Characterization and function analysis of a novel gene, Hc-maoc-1, in the parasitic nematode Haemonochus contortus
Parasites & Vectors volume 10, Article number: 67 (2017)
Enoyl-CoA hydratase (MAOC) is required for the biosynthesis of the fatty acid-derive side chains of the ascaroside via peroxisome β-oxidation in the free-living nematode Caenorhabditis elegans. The derivative of dideoxy-sugar, ascarylose is used as dauer pheromones or daumones to induce development of the stress-resistant dauer larvae stage.
Hc-maoc-1 gene was obtained by searching the Wellcome Trusts Sanger Institute’s H. contortus genomic database. qRT-PCR was performed to analyse the transcriptional levels of Hc-maoc-1 with different developmental stages as templates. IFA was carried out to determine the expression pattern in L3 larvae and micro-injection was used to verify the promoter activity of 5′-flanking region of Hc-maoc-1. Overexpression and RNAi experiments were applied in N2 strain to ascertain the gene function of Hc-maoc-1.
The full-length cDNA of Hc-maoc-1 was 900 bp in length, which contained eight exons separated by seven introns and possessed the Hotdog domain and the MaoC-like domain, together with several other residues and a hydratase 2 motif. It was transcribed throughout the lifecycle and peaked in the fourth-stage larvae (L4) of H. contortus; however, its transcription level decreased in diapausing L4. The protein expression and location of Hc-MAOC-1 were mainly in the intestine of L3 larvae. Overexpression of Ce-maoc-1 and Hc-maoc-1 in C. elegans showed extended lifespan and increased body size. The protein Ce-MAOC-1 and Hc-MAOC-1 were localized in the intestine with a punctate pattern. In C. elegans, knockdown of Ce-maoc-1 conferred shortened lifespan and body lengths, decreased brood size and increased lipid storage.
Caenorhabditis elegans was used as a model organism to ascertain the function of Hc-maoc-1 in H. contortus. Our results showed the similar characteristics and functions with Ce-maoc-1 and provided evidences of the potential functions of Hc-maoc-1 in biosynthesis of daumones in H. contortus.
The gastric nematode Haemonchus contortus infects small ruminants (sheep and goats) worldwide, and causes great production losses. Infection by the infective third stage larvae (iL3) is seasonal. Haemonchus contortus may enter diapause to improve its population viability in harsh environment. Diapause is widespread in nematodes . Diapause, a form of arrested development, occurs in the early fourth stage of H. contortus in abomasa of ruminants . It is a strategy for this parasitic nematode to adapt to adverse environmental conditions such as low temperature in winter, low oxygen or immunoreaction of the infected host [1–3].
Dauer is a specialized stage in the free-living nematode Caenorhabditis elegans. It is regarded as a form of arrested L3 . L2 stage of C. elegans would be induced to enter the arrest stage when larvae encounter hostile environments such as scarce food, high population density or high temperature [4–6]. The formation of dauer and recovery in C. elegans are precisely controlled by a constitutively produced ascaroside pheromones. Pheromones are regarded as the derivative of the dideoxy-sugar, ascarylose, which are consist of dideoxyhexose ascarylose and various short chain fatty acid moieties [7, 8]. Four peroxisomal enzymes participate in the ascaroside biosynthesis: acyl-CoA oxidase (ACOX-1), enoyl-CoA hydratase (MAOC-1), (3R)-hydroxyacyl-CoA dehydrogenase (DHS-28), and 3-ketoacyl-CoA thiolase (DAF-22) [9–12]. Nematodes carrying the gene daf-22 mutants have been verified that are non-functioning in the biosynthesis of the dauer pheromone and male-attracting signals [13, 14]. ACOX-1 plays a role in ascaroside biosynthesis as a model: β-oxidation shortens long-chain ω/(ω-1)-ascarosides to short-chain ω/(ω-1)-ascarosides . MAOC-1 and DHS-28 are homologues to human MFE-2 and control biosynthesis of different ascarosides in C. elegans . Although the role of peroxisome in the dauer stage has been well studied, and great progress has been made in understanding its molecular mechanisms in C. elegans, the analogous process in the H. contortus iL3 is poorly understood. Caenorhabditis elegans and H. contortus evolutionarily belong to the clade V . So, it is proposed that mechanisms used to determine entry into dauer in C. elegans and diapause in H. contortus are similar .
