The H. contortus Haecon-5 strain was maintained by goats, which were infected orally with 5000 L3s. Free-living stages including eggs, first-stage larvae (L1s), second-stage larvae (L2s), and L3s were harvested from faeces of infected goats. Parasitic stages (L4s and adults) were collected from the abomasa of infected goats euthanized at 8 and 30 days, respectively, and these two developmental stages were washed extensively in 0.85% sodium chloride, and male and female worms were carefully separated under a microscope prior to storage at −80 °C.
Nucleic acid and protein preparation and gene cloning
Total RNA was extracted from individual developmental stages of H. contortus using TRIzol reagent (Simgen, China). All RNA samples were treated with DNase I to remove genomic DNA (gDNA), and then their integrity and yields were verified by electrophoresis and spectrophotometric analysis. Complementary DNA (cDNA) was synthesized from RNA (1 μg) using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan) for coding sequence (CDS) amplification and real-time PCR. RNA was stored at −80 °C and DNA was stored at −20 °C until use.
The whole-worm proteins were prepared by grinding adult worms in phosphatase inhibitor and protein lysate (Roche Molecular Biochemicals, Switzerland), which were fractionated into soluble and insoluble materials after centrifugation at 10,000×g for 3 min at 4 °C. Then the soluble material was stored at −80 °C with protease inhibitor (Thermo Fisher, USA) added, followed by western blot analysis.
The sequences of full-length cDNA and gDNA, were retrieved from the transcriptomic and genomic datasets of H. contortus. The CDS was amplified from cDNA with the primer pair Hc-daf-5-cF/Hc-daf-5-cR (Additional file 1: Table S1) using the following protocol: 98 °C/10 min, then 98 °C/10 s, 55 °C/5 s and 72 °C/2 min for 35 cycles, and 72 °C/10 min. The PCR product was inserted into pMD-19T and sequenced in both directions directly by Tsingke Biological Technology, China.
The CDS of Hc-daf-5 were conceptually translated into predicted amino acids using DNASTAR software (http://www.dnastar.com/). The predicted amino acid sequence of Hc-DAF-5 was compared with the sequences in non-redundant protein databases using BLASTP from the National Center for Biotechnology Information (NCBI) to confirm the homologous sequences.
The structural domains (Dach and SDS boxes) of Hc-DAF-5 were aligned with its homologues from 11 species (Ancylostoma ceylanicum, Brugia malayi, Caenorhabditis briggsae, C. elegans, Danio rerio, Drosophila melanogaster, Equus caballus, Homo sapiens, Mus musculus, Nippostrongylus brasiliensis, and Toxocara canis) [6,7,8,9,10,11,12,13,14,15,16,17,18,19,20] (Additional file 1: Table S2) using the BioEdit program. For Hc-DAF-5, the coiled coil region was predicted by Expasy (https://embnet.vital-it.ch/software/COILS_form.html).
The full-length protein sequences from eight species (C. briggsae, C. elegans, D. rerio, D. melanogaster, H. sapiens, M. musculus, N. brasiliensis, and T. canis) (Additional file 1: Table S2) were aligned and used for phylogenetic analyses by means of the neighbour-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) methods. Confidence limits were assessed using a bootstrap procedure employing 1000 pseudo-replicates in MEGA .
Transcriptional analyses of Hc-daf-5 in different stages of H. contortus by real-time PCR
Real-time PCR was carried out using the specific primers Hc-daf-5-qF/Hc-daf-5-qR (Additional file 1: Table S1) to determine the mRNA levels in different developmental stages of H. contortus including eggs, L1s, L2s, L3s, both sexes of L4s, and adult worms. Total RNA of each stage was isolated with TRIzol reagent according to the manufacturer’s instructions and treated with DNase I to remove gDNA before synthesis of cDNA. The real-time PCR conditions were set as follows: one cycle at 50 °C/2 min and 95 °C/30 s, 40 cycles at 95 °C/15 s, 60 °C/15 s, and 72 °C/20 s, and one cycle at 60 °C/1 min, 95 °C/15 s, and 60 °C/15 s. Each sample was tested in triplicate, with β-tubulin of H. contortus (GenBank: M76493) as a reference gene (using specific primers Hctubulin-qF and Hctubulin-qR; Additional file 1: Table S1), and the average threshold (Ct) was taken to compare the relative quantities with Am (Am = 1) using the 2−△△Ct method . This assay was carried out three times. One-way analysis of variance (ANOVA) in GraphPad Prism 6 was adopted for statistical analysis. P-values were calculated using the Tukey post hoc test, and P < 0.05 was considered statistically significant.
