Identification and preliminary characterization of Hc-clec-160, a novel C-type lectin domain-containing gene of the strongylid nematode Haemonchus contortus

Background The strongylid parasite Haemonchus contortus causes severe anemia in domestic animals worldwide. Effective preventive and therapeutical agents are lacking, because of drug resistance and that little is known about the molecular mechanism of the interaction between H. contortus and host cells. Methods A new gene, Hc-clec-160, was discovered with RT-PCR. Transcriptional levels of Hc-clec-160 and Ce-clec-160 throughout different growth phases of corresponding nematodes were assayed by qPCR. Immunofluorescence staining of paraffin section were performed to determine the protein localization in adult worms of H. contortus. To monitor the promoter capacity of the 5'-flanking region of Ce-clec-160, micro-injection was used. Overexpression and RNAi constructs was carried out in the N2 strain of Caenorhabditis elegans to find out the gene function of Hc-clec-160. Results The full-length cDNA of 1224 bp of Hc-clec-160 was cloned by RT-PCR. The corresponding gene contained twelve exons. Its transcripts peaked in male adult worms. Hc-CLEC-160 was predicted to have a Willebrand factor type A (vWA) domain and a C-type lectin domain. The proteins were not detected by expression in C. elegans or paraffin section experiments in adult of H. contortus. Knockdown of Ce-clec-160 expression in C. elegans by RNAi resulted in shortened body length and decreased brood size. Conclusions In this experiment, a new gene Hc-clec-160 was obtained in H. contortus and its function was addressed using a model organism: C. elegans. Our study showed that Hc-clec-160 possesses characteristics similar to those of Ce-clec-160 and plays an important role in the growth and reproduction of this parasitic nematode. Electronic supplementary material The online version of this article (10.1186/s13071-018-3005-3) contains supplementary material, which is available to authorized users.

protein C [1,2]. They often oligomerize, which increases their avidity for multivalent ligands. CTLs bind to various types of glycans with high affinity. CTLs include selectins, collectins, endocytic receptors and proteoglycans. Some CTLs are exocrine whereas others are transmembrane proteins. They play a role in adhesion and signaling receptors in many pathways, including homeostasis and innate immunity, and are crucial in inflammatory responses and leukocyte and platelet trafficking. It is difficult to predict the glycan structures that bind to a particular CTL due to the relatively shallow CRD of CTL with few contacts to sugars motifs.
CTLs have been identified in some parasitic nematodes such as Trichostrongylus colubriformis, Onchocerca volvulus, Haemonchus contortus and Teladorsagia circumcincta [3]. They can significantly regulate host immune response [4][5][6]; however, their functions in H. contortus are poorly understood. Haemonchus contortus is one of the most economically important parasites of small ruminants (sheep and goats) worldwide, and can lead to anemia, weight loss and death of the host (sheep and goats) [7]. It can also cause immunosuppression and reduce the level of immunity in the host [8]. Caenorhabditis elegans is the most widely used model nematode in drug research [9], vaccine discovery [10] and helminth resistance studies [11][12][13].
In our study, we identified and characterized a novel gene of Hc-clec-160 in H. contortus, which is an ortholog of C. elegans and possesses one CTLD. Understanding the structure and function of Hc-clec-160 may provide useful information for deciphering how this parasite interacts with its hosts and crucial elements influencing its growth, development and reproduction.

Parasites and animals
Two female sheep, 4-5 months-old and maintained under helminth-free conditions for 30 days, were infected orally with 8000 L3s of H. contortus ZJ strain each, after being dewormed twice. Three weeks later, sheep feces were collected and helminth eggs detected by microscopy. First-(L1), second-(L2) and third-stage larvae (L3) were harvested at the 1st, 3rd and 7th day, respectively, after incubation of the collected eggs at 28°C . Adults of H. contortus (ZJ strain) were obtained from abomasa of sheep in a Hu Zhou Slaughterhouse. They were washed several times in phosphate-bufferred saline (PBS), pH 7.4. The clean adult worms were stored in liquid nitrogen. Caenorhabditis elegans wild strains (N2) were cultivated on NGM plates and fed on Escherichia coli (OP 50 strain) at 20°C as previously described [14].
Isolation of the Hc-clec-160 gene A homolog (>HCISE02108900.t1) was identified by using the Ce-clec-160 gene by searching the Sanger Institute genomic database for H. contortus (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/h_contortus). Specific primers (Hc-clec-160 1F and Hc-clec-160 1R) (Table 1) were designed according to the CDs sequence. The PCR reaction program included: one cycle at 94°C for 3 min; 30 cycles at 94°C for 30 s, 57°C for 30 s and 72°C for 80 s; and finally one cycle at 72°C for 10 min. The amplified fragments were cloned into the pMD18-T vector (Takara Biotechnology Co., Ltd., Dalian, China) and sent to Shanghai BioSune Co., Ltd. (Shanghai, China) for sequencing.