The purpose of the current study was to characterize Hc-MAOC-1 and to explode its function in H. contortus. In this study, we first characterized the complete cDNA of Ce-maoc-1 orthologue in H. contortus and named as Hc-maoc-1. The localization of protein Hc-MAOC-1 was ascertained by indirect immunofluorescence assay (IFA). The 5′-flanking region of Hc-maoc-1 was confirmed to have promoter activity in C. elegans and the coding region of Hc-maoc-1 was also expressed in C. elegans to see whether it can influence the growth and development. Ce-maoc-1 RNAi was performed in C. elegans to confirm the function in peroxisomal β-oxidation and accumulation of fat droplets in intestine.
Nematode strains and animals
Diapause, L4, and adults of H. contortus (ZJ strain) were collected from sheep abomasa (sheep abomasa were obtained from the Hu Zhou Slaughter house) and stored in liquid nitrogen until use. Adults of H. contortus were washed by PBS from the abomasal mucosa. The ingesta, washings and abomsal mucosa were digested in peptic-HCl and then the diapause worms were detected and collected under an anatomical lens (Motic, Fujian, China). L1, L2 and L3 were collected after 1, 3 and 7 days of incubation of collected eggs at 28 °C. Exsheathment of L3 worms (×L3s) were carried out with NaClO as previously described . A C. elegans strain of Bristol N2 was maintained on Nematode Growth Media (NGM) agar plates at 20 °C . Caenorhabditis elegans worms were fed with Escherichia coli (OP 50 strain). Worm collections were facilitated with an anatomical lens (Motic, Fujian, China).
Isolation of Hc-maoc-1 gene and acquisition 5′-flanking region
The amino acid sequence of Ce-maoc-1 gene was used to search the Sanger Institute’s H. contortus genomic database (http://www.sanger.ac.uk) using BLASTP algorithm. A protein sequence (HCISE00990300.t1_1) with 68% similarity to Ce-MAOC-1 (NP_495494.1) was identified. The coding sequence of Hc-maoc-1 was amplified using the primer pair Hc-maoc-1 F and Hc-maoc-1R (Additional file 1: Table S1). The PCR reaction procedure was denaturation at 94 °C for 1 min, followed by 35 cycles of 94 °C for 50 s, 52 °C for 40 s, 72 °C for 1 min, with a final extension at 72 °C for 10 min. The purified PCR products were then cloned into pMD18-T vector and sequenced. Gene-specific primers (Additional file 1: Table S1) were designed based on the sequence of 2,000 bp sequences upstream of the Hc-maoc-1 and used to amplify the upstream region from total genomic DNA of adult H. contortus. The PCR reaction procedure was: denaturation at 94 °C for 1 min, followed by 35 cycles of 94 °C for 50 s, 63 °C for 40 s, 72 °C for 2 min, and a final extension at 72 °C for 10 min. The purified PCR products were then cloned into the pMD18-T vector and sequenced.
Homologues of Hc-maoc-1 gene were identified using the BLASTp at the Nation Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/Blast). Amino acid sequences were aligned using Clustal W software . Protein motifs were identified by scanning the databases of PROSITE and Pfam (www.ebi.ac.uk/interpro). Phylogenetic analyses were carried out using neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods, respectively, based on the Jones-Taylor-Thornton (JTT) model in the Molecular Evolutionary Genetic Analysis (MEGA V7) .
Immunofluorescence localisation of Hc-MAOC-1 in the third stage of H. contortus
The full-length cDNA of Hc-maoc-1 was amplified by PCR with primers listed in Additional file 1: Table S1, with restriction sites for endonucleases Kpn I and Hind III underlined. The PCR products were cloned into pET-30a to construct the prokaryotic expression vector pET-30a-Hc-maoc-1, which was then transformed into BL21 (DE3) cells. Recombinant Hc-MAOC-1 (rHc-MAOC-1) expression was induced by 1 mM isopropy β-D-1-thiogalactopyranoside (IPTG) at 37 °C and was purified by affinity chromatography using a Ni-NTA agarose column system (Qiagen, Shanghai, China), according to manufacturer’s protocol. Anti-Hc-MAOC-1 polyclonal antibody was produced in a New Zealand white rabbit. Briefly, rHc-MAOC-1 was subcutaneously injected into the rabbit at a concentration of 500 μg/kg every 2 weeks for 3 times. Serum was collected at 7 days after final immunization and antibody titer of each serum was determined by enzyme-linked immunosorbent assay (ELISA).