Production of polyclonal antibody against recombinant Hc-DAF-5 and immunoblot analysis
A pair of specific primers Hc-daf-5-pF/Hc-daf-5-pR (Additional file 1: Table S1) containing double restriction sites was designed according to the CDS of Hc-daf-5, and it was employed to amplify the CDS of Hc-daf-5 through PCR. Then the amplified sequence was cloned into the prokaryotic expression vector to create the expression plasmid pET-28a-Hc-daf-5, which was transformed into BL21 (DE3) cells of Escherichia coli, followed by 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) induction at 16 °C for 12 h to produce recombinant rHc-DAF-5. Next, the recombinant rHc-DAF-5 was purified using a Ni Sepharose column system (GE, USA) according to the manufacturer’s protocol. Later, the purified rHc-DAF-5 was used in immunizing New Zealand white rabbit to produce anti-Hc-DAF-5 polyclonal antibody. Finally, the titer and specificity of anti-Hc-DAF-5 polyclonal antibody were determined by enzyme-linked immunosorbent assay (ELISA) and western blot.
Western blot was performed as follows. Proteins were resolved by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto an Immobilon®-PSQ membrane (Merck Millipore Ltd.). Then the immunoblot membrane was blocked with blocking buffer [1% (w/v) bovine serum albumin (BSA) (BioFroxx, China)] in phosphate-buffered saline with 20% Tween-20 (PBST)] for 6 h at 4 °C, washed three times with PBST, and incubated with Hc-DAF-5 antiserum (1:1000 in PBST) overnight at 4 °C. Next, samples were washed six times in PBST and subsequently incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:1000, Beyotime Biotechnology, China) for 2 h at 37 °C, followed by washing an additional five times. Finally, immunodetection was performed by chemiluminescence (WesternBright ECL kit; K-12045-D10, China), and images were acquired using the ChemiDoc XRS+ system (Bio-Rad, USA).
Evaluation of protein expression in H. contortus via immunofluorescence assay
Fresh male and female adults of H. contortus were fixed in 4% paraformaldehyde at 4 °C for 24 h and then consecutively dehydrated in an ethanol series (75% for 4 h, 85% for 2 h, 90% for 2 h, 95% for 1 h, and 100% for 30 min twice). The dehydrated worms were then incubated in xylene/absolute ethanol (1:1) solution for 5 min and xylene for 10 min, and then embedded in paraffin. Next, 4 μm-thick paraffin-embedded sections were subjected to immunofluorescence staining. For immunofluorescence assay, the sections were treated with EDTA buffer at 100 °C for 10 min. After blocking with 5% BSA for 20 min, anti-Hc-DAF-5 polyclonal antibody and goat anti-rabbit IgG antibody diluted at 1:100 were sequentially added and incubated at 4 °C overnight and at 37 °C for 50 min. Then the sections were stained with 4′6-diamidino-2-phenylindole (DAPI) for 5 min at 37 °C in a dark place. After thorough washing with PBS, the sections were observed under a fluorescence microscope (Olympus CX21, Japan).
RNA interference (RNAi) in H. contortus by soaking in siRNA
The CDS of Hc-daf-5 was used to design the siRNA sequences with the siRNA Design Tools program, and siRNA oligos (Additional file 1: Table S3) were chemically synthesized by Shanghai GenePharma Co., Ltd.
For RNAi, L3s were exsheathed and washed five times in 0.9% NaCl solution, followed by centrifugation at 600×g with diethyl pyrocarbonate (DEPC)-treated water three times. In each silencing assay, 50 μl of nematode suspension (about 5000 larvae) was placed into a 96-well plate. Then 10 μl of Lipofectamine 2000 (Thermo Fisher, USA) was incubated with 5 μl of Earle's Balanced Salt Solution (EBSS) (pH 5.2) containing 2.5 μg/μl amphotericin, 100 μg/μl streptomycin, and 100 IU/ml penicillin (Gibco, USA) at 25 °C for 5 min. RNasin (0.2 U) and siRNA solutions were added for incubation for 15 min. Three siRNAs of Hc-daf-5 were mixed in equal amounts, and the final concentration of each siRNA was adjusted to 1 μM, while the final concentration of negative control siRNA was 3 μM, with nuclease-free water as the blank control.