Polyclonal antibody preparation
Specific primers with double restriction sites (Hc-clec-160 2F and Hc-clec-160 2R) ( Table 1) were designed according to the full-length cDNA of the Hc-clec-160 gene sequence. Hc-clec-160 was cloned into the prokaryotic expression vector pET-30a to construct the plasmid pET-30a-Hc-clec-160. The vector was transformed into BL21 (DE3) cells of E. coli to produce recombinant Hc-CLEC-160 (rHc-CLEC-160), followed by treatment with 1 mM isopropy β-D-1-thiogalactopyranoside (IPTG) at 37°C for 2 h and purification by affinity chromatography using a Ni-NTA agarose column (Qiagen, Shanghai, China) as per the manufacturer's instructions. Anti-rHc-CLEC-160 polyclonal antibodies were produced by immunizing a New Zealand white rabbit and their titer and specificity were determined by enzyme-linked immunosorbent assay (ELISA).

Immunofluorescence staining of H. contortus adult paraffin sections
Male and female adults of H. contortus were fixed in 4% paraformaldehyde (PFA) at 4°C for 2 days. After being washed with tap water for 12 h, they were consecutively dehydrated in an ethanol series [50%, 75%, 80%, 95%, 100% (twice), 10 min at each step]. The dehydrated worms were incubated in xylene:absolute ethanol (1:1) solution and xylene and then embedded in paraffin. Paraffin sections of 5 μm were stained with hematoxylin and eosin (H&E) and analyzed histologically. The remaining sections were subjected to immunofluorescence staining. Slices were treated with 0.01 M citrate buffer at 100°C for 20 min for antigen repair. After overnight blocking with 2% BSA at 4°C, polyclonal antibody and goat anti-rabbit IgG H&L (Alexa Fluor® 488) diluted 1:100 in PBS were sequentially added and incubated at 37°C for 1 h each. The sections were stained with 4',6-diamidino-2-phenylindole (DAPI) for 40 min at 37°C and then observed by fluorescence microscopy.
Ce-clec-160 promoter transformation of C. elegans The Ce-clec-160 promoter was amplified by PCR using primers Ce-clec-160 pF1 and Ce-clec-160 pR1 (Table 1) containing restriction sites SalI or XbaI. The primers were designed according to the 2000 bp upstream of the start codon of Ce-clec-160. The PCR products were cloned into the multiple cloning sites upstream of the gfp gene of pPD95.77 eukaryotic expression vector, resulting in the plasmid pPD95.77-Ce-clec-160-prom. The latter was microinjected into the gonads of C. elegans along with another plasmid pRF4 carrying the rol-6 gene at a final concentration of 50 μg/ml each. The GFP expression of F2 progeny with the phenotype of roller was observed by fluorescence microscopy.

Expression of Hc-clec-160 in C. elegans
Microinjection was performed as previously described [17]. Hc-clec-160 was amplified based on the specific PCR primers Hc-clec-160 3F and Hc-clec-160 3R listed in Table 1 with XbaI/KpnI restriction enzyme sites. The amplified fragments were cloned into the plasmid pPD95.77 vector between the Ce-clec-160 promoter region and the gfp region. The recombinant plasmid CeP-pPD95.77-Hc-clec-160 with CeP-pPD95.77-Ce-clec-160 as a control was microinjected into N2 strains as described above. The F2 progeny with the roller phenotype were collected for further analysis.

Hc-clec-160 RNA interference
Ce-clec-160 and Hc-clec-160 genes were amplified by PCR with specific primers Hc-clec-160 4F-Hc-clec-160 4R and Ce-clec-160 F-Ce-clec-160 R, respectively ( Table  1). The primer pairs contained the restriction sites for XbaI/HindIII and SmaI/HindIII, respectively. The amplified fragments were cloned into the L4440 vector, resulting in the recombinant plasmids L4440-Ce-clec-160 and L4440-Hc-clec-160. The latter were transformed into E. coli HT115 (DE3) strain with the empty L4440 vector as a negative control. A feeding RNAi experiment was performed according to the classic bacterial feeding manuals [18] with three repeats. The transcriptional levels of RNAi worms were detected by qPCR and the RNAi worms were selected for further phenotype analysis.

