. Ace-HPI had a higher expression level in the adult stage, immunolocalized to the cephalic gland and esophagus and could inhibit platelets aggregation, supporting its possible role in blood feeding. These results can provide a basis for further studies on the biological function of this protein and its possible role as a vaccine candidate that might interfere with hookworm-induced intestinal bleeding and iron deficiency anemia in the host.

To date, two CAP superfamily members secreted from A. caninum, NIF and HPI, have been related to specific functions. NIF function to block neutrophil adhesion to endothelial cells by binding to the integrin receptor CD11a/CD18 expressed on the neutrophil surface (Moyle et al., 1994). Ac-HPI inhibit platelets function through a blockade of the collagen receptor integrin GPIa/IIa (α2β1) and the fibrinogen receptor integrin GPIIb/IIIa (αIIbβ3) (Chadderdon and Cappello, 1999; Del Valle et al., 2003). Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, and Ac-ASP-6 are also members of CAP superfamily that are secreted from adult A. caninum from distinct locations, including pharyngeal and esophageal glands; the cuticle; intestinal microvilli; and excretory and cephalic glands, respectively (Zhan et al., 2003). However, the function of these proteins is still unclear. Real time PCR revealed that Ace-HPI was expressed in both adult and L3 stages, but its expression in adults showed the highest values, which may be explained by its role in blood-feeding of adults. As well as, Ace-HPI transcription level significantly increased in L3 after serum stimulation (ssL3). This result suggests that Ace-HPI protein may be related to the pathogenic mechanism of larval activation. Immunolocalization studies using specific Ace-HPI polyclonal antibodies showed that Ace-HPI was localized to the esophagus and cephalic glands of the adult. By contrast Ac-HPI that was only localized to cephalic gland and was detected in A. caninum adult extract and ES products, suggesting its release at the site of attachment in the intestine (Del Valle et al., 2003). In consistent with Ace-HPI localization, anticoagulant peptide-5 from A. caninum (AcAP5) localized to the adult 7