The indirect immunofluorescence assay (IFA) was performed as described elsewhere . L3 larvae were digested in proteinase K (0.2 mg/ml) at 37 °C for 1 h followed by incubated in 0.1% trition-100 at room temperature overnight. After three washes with 0.01 mM phosphate-buffer saline (PBS), worms were incubated in 1 ml PBS containing 1.5% bovine serum albumin (BSA). After three washes, anti-Hc-MAOC-1 rabbit antibody (dilution 1:1,000) and Alexa Fluor® 488 nm goat anti-rabbit IgG antibody (Invitrogen™) (dilution 1:100) were incubated with worms as primary and secondary antibodies at 37 °C for 1 h, respectively. Worms were then strained with 4′,6-diamidino-2-phenylindole (DAPI) at room temperature for 30 min and viewed by fluorescent microscope (Olympus IX71, Tokyo, Japan).
Transcriptional analysis of Hc-maoc-1 in different developmental stages and relative genes in Ce-maoc-1RNAi C. elegans
Quantitative reverse transcription PCR (qRT-PCR) with specific primers was carried out to determine the relative abundance of Hc-maoc-1 transcripts in all key stages (i.e. L1s, L2s, iL3s, female L4s, male L4s, female adults and male adults) of H. contortus and some transcripts of candidate gene in Ce-maoc-1RNAi (RNA interference) C. elegans. In brief, total RNA was extracted separately from worms at different developmental stages of H. contortus and Ce-maoc-1RNAi C. elegans employing Trizol reagents (Invitrogen, Shanghai, China), followed by treatment by DNase I (Toyobo, Shanghai, China). First strand cDNA was obtained using ReverTra Ace-α (TOYOBO, Shanghai, China). Gene expression levels were determined by qRT-PCR (25 μl) using SYBR® Green qPCR Master Mix (TOYOBO) and an ABI 7300 thermal cycle. The qRT-PCR reaction procedure was 95 °C for 15 s, 60 °C for 15 s and 72 °C for 30 s for 40 cycles. The dissociation curve was generated under the following conditions: 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s and 60 °C for 15 s. Each sample was employed in triplicate using tubulin as an internal loading control using specific primers (Additional file 1: Table S1). The mean threshold cycle (Cq) values were used for further analysis.
Hc-maoc-1 promoter transformation of C. elegans
The 5′-flanking region of Hc-maoc-1 was used as Hc-maoc-1 promoter and amplified by PCR with primers listed in Additional file 1: Table S1 with restriction sites Pst I and Xba I underlined. The purified products were cloned into the upstream of the pPD95.77 expression vector’s gfp region to verify the ability of initiating the green fluorescent protein (GFP) expression in C. elegans. This recombinant plasmid, named as pPD95.77Hc-maoc-1-prom, was injected into wild type (N2) hermaphrodites using standard gonadal microinjection as described . Another plasmid pRF4 carrying a dominant mutant allele of rol-6 gene was co-injected with the recombinant plasmid each at a final concentration of 50 μg/ml to detect GFP expressed in these transformed C. elegans. The parental pPD95.77Ce-maoc-1-prom and pRF4 plasmid mixtures were used as controls. The F2 and later generations with a roller phenotype were selectively examined for the expression of GFP by fluorescent microscope (Olympus IX71, Tokyo, Japan).
Expression of Hc-maoc-1 in N2 strain of C. elegans
The full-length cDNA of Hc-maoc-1 was amplified by PCR with primers containing Not I/Sma I sites (underlined). The purified PCR products were cloned into pPD95.77 between the 1,574 bp Ce-maoc-1 promoter region and gfp region. The amplification of Ce-maoc-1 promoter region was performed by PCR from the wild strain genome with the primers containing the restriction site BamH I and Kpn I (underlined). The recombinant plasmid was designated as CeP-pPD95.77-Hc-maoc-1 (pPD95.77 vector with Ce-maoc-1gene promoter region and whole cDNA region of Hc-maoc-1 gene). The construct CeP-pPD95.77-Ce-maoc-1 (pPD95.77 vector with Ce-maoc-1gene promoter region and whole cDNA region of Ce-maoc-1 gene) was used as a control. The full-length cDNA of Ce-maoc-1 (NP_495494.1) was amplified with primers containing Not I/Sma I (underlined). The PCR products were cloned into pPD95.77 as described above. The recombinant plasmids were microinjected into N2 strain as described above. Transgenic worms with the roller phenotype were selected for examination of GFP activity, lifespan, brood size and body size. Measurement of lifespan, brood size and body size was performed according to Morck, et al. [24, 25]. Body size of worms were gauged from the nose to the tail tip with the free Java image processing program Image J . Larvae was placed singly onto fresh plates and incubated at 20 °C until they had laid the first few eggs. The hermaphrodites were then transferred onto fresh plates daily to prevent overcrowding until egg that laid ceased. Three days after the parents’ removal, the progeny were counted. Five synchronized worms were subsequently plated on these plates for 2 h to lay eggs to measure the lifespan. The adult worms were removed, and then eggs allowed to hatch to larva. Larvae was placed singly onto fresh plates and monitored once daily until death. The animals were transferred once daily while producing eggs to keep them separate from their progeny. Animals were scored as dead when they no longer responded with movement to light prodding of the head. All primers used are listed in Additional file 1: Table S1.