The knockdown experiments were carried out as described previously . In brief, three groups of xL3s (5000 in each group), which were kept in 80 μl of EBSS (pH 5.2) and supplemented with respective siRNAs, were soaked for 72 h. Approximately 100 larvae in each group were transferred to a fresh culture medium with EBSS for another 5 days to assess their development. The remaining larvae were subjected to RNA extraction.
All RNAs were extracted using TRIzol reagent according to the manufacturer’s instructions. Then cDNA was synthesized using the PrimeScript RT reagent kit with gDNA Eraser (Takara, China). Each 10 μl of reaction was conducted with the same amount of cDNA from each sample. The 18S gene of H. contortus was taken as the endogenous control using the primer pair Hc-18S-qF/Hc-18S-qR (Additional file 1: Table S1). Primers (Hc-daf-5-qF/Hc-daf-5-qR) used in detecting transcriptional profiles were also used here for the detection of transcriptional changes of Hc-daf-5 in worms treated with siRNA. The amplification efficiency of the primers was tested by a standard curve assay, and linear regression analysis showed similar slopes of all tested primers. The real-time PCR was performed on an ABI 7100 thermal cycler (Applied Biosystems, Germany) as per the following conditions: one cycle at 50 °C/2 min and 95 °C/30 s, 40 cycles at 95 °C/15 s, 60 °C/15 s, and 72 °C/20 s, and a cycle at 60 °C/1 min, 95 °C/15 s, and 60 °C/15 s. The fold change of Hc-daf-5 expression after RNAi was calculated by the 2−ΔΔCt method. ΔΔCt = [(Ct RNAi, Hc-daf-5) − (Ct RNAi, Hc-18S)] − [(Ct Blank, Hc-daf-5) − (Ct Blank, Hc-18S)] .
Assessing the interaction between Hc-DAF-5 and Hc-DAF-3 by bimolecular fluorescence complementation (BiFC)
The BHK21 cells were grown and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The plasmids pbJun-HA-KN151 and pbFos-Myc-LC151 were used as original plasmids for plasmid construction. In brief, the CDS fragment of Hc-daf-3 was amplified with the primer pair Hc-daf-3-fF/Hc-daf-3-fR (Additional file 1: Table S1) and cloned into pbJun-HA-KN151 between the NheI and XhoI sites to replace bJun, thus generating the pHcDAF3-HA-KN151 plasmid. Meanwhile, the CDS fragment of Hc-daf-5 was amplified with the primer pair Hc-daf-5-fF/Hc-daf-5-fR (Additional file 1: Table S1) and cloned into pbFos-Myc-LC151 between the NheI and PvuI sites to replace bFos, so as to generate the pHcDAF5-Myc-LC151 plasmid. The BHK21 cells were seeded onto six-well plates and grown to 70–80% confluence for transfection. To examine the interactions between Hc-DAF-3 and Hc-DAF-5, the plasmids pHcDAF3-HA-KN151 and pHcDAF5-Myc-LC151 were co-transfected using Lipofectamine 2000 (Thermo Fisher, USA). Then the cells were incubated at 37 °C with 5% CO2 for a further 20 h, and fluorescence was subsequently detected at 580–680 nm and imaged.
In addition to the full-length constructs of Hc-DAF-3, a DNA fragment encoding the Hc-DAF-3-MH2 domain (645 bp; 484–697 amino acids [aa]) was amplified with the primer pair Hc-daf-3-MH2-F/Hc-daf-3-fR (Additional file 1: Table S1), sequenced, and cloned into pHcDAF3-MH2-HA-KN151. Similarly, a DNA fragment encoding the Hc-DAF-5-SDS domain (900 bp; 1–300 aa) was amplified with the primer pair Hc-daf-5-fF/Hc-daf-5-SDS-R (Additional file 1: Table S1), sequenced, and cloned into pHcDAF5-SDS-Myc-LC151. Finally, the plasmids pbFos-Myc-LC151 and pbJun-HA-KN151, which express bJun and bFos, respectively, were co-transfected as positive controls, while pHA-KN151 and pHcDAF5-SDS-Myc-LC151 were set as negative controls.