Infection, Genetics and Evolution 77 (2020) 104102

Y. Huang, et al.

Declaration of Competing Interest

Hawdon, J.M., Jones, B.F., Hoffman, D.R., Hotez, P.J., 1996. Cloning and characterization of Ancylostoma-secreted protein. A novel protein associated with the transition to parasitism by infective hookworm larvae. J. Biol. Chem. 271, 6672–6678. Hayashi, H., Kyushiki, H., Nagano, K., 2012. Anopheline anti-platelet protein from a malaria vector mosquito has anti-thrombotic effects in vivo without compromising hemostasis. Thromb. Res. 129, 169–175. Hotez, P., 2009. Mass drug administration and integrated control for the world’s highprevalence neglected tropical diseases. Clin. Pharmacol. Ther. 85, 659–664. Jiraanankul, V., Aphijirawat, W., Mungthin, M., Khositnithikul, R., Rangsin, R., Traub, R.J., Piyaraj, P., Naaglor, T., Taamasri, P., Leelayoova, S., 2011. Incidence and risk factors of hookworm infection in a rural community of central Thailand. Am. J. Trop. Med. Hyg. 84, 594–598. Jones, B.F., Cappello, M., 2004. Hookworm infection: molecular mechanisms of disease and targets for control. Drug Discov. Today Dis. Mech. 1, 217–222. Jr, S.G., Nossel, H.L., 1971. Anticoagulant activity of dog hookworm. Am. J. Phys. 220, 922–927. Kaya, D., Yoshikawa, M., Nakatani, T., Tomo-Oka, F., Fujimoto, Y., Ishida, K., Fujinaga, Y., Aihara, Y., Nagamatsu, S., Matsuo, E., Tokoro, M., Ouji, Y., Kikuchi, E., 2016. Ancylostoma ceylanicum hookworm infection in Japanese traveler who presented chronic diarrhea after return from Lao People's Democratic Republic. Parasitol. Int. 65, 737–740. Keiser, J., Utzinger, J., 2008. Efficacy of current drugs against soil transmitted helminth infections. J. Amer. Med. Assoc. 299, 1937–1947. Keymer, A., Bundy, D., 1989. Parasitology. Seventy-five years of solicitude. Nature 337, 114. Liu, L.X., Weller, P.F., 1992. Intravascular filarial parasites inhibit platelet aggregation. Role of parasite-derived prostanoids. J. Clin. Invest. 89, 1113–1120. Ma, D., Francischetti, I.M.B., Ribeiro, J.M.C., Andersen, J.F., 2015. The structure of hookworm platelet inhibitor (HPI), a CAP superfamily member from Ancylostoma caninum. Acta Crystallogr. 71, 643–649. Mcmorran, B.J., Marshall, V.M., De, G.C., Drysdale, K.E., Shabbar, M., Smyth, G.K., Corbin, J.E., Alexander, W.S., Foote, S.J., 2009. Platelets kill intraerythrocytic malarial parasites and mediate survival to infection. Science 323, 797–800. Mieszczanek, J., Harrison, L.M., Vlasuk, G.P., Cappello, M., 2004. Anticoagulant peptides from Ancylostoma caninum are immunologically distinct and localize to separate structures within the adult hookworm. Mol. Biochem. Parasitol. 133, 319–323. Morante, T., Shepherd, C., Constantinoiu, C., Loukas, A., Sotillo, J., 2017. Revisiting the Ancylostoma caninum secretome provides new information on hookworm-host interactions. Proteomics 17. Moroi, M., Jung, S.M., 1997. Platelet receptors for collagen. Thromb. Haemost. 78, 439–444. Moyle, M., Foster, D.L., McGrath, D.E., Brown, S.M., Laroche, Y., De Meutter, J., Stanssens, P., Bogowitz, C.A., Fried, V.A., Ely, J.A., Soule, H.R., Vlasuk, G.P., 1994. A hookworm glycoprotein that inhibits neutrophil function is a ligand of the integrin CD11b/CD18. J. Biol. Chem. 269, 10008–10015. Ngui, R., Lim, Y.A.L., Traub, R., Mahmud, R., Mistam, M.S., 2012. Epidemiological and genetic data supporting the transmission of Ancylostoma ceylanicum among human and domestic animals. PLoS Neglect. Trop. Dis. 6, e1522. Pullan, R.L., Smith, J.L., Jasrasaria, R., Brooker, S.J., 2014. Global numbers of infection and disease burden of soil transmitted helminth infections in 2010. Parasit. Vectors 7, 37. Sixma, J.J., van Zanten, H., Saelman, E.U., Verkleij, M., Lankhof, H., Nieuwenhuis, H.K., de Groot, P.G., 1995. Platelet adhesion to collagen. Thromb. Haemost. 73, 454–459. Traub, R.J., 2013. Ancylostoma ceylanicum, a re-emerging but neglected parasitic zoonosis. Int. J. Parasitol. 43, 1009–1015. Traub, R.J., Inpankaew, T., Sutthikornchai, C., Sukthana, Y., Thompson, R.C., 2008. PCRbased coprodiagnostic tools reveal dogs as reservoirs of zoonotic Ancylostomiasis caused by Ancylostoma ceylanicum in temple communities in Bangkok. Vet. Parasitol. 155, 67–73. Wei, K.Y., Yan, Q., Tang, B., Yang, S.M., Zhang, P.B., Deng, M.M., Lv, M.H., 2017. Hookworm infection: a neglected cause of overt obscure gastrointestinal bleeding. Korean J. Parasitol. 55, 391–398. Wiśniewski, M., Jaros, S., Bąska, P., Cappello, M., Długosz, E., Wędrychowicz, H., 2016. Hamsters vaccinated with Ace-mep-7 DNA vaccine produced protective immunity against Ancylostoma ceylanicum infection. Exp. Parasitol. 163, 1–7. Zhan, B., Liu, Y., Badamchian, M., Williamson, A., Feng, J., Loukas, A., Hawdon, J.M., Hotez, P.J., 2003. Molecular characterisation of the Ancylostoma-secreted protein family from the adult stage of Ancylostoma caninum. Int. J. Parasitol. 33, 897–907.