RNA interference feeding experiments
Ce-maoc-1 and Hc-maoc-1 cDNAs were cloned into L4440 vector to generate Ce-maoc-1- and Hc-maoc-1-specific RNAi vectors. The recombinant plasmids were then transformed into E. coli strain HT115 (DE3) cells, an RNase III-deficient Escherichia coli strain with isopropyl-β-D-thiogalac-topyranoside-inducible T7 polymerase activity. Primers used for PCR analysis were listed in Additional file 1: Table S1. RNAi plates were prepared according to Kwon and Narasimhan . Young adult C. elegans were incubated in the RNAi plates with E. coli transformed either with Ce-maoc-1- or Hc-maoc-1-specific RNAi vector overnight. Parental L4440 vector transformed into the E. coli strain HT115 was used as negative controls.
Oil-red-O staining was performed as previously described . Briefly, 200–300 C. elegans adult worms were collected from the NGM plates and washed with PBS (pH 7.4) three times and settled by gravity. Worms were incubated for 1 h at room temperature with constant rocking in a1:1 ratio of PBS and 2× the Modified Ruvkun’s witches brew (MRWB) buffer with 2% paraformaldehyde (PFA) for permeabilizing the worm cuticle. The 2× MRWB buffer contained 160 mM KCl, 40 mM NaCl, 14 mM Na2EGTA, 1 mM spermidine-HCl, 0.4 mM spermine, 30 mM Na-PIPES pH 7.4 and 0.2% β-mercaptoethanol. Worms were then washed with PBS once to remove PFA after being settled by gravity. The worms were subsequently dehydrated in 60% isopropanol for 15 min and strained in 60% Oil-Red-O overnight at room temperature with constant agitation. The stained worms were washed once in PBS containing 0.01% Triton X-100. Afterwards they were mounted on slides and viewed using a microscope with differential interference contrast optics (Nikon, Tokyo, Japan).
Statistical analysis for Hc-maoc-1 mRNA transcription levels and parameters of C. elegans were performed using one-way ANOVA in Excel. P-values < 0.05 were considered statistically significant. Graphs were made by GraghPad Prism 5.
Characterization of cDNA and phylogenetic analysis of amino acid sequence
The full length of Hc-maoc-1 (from the ATG initiation codon to the TGA stop codon) was 1,777 bp and contained 8 exons separated by 7 introns (Fig. 1). The gene structure was more complicated compared to Ce-maoc-1 (Fig. 1). The full-length cDNA of Hc-maoc-1 was 900 bp in length and encoded a 299-amino-acid protein with a mass of 32.8 KDa, which had 53–84% similarity to homologs from Caenorhabditis elegans (NP_495494.1), Ancylostoma ceylanicum (EPB80627.1), Oesophagostomum dentatum (KHJ91714.1), Dictyocaulus viviparus (KJH49452.1) and Pristionchus pacificus (KKA69622.1), as well as Homo sapiens (NP_001278957.1). The alignment with C. elegans and H. sapiens showed that Hc-MAOC-1 contained two conservative functional domains, named as the HotDog domain (1–154 bp) and MaoC-like domain (140–282 bp) (Fig. 2). There were several conserved catalytic sites (Asp-187, His-192 and Gly-202) and a conserved hydratase 2 motif (Y-R-L-S-G-D-X-N-XL-H-I-D-P-X-X-A) (Fig. 2). A peroxisome targeting signal 1 (PST1) was observed in the C-domain of Hc-MAOC-1 (Fig. 2). The hypothetical Hc-MAOC-1 protein sequence was phylogenetically analyzed with homologous proteins from 9 nematodes and Homo sapiens (Fig. 3). The phylogenetic tree showed that Hc-MAOC-1 was in the same clad with C. elegans, which indicated a highly similarity between Hc-MAOC-1 and Ce-MAOC-1 in amino acids.