Results
Characterization of cDNA and phylogenetic analysis of amino acid sequences Identification and sequence analysis of the Hc-clec-160 gene revealed 1224 bp that encoded a 408-amino-acids protein with a predicted mass of 45.2 kDa. Hc-clec-160 of H. contortus contained 12 exons separated by 11 introns, which was structurally similar to Ce-clec-160 of C. elegans. Structural analysis revealed that Hc-CLEC-160 has 2 conserved domains: the von Willebrand factor type A (vWA) domain and the CTL conserved domain, carbohydrate-recognition domain (CRD) (Fig. 1a). The protein sequences of Hc-clec-160 and another 8 nematodes were phylogenetically analyzed. The phylogenetic tree revealed that Hc-CLEC-160 clustered within the clade containing C. elegans, which suggest that their evolutionary relationship is relatively close (Fig. 1b).
Transcriptional level of clec-160 throughout the life-cycles of H. contortus and C. elegans and its protein localization The transcriptional level of clec-160 was performed throughout the life-cycles of H. contortus and C. elegans by qPCR. The result showed that adults had highest levels followed by L3 (Fig. 2). In addition, the transcriptional levels between male and female adults were particularly different (t-test: t (16) = 19.81, P < 0.0001) with adult males showing much higher transcriptional levels than adult females (Fig. 2a). The results of H&E staining of the sections showed that, regardless of the sex of adults H. contortus, parasite morphology was well maintained after sectioning and the tissue boundaries were clear (Additional file 1: Figure S1). Immunofluorescence staining of paraffin sections to detect protein localization was carried out, but no fluorescence staining was observed.
Expression of Hc-clec-160 in C. elegans The entire coding regions of Hc-clec-160 fused in frame with the gfp gene was expressed in transgenic C. elegans to detect its expression in vivo. Ce-clec-160 5'-flanking region was used as a promoter. Activity analysis of the promoter showed that GFP was mainly localized in the distal and anterior part of the intestine (Fig. 3). The reconstructed plasmid Cep-PD95.77-Hc-clec-160 and Cep-pPD95.77-Ce-clec-160 was transformed into the gonads of C. elegans by microinjecting as described above. However, neither Hc-clec-160 nor Ce-clec-160 expression worms showed GFP signal in any stage of C. elegans.