The authors declare that they have no conflicts of interest. Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (Grant No. 31672541) and the Science and Technology Planning Project of Guangdong Province, China (Grant No. 2014A020214005). References Bartsch, S.M., Hotez, P.J., Asti, L., Zapf, K.M., Bottazzi, M.E., Diemert, D.J., Lee, B.Y., 2016. The global economic and health burden of human hookworm infection. PLoS Neglect. Trop. Dis. 10, e0004922. Bednar, B., Condra, C., Gould, R.J., Connolly, T.M., 1995. Platelet aggregation monitored in a 96 well microplate reader is useful for evaluation of platelet agonists and antagonists. Thromb. Res. 77, 453–463. Briggs, M.S., Gierasch, L.M., 1986. Molecular mechanisms of protein secretion: the role of the signal sequence. Adv. Protein Chem. 38, 109–180. Calvete, J.J., 1995. On the structure and function of platelet integrin αIIbβ3, the fibrinogen receptor. Proc. Soc. Exp. Biol. Med. 208, 346–360. Carroll, S., 1984. The anticoagulant effects of hookworm, Ancylostoma ceylanicum; observations in human and dog blood in vitro and infected dogs in vivo. Thromb. Haemost. 51, 222–227. Chadderdon, R.C., Cappello, M., 1999. The hookworm platelet inhibitor: functional blockade of integrins GPIIb/IIIa (αIIbβ3) and GPIa/IIa (α2β1) inhibits platelet aggregation and adhesion in vitro. J. Infect. Dis. 179, 1235–1241. Chen, J., Xu, M.J., Zhou, D.H., Song, H.Q., Wang, C.R., Zhu, X.Q., 2012. Canine and feline parasitic zoonoses in China. Parasit. Vectors 5, 152. Conlan, J.V., Khamlome, B., Vongxay, K., Elliot, A., Pallant, L., Sripa, B., Blacksell, S.D., Fenwick, S., Thompson, R.C., 2012. Soil-transmitted helminthiasis in Laos: a community-wide cross-sectional study of humans and dogs in a mass drug administration environment. Am. J. Trop. Med. Hyg. 86, 624–634. Crab, A., Noppe, W., Pelicaen, C., Van Hoorelbeke, K., Deckmyn, H., 2002. The parasitic hematophagous worm Haemonchus contortus inhibits human platelet aggregation and adhesion: partial purification of a platelet inhibitor. Thromb. Haemost. 87, 899–904. Del Valle, A., Jones, B.F., Harrison, L.M., Chadderdon, R.C., Cappello, M., 2003. Isolation and molecular cloning of a secreted hookworm platelet inhibitor from adult Ancylostoma caninum. Mol. Biochem. Parasitol. 129, 167–177. Du, X., Ginsberg, M.H., 1997. Integrin alpha IIb beta 3 and platelet function. Thromb. Haemost. 78, 96–100. Fu, Y., Wang, M., Yan, X., Abdullahi, A.Y., Hang, J., Zhang, P., Huang, Y., Liu, Y., Sun, Y., Ran, R., Li, G., 2018. Tm-shift detection of dog-derived Ancylostoma ceylanicum and A. caninum. Biomed. Res. Int. 2018, e7617094. Furmidge, B.A., Horn, L.A., Pritchard, D.I., 1996. The anti-haemostatic strategies of the human hookworm Necator americanus. Thromb. Haemost. 112, 81–87. Geiger, S.M., Fujiwara, R.T., Freitas, P.A., Massara, C.L., Carvalho, O.D.S., CorrêaOliveira, R., Bethony, J.M., 2011. Excretory-secretory products from hookworm L3 and adult worms suppress proinflammatory cytokines in infected individuals. J. Parasitol. Res. 2011, 1–8. Gibbs, G.M., Roelants, K., O’Bryan, M.K., 2008. The cap superfamily: cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins-roles in reproduction, cancer, and immune defense. Endocr. Rev. 29, 865–897. Goud, G.N., Zhan, B., Ghosh, K., Loukas, A., Hawdon, J., Dobardzic, A., Deumic, V., Liu, S., Dobardzic, R., Zook, B.C., Jin, Q., Liu, Y., Hoffman, L., Chung-Debose, S., Patel, R., Mendez, S., Hotez, P.J., 2004. Cloning, yeast expression, isolation, and vaccine testing of recombinant Ancylostoma-secreted protein (ASP)-1 and ASP-2 from Ancylostoma ceylanicum. J. Infect. Dis. 189, 919–929. Goud, G.N., Bottazzi, M.E., Zhan, B., Mendez, S., Deumic, V., Plieskatt, J., Liu, S., Wang, Y., Bueno, L., Fujiwara, R., Samuel, A., Ahn, S.Y., Solanki, M., Asojo, O.A., Wang, J., Bethony, J.M., Loukas, A., Roy, M., Hotez, P.J., 2005. Expression of the Necator americanus hookworm larval antigen Na-ASP-2 in Pichia pastoris and purification of the recombinant protein for use in human clinical trials. Vaccine 23, 4754–4764.

8