Prokaryotic expression of Hc-maoc-1 and the preparation of its polyclonal antibody
The recombinant plasmid pET-30a-Hc-maoc-1 highly expressed in E. coli BL21 as shown by SDS-PAGE (Additional file 2: Figure S1a). Western blot analysis showed that the purified rHc-MAOC-1 with His tag could be recognized by monoclonal antibody to His tag (Additional file 2: Figure S1b). Western blot was subsequently performed to confirm the polyclonal antibodies could recognize natural Hc-MAOC-1 protein and showed that the natural protein of H. contortus reacted with the polyclonal antibodies (Additional file 3: Figure S2).
Immunofluorescence localisation of Hc-MAOC-1
Immunofluorescence localisation revealed that the protein Hc-MAOC-1was present in all cells in H. contortus × L3s. The protein Hc-MAOC-1 was detected in the intestinal region and the pharyngeal region as well. Punctiform expression of Hc-MAOC-1 in the intestine region may be the mainly expression pattern of Hc-MAOC-1 in H. contortus (Fig. 4).
Transcriptional level of Hc-maoc-1 in different developmental stages
Hc-maoc-1 was transcribed at detectable levels at L1, L2, L3, diapause and adults, with a peak in L4 stages. The transcriptional level of Hc-maoc-1 in diapause stage compared with L1, L2, L3, and adults was significantly downregulated (t-test: t (4) = 26.79, P < 0.0001; t (4) = 10.37, P = 0.0002; t (4) = 4.368, P = 0.006; t (4) = 18.47, P < 0.0001; t (4) = 17.82, P < 0.0001) (Fig. 5).
Promoter activity analysis of the 5’-flanking region of Hc-maoc-1
The reconstructed plasmid described above was transformed into the N2 strain of C. elegans. The plasmid pPD95.77Ce-maoc-1-prom was microinjected as a control. Transgenic lines showing the roller phenotype were selected. GFP was expressed in the intestine, but the level of GFP expression was obviously enhanced in the distal, middle and anterior part of the intestine region in the transgenic Hc-maoc-1-promoter::gfp worms (Fig. 6). The GFP expression in the transgenic Ce-maoc-1-promoter::gfp worms was throughout the intestine, and the level of GFP expression was clearly enhanced in similar part of the intestine. However, compared with the Hc-maoc-1-promoter::gfp worms, the general expression level of GFP was extremely improved (Fig. 7). The pattern of GFP expression was observed in all developmental stages of C. elegans transformed with Hc-maoc-1-promoter or Ce-maoc-1-promoter (only adults shown).
Expression of Hc-maoc-1 in N2 strain of C. elegans
The full-length cDNA regions were overexpressed in transgenic C. elegans to detect the gene function of Hc-maoc-1 in vivo. Ce-maoc-1 5′-flanking region was used as promoter. The results showed that the expression of Hc-maoc-1::gfp fusion protein could be observed in the whole intestine with a punctate pattern (Fig. 8). Worms overexpressing Hc-maoc-1 showed an extended lifespan and significantly lengthened body length (t-test: t (18) = 2.476, P = 0.0234), but there was no significant difference in brood size with N2 (t-test: t (6) = 0.2440, P = 0.4077) (Fig. 9).
The full-length cDNA region of Ce-maoc-1 was overexpressed as a control. The expression of Ce-maoc-1::gfp fusion protein in transgenic lines showed the similar results with Hc-maoc-1::gfp fusion protein (Fig. 10). In addition, there was a significant increase in the body length and lifespan compared to N2 and no different in brood size (t-test: t (18) = 8.685, P < 0.0001; t (6) = 0.7884, P = 0.2329) (Fig. 9).