Discussion
In this study, the full-length cDNA sequences of two genes (Hc-clec-160 and Ce-clec-160) were determined. CTLs are highly representative of all studied metazoan phyla to date. Many proteins in the CTL superfamily contain multiple CRDs, along with additional non-lectin domains. CTLDs were classified to seventeen groups; Hc-clec-160 was similar to the group XIII DGCR2 (DGCR2/ DD/ Sez 12), which is localized in the DiGeorge syndrome (OMIM 188400) critical region [1]. In von Willebrand factor, the vWF is the archetype for a protein superfamily. The structure of vWF was discovered in various plasma proteins: complement  [19,20]. Most of the proteins containing vWF domains are exocrine. The vWF-containing proteins are involved in many biological functions such as cell adhesion, pattern formation, migration, homing, and signal transduction, concerning interactions with a large number of ligands. It is plausible that the interaction between lectins of gastrointestinal nematodes and complex mucin oligosaccharides can affect worm infection [21]. It has been reported recently that a CTL named cpclec from Cryptosporidum parvum plays an important role in binding sulfated proteoglycans of host cells [22,23]. These findings suggest that Hc-CLEC-160 may have the ability to adhere and bind to host cells.
The relative abundance of Hc-clec-160 transcripts throughout different developmental stages determined by qPCR showed that they had generally low abundance in all life-cycle stages except for adult males (Fig. 2a). High transcriptional levels of Hc-clec-160 in the male adult stage suggests that Hc-clec-160 may be related to the mating and reproduction of H. contortus. Furthermore, higher Hc-clec-160 transcriptional levels in L3s and L4s than in eggs, L1s and L2s, indicate that Hc-clec-160 is likely to play a crucial part in the parasitic stages, i.e. L3s and L4s, for adhesion to the host cells. The microinjection of Ce-clec-160 promoter results indicated that this promoter was capable of promoting the GFP expression in C. elegans with GFP proteins being mostly located in the anterior and distal part of the intestine.
As for paraffin section immunofluorescence staining and microinjection transgene assays, the possible causes for the lack of fluorescence in H. contortus or GFP in C. elegans could be summarized as follows. First, the polyclonal antibodies can be detected by western blot rather than the immunofluorescence staining of sections, which may be because the structure of the parasite antigen immobilized by paraformaldehyde is different from the linear structure in the western blot experiment. Secondly, it is possible that the antigen of CTL was damaged during the production of the slices, resulting in the fact that the antibody cannot specifically recognize and bind to it. Thirdly, the GFP expressed in the worms microinjected with Ce-lec-160 promoter, indicating that the promoter was able to efficiently initiate the expression of GFP. However, the ability to drive the expression of clec-160 gene was weak. The most likely reason for the failures of these two experiments is due to the rather low expression levels of clec-160 in H. contortus and C. elegans.
The mechanism of RNAi is divided into two procedures. First, dsRNA (double stranded RNA) is processed into small ds RNA (siRNA) of 21 to 23 nt. Then, the antisense strands of siRNA induce a ribosome called RNA-induced Post-embryonic development in Ce-clec-160 knockdown C. elegans worms. a Brood size of twenty worms for three repeats of Ce-clec-160 RNAi worms, using L4440 "empty" vector as a negative control. b Body length of twenty worms for three repeats of Ce-clec-160 RNAi worms. c Body width of twenty worms for three repeats of Ce-clec-160 RNAi worms. *P < 0.05, ***P < 0.001. Abbreviation: ns, difference not significant silencing complex (RISC), which can target homologous mRNA to silence the gene. Hc-clec-160 and Ce-clec-160 were ligated into the L4440 plasmid vector with a bidirectional T7 promoter at both ends of the inserted gene. L4440-Hc-clec-160 and L4440-Ce-clec-160 were transformed into E. coli HT115 (DE3) to induce dsRNA with IPTG. Later on, large numbers of RNAi knockdown worms were produced by feeding C. elegans on dsRNA-expressing bacteria. The abundance of Ce-clec-160 transcripts as a result of RNA interference was determined by qPCR. The relative abundance of mRNA indicates that Hc-clec-160 can partially silence Ce-clec-160, further confirming the genetic structure and possible functional similarities between the two homologous genes. Furthermore, the Ce-clec-160 (RNAi) phenotype showed much less abundant progeny and shorter body length than negative control worms, which indicates that Ce-CLEC-160 plays a significant role in the growth and development, and reproduction of C. elegans.
Helminth parasites possess the ability of modulating the host defense system in order to establish long-term infections in mammalian hosts [24,25]. The glycans of schistosome eggs can be recognized by L-SIGN [26], DC-SIGN [27] and other members of the CTL family of its hosts, suggesting that the CLRs constitute an ancient host innate immune pattern recognition system. The parasite itself also has CTLs [28,29], raising the possibility that they may affect host immunity [25,30]. CTLs can also perform other functions within the nematode phylum. Most notably, C. elegans is predicted to have a minimum of 278 CTL-like genes in its genome and some have been experimentally certified to be upregulated and protective in bacterial infections. One example is CLEC-50 [31].
Hc-CLEC-160 is a new protein and, to our knowledge, its function has not been studied so far. In this study, we cloned the Hc-clec-160 gene and first analyzed its structures. The possible roles of Hc-clec-160 in H. contortus attachment to and invasion of host cells, and also in the escape of host immunity, remain to be determined experimentally. It is very challenging, if not impossible, to directly study H. contortus growth and development and reproduction in vitro due to the necessity of culturing L3s, L4s and adults. Nevertheless, the predicted structural features and characteristic of Hc-clec-160 gene reported in the present study along with the known functions of CTLand CTLD-containing proteins in other organisms including the model nematode C. elegans implicate that Hc-clec-160 may play a significant role in H. contortus invasion and the host-parasite cell interactions.

Conclusions
In the present study, a novel gene Hc-clec-160 encoding a cDNA of 1224 bp in H. contortus was identified. The gene possesses one conserved domain of carbohydrate-recognition domains (CRDs) and contains 12 exons. A possible promoter in the 2000 bp sequence upstream of the 5'-flanking region was demonstrated by microinjection experiments. Furthermore, partially silencing the Ce-clec-160 in N2 worms of C. elegans achieved by RNAi with Hc-clec-160 showed that Hc-clec-160 shared similar characteristics and functions with Ce-clec-160 and plays a crucial part in the development and reproduction of H. contortus.

Additional file
Additional file 1: Figure S1.