Structural and functional characterization of a novel gene, Hc-daf-22, from the strongylid nematode Haemonchus contortus

Recently, the whole genome of H. contortus was sequenced [23]; however, the precision and function annotation of the genes still need to be improved. In the present study, a new gene of H. contortus, Hc–daf–22, was isolated and characterized both in relation to gene structure and function. Hc–daf–22 is composed by 16 exons separated by 15 introns, and this composition is identical to human [24] and mouse [25] SCPx genes. Comparison of amino acid sequences of Hc-DAF-22 among different species revealed that the thiolase domain and SCP2 domain were conserved in nematodes and mammals. The C-terminal of Hc-DAF-22 contained a tripeptide (Ala-Lys-Ile), which has already been proven to be functional in porcine species [26], in contrast to the peroxisomal-targeting signal 1 (PTS1; typically Ser-Lys-Leu [27, 28] that are conserved among yeasts, plants, insects and mammals. Moreover, the C-terminal of Hc-DAF-22 was also found containing the amino acids that are important for SCP2 sterol transfer function (Fig. 3). The SCPx domain in the N-terminal of Hc-DAF-22 was more close to C. elegans compared to other species; however, thiolase and SCP-2 domains in C. elegans are coded by two separate genes and not fused together as one protein [29]. These indicate that the SCPx domain is conserved through most species, while the fusion of SCP2 domain in the Hc–daf–22 gene indicates a more advanced evolutionary position of the parasitic H. contortus in relation to the free-living C. elegans. Due to the similarities in structure and function with Ce–daf–22 and other SCPx genes, Hc–daf–22 may act as a peroxisomal protein and play an important role in worm development.

We obtained the 5?-flanking region of Hc–daf–22 by genome walking to analyze sequence character and verify its promoter activity with the 5?-flanking region of Ce–daf–22 as a control. Results from our analysis showed that the 5?-flanking region of Hc–daf–22 contained a TATA box, GATA-1, CCAAT binding factor, CAC-binding protein, Unc-86, AP-1, AP-2, and NF?B sites, some of which can also be found in the promoter region of the SCPx gene in humans [30]. The micro-injection results indicated that this region shows promoter activity which could drive the GFP expression in the transformed lines in N2 strain mostly in the intestine area. However, compared to the control lines, the level of GFP expression was slightly low. Since the promoter region was forced to function in an alien environment of C. elegans, the driving ability could be affected and causing the relatively low GFP levels. In addition, the difference of gene structure may also lead to the difference between two promoters. Hc–daf–22 contained two functional domains that are SCPx and SCP2 domains, while the Ce–daf–22 only has one SCPx domain; therefore, it was reasonable to infer that Hc–daf–22 function is more complicated than Ce–daf–22 which may lead to a different driving pattern of the promoter. There are other possible explanations such as the first exon of Hc–daf–22 has a distinct signal which may lead to different driving pattern of the promoter just like the first exon of human SCPx gene which has influence on the promoter region [31].

To demonstrate the gene function of Hc–