RNAi in C. elegans
Hc-maoc-1 transcription was significantly downregulated in diapause-L4s (Fig. 5). To gauge the effect of Hc-maoc-1 knockdown in H. contortus, Hc-maoc-1-L4440 and Ce-maoc-1-L4440 were constructed and transformed into the HT115 (DE3) cells to perform RNAi in C. elegans. qRT-PCR was performed to determine the relative abundance of maoc-1 transcripts in RNAi worms. The results showed that both Hc-maoc-1 and Ce-maoc-1 RNAi could successfully downregulate the Ce-maoc-1 transcripts in C. elegans (t-test: t (6) = 10.91, P < 0.0001; t (6) = 15.84, P < 0.0001) (Fig. 11). The relevant genes in the peroxisome β-oxidation also showed significant downregulation in the Ce-maoc-1 RNAi worms (t-test: t (4) = 15.75, P < 0.0001; t (4) = 9.646, P = 0.0003; t (4) = 5.036, P = 0.0037; t (4) = 5.644, P = 0.0024) (Fig. 11).
Ce-maoc-1 RNAi worms showed a significant reduction in the brood size and shortened body length during their life cycle (t-test: t (4) = 8.184, P = 0.0012; t (10) = 13.73, P < 0.0001). The lifespan of Ce-maoc-1 RNAi worms were also shortened comparing to the control (Fig. 12). 7 days Ce-maoc-1 RNAi worms all began to have obvious big fat droplets deposition, which was further confirmed by the Oil-red-O strain (Fig. 13).
Haemonchus contortus as a pathogen in ruminants causes great production losses in agricultural industry. Diapausing in the early L4 stage is a strategy for H. contortus to survive harsh environments . In C. elegans, dauer-inducing pheromone is regarded as the derivatives of the dideoxy-sugar and a signaling molecule inducing L2 larvae to enter the arrested stage [4, 5, 28]. Peroxisome β-oxidation is considered as the pathway to biosynthesize daumones in C. elegans [9, 11, 13]. Four peroxisomal enzymes, acyl-CoA oxidase (ACOX) , enoyl-CoA hydratase (MAOC-1) , (3R)-hydroxyacyl-CoA dehydrogenase (DHS-28) and 3-ketoacyl-CoA thiolase (DAF-22) [9, 11, 29], have been identified to catalyze the four consecutive reactions in the biosynthesis of the menthyl-branched fatty acid moieties of daumones in C. elegans. MaoC-like hydratese (MaoC) has been proved to possess the activity of (R)-specific enoyl-CoA hydratase ((R)-hydratese) in associating with the β-oxidation and the polyhydroxyalkanoate (PHA) biosynthetic pathways in the fadB mutant E. coli strain . The eukaryotic (R)-hydrateses have similar size, containing about 300 amino acid residues .
In this study, we cloned and analysed homologous sequence of C. elegans, Hc-maoc-1. The full-length cDNA of Hc-maoc-1 was 900 bp encoding a protein (Hc-MAOC-1) of 299 amino acids, with a calculated molecular mass of 32804.50 Daltons. Hc-maoc-1 contained eight exons separated by seven introns, which was more complicated than C. elegans maoc-1. Sequence and structural analysis showed that Hc-MAOC-1 possesses two conserved domains: HotDog domain and MaoC-like domain (Fig. 2). Hot dog fold, an ancient ubiquitous domain, had been observed in about sixty proteins. The hot dog fold was first described from the β-hydroxydecanoyl thiol eater dehydratase in E. coli  and the 4-hydroxybenzoyl-CoA thioesterase in Pseudomaonas sp. Strain CBS-3 . The motif (Y-R-L-S-G-D-X-N-X-L-H-I-D-P-X-X-A) located in the conserved region was recognized as the hydratase 2 motif first identified on human peroxisomal multifunctional enzyme type 2 , and highly conserved in sequence and structure levels . Several residues (Asp-187, His-192 and Gly-202) in the conserved domain had been considered as the important catalytic site in MaoC and its similar eukaryotic hydratases . The peroxisomal targeting signal I (PTS1) in the C-domain of Hc-MAOC-1 and Ce-MAOC-1 was recognized by cytosolic receptors [PEX5 (peroxin)] which escort their cargo protein into the peroxisome . Homologous analysis showed that there was 68% and 53% similarity with homologs from MAOC-1 of C. elegans (NP_495494.1) and MFE-2 of Homo sapiens (NP_001278957.1), respectively. Additionally, phylogenetic analyses showed that MAOC-1 homologs existed widely in parasitic nematodes, including A. ceylanicum (EPB80627.1), O. dentatum (KHJ91714.1) and D. viviparus (KJH49452.1) (Fig. 3). These findings suggest that MAOC-1 is a relativity conserved protein and might possess similar functions with various parasitic nematodes or free living nematodes.
In the adult nematode, the distribution of peroxisomes was primarily in the epithelial cells of the digestive tract and in the pharyngeal gland . The distribution of Hc-MAOC-1 in the structure and tissues of free living stage L3 H. contortus was investigated by IFA [22, 38]. As the results show, Hc-MAOC-1 was detected in the intestinal cells with the pattern of punctate fluorescent staining. Additionally, the localization of Hc-MAOC-1 was also presented in all body cells and might also be in the pharyngeal gland (Fig. 4).
The 5′-flanking region of Hc-maoc-1 was cloned into pPD95.77 to verify its promoter activity and compare it to the 5′-flanking region of Ce-maoc-1. The results showed that Hc-maoc-1-promoter::gfp could expressed throughout the intestine of C. elegans (Fig. 6), which indicated that the region possess promoter activity. However, the expression intensity of Hc-maoc-1-promoter::gfp was weaker than that of Ce-maoc-1-promoter::gfp in transgenic worms (Fig. 7). Because the promoter region was being forced to function in an alien environment, the relatively GFP levels were lower than control. The micro-injection results also indicated that GFP was localized in the distal, middle and especially anterior part of the intestine. The similar results might be due to possess the same domain: Hotdog and MaoC-like domains and similar gene structure.
Both overexpressing Hc-MAOC-1 and Ce-MAOC-1 showed the punctate expression pattern throughout the intestine. The punctate expression pattern might be a result of the peroxisome targeting signal PTS1 (Fig. 2) . In previous studies, the peroxisome acox gene was identified to be expressed mainly in the intestine with a punctate pattern and could be abolished by prx-5 RNAi . Additionally, both daf-22 and dhs-28 were expressed mainly in the intestine in C. elegans and the intestine was considered as the organ to produce the ascarosides . The results of localization in H. contortus and C. elegans suggested that the gene Hc-maoc-1 encode enyol-CoA hydratase in H. contortus and might have the same function with C. elegans.
qRT-PCR performed to determine the relative abundant of Hc-maoc-1 transcription in different developmental stages showed that Hc-maoc-1 transcripts were detected throughout the life-cycle, but had a relative higher abundance in L4 and reduced abundance in diapause stage (Fig. 5). The transcriptional level of Hc-maoc-1 was detected with a peak in L4 stages. As we know, MAOC-1 is an enzyme in the peroxisome β-oxidation, which participates in the fat acid metabolism and daumone biosynthesis . High transcriptional level of Hc-maoc-1 in L4 stage might be related to the transition from free living stage to parasitic stage. In the parasitic stage, H. contortus began to feed on blood. Since diapause stage was deemed as one form of arrested development , the transcriptional level becomes lower than non-diapause stages. The results might hint Hc-maoc-1 played an important role in the early fourth stage for H. contortus to entry into the diapause stages . All these results indicated that Hc-MAOC-1was related to the development of H. contortus especially in the L4 and dauer stages.
Peroxisomal metabolism was necessary for postnatal growth, normal maturation and the organisms of embryonic development and organogenesis . Mutations in the human orthologues of the genes in this pathway could cause severe peroxisomal diseases, and patients suffering from this disease showed neurodegeneration and neonatal death . The percentage of adult progeny could be significantly reduced by RNAi inactivation of pex5, pex12, pex13 and pex19 in C. elegans . The function of MAOC-1 in peroxisome β-oxidation pathway in very long-chain fatty acid catabolism modulating lipid droplet size was evolutionarily conserved . In this study, there were no significant differences in brood size between C. elegans worms overexpressing the Hc-maoc-1 or Ce-maoc-1 and N2. However, the Ce-maoc-1 and Hc-maoc-1 transgenic lines had a lengthened lifespan compared with N2 and lengthened body-length. The results might indicate that Hc-maoc-1 played a more important role in the lifespan and body size, but the role could not be obvious like Ce-maoc-1. By contrast, the Ce-maoc-1RNAi worms shortened body length and lifespan and had a significant reduction in brood size. These results might indicate specific functions of Ce-maoc-1 in reproduction, growth and lifespan. Additionally, obvious fat droplet was observed in the gut of Ce-maoc-1 RNAi worm and identified by the Oil-red-O strain, consistent with Zhang, et al. (Fig. 13) . The significant reduction of relative abundance of four enzymes in peroxisome in the Ce-maoc-1RNAi worms suggested the deletion of MAOC-1 in β-oxidation disturb the normal reactions (Fig. 11). The relative quantification of transcriptional levels showed that Hc-maoc-1 could successfully partially silence the Ce-maoc-1. The results may suggest that deletion of Hc-maoc-1 might also influence reproduction, growth and lifespan.
Our data collectively showed a new gene Hc-maoc-1that is a homology to Ce-maoc-1. We also ascertained the localization in key developmental stages of H. contortus and assessed transcriptional levels of this gene in all stages including dauer stage. Using C. elegans as a model to detect the functions of Hc-maoc-1 showed similarities in functions between Hc-maoc-1 and Ce-maoc-1. Our findings provided new evidence that H. contortus might possess the similar molecular mechanism via peroxisome β-oxidation to control dauer phenomenon.
In this study, a new gene Hc-maoc-1 was identified which encodes an Enoyl-CoA hydratase in H. contortus and was the homologue of Ce-maoc-1 and human MFE-2. The expression pattern of Hc-maoc-1 in H. contortus was confirmed mainly in the intestine by IFA and a peak transcriptional level in L4 stage. Micro-injection was performed to verify the promoter activity of 5′-flanking region of Hc-maoc-1 with 5′-flanking region of Ce-maoc-1 as the control. RNAi inactivation of Hc-maoc-1 was verified it could partially silence the endogenous Ce-maoc-1 in N2 worms. Measurements with lifespan, brood size and body length showed the similar function to Ce-maoc-1. Hc-maoc-1 was identified which have the similar characteristics and function, and might play an important role in peroxisomal β-oxidation and development in H. contortus.
Bovine serum albumin
- Cq :
Enzyme-linked immunosorbent assay
Green fluorescent protein
Indirect immunofluorescence assay
Infective third-stage larvae
Modified Ruvkun’s witches brew
- N2 :
Nematode growth media
- PCR RNAi:
Peroxisomal targeting signal I
Quantitative reverse transcription
Exsheathment of L3 worms
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We would like to thank Dr. Yang CL (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences), Dr. Wang XC (the National Institute of Biological Science, Beijing) and Dr. Ren CH (Academy of Military Medical Science, Beijing) for their assistance in transgenic technique in C. elegans and for donating of vectors we needed.
This project was supported by grants from the National Key Basic Research Program (973 Program) of China (No. 2015CB150300), the National Natural Science Foundation of China (No. 31372426), the Natural Science Foundation of Zhejiang Province (LY14C180002), and the Science and Technology Project of Jiaxing (No. 2016AY23025).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files. The sequence of Hc-maoc-1 had been submitted to the GenBank database under accession number KY348613.
Haojie Ding completed the isolation of Hc-maoc-1, IFA and RNAi experiments. Hengzhi Shi finished the fat stain and promoter activity analysis. Yu Shi prepared the Anti-Hc-MAOC-1 Polyclonal antibody and nematode. Xiaolu Guo did the overexpression test. Xiuping Zheng carried out the relative quantification PCR of RNAi. Qianjin Zhou did the gene structural and phylogenetic analysis. Xueqiu Chen analysed all data and performed the Transcriptional analysis of Hc-maoc-1 in different development stages. Aifang Du and Yi Yang were the corresponding author. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All animals were treated in strict accordance with the recommendations of the Guide for the regulation for the Administration of Affairs concerning Experimental Animal of the People’s Republic of China. The experiments were approved by Zhejiang University Experimental Animal Ethics Committee (Permit Number: ZJU201308-1-10-072).
Primers used in all experiments. (DOCX 15 kb)
a Hc-maoc-1-pET30a transformed Into E. coli (BL21) and induced in 37 °C in different temperature. Lane M: Marker; Lane 1: 0 h; Lane 2: 2 h; 3: 4 h; Lane 4: 6 h; Lane 5: 8 h; Lane C: Control (pET30a empty). b Western blot (recombine protein recognized by goat-anti-mouse His Tag antibody). Lane M: Marker; Lane 1: Hc-MAOC-1 purified protein with His Tag. (PDF 427 kb)
Figure S2. Western blot: Hc-MAOC-1 polyclonal antibody recognized the whole worm protein of H. contortus. (PDF 374 kb)
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Ding, H., Shi, H., Shi, Y. et al. Characterization and function analysis of a novel gene, Hc-maoc-1, in the parasitic nematode Haemonochus contortus . Parasites Vectors 10, 67 (2017). https://doi.org/10.1186/s13071-017-1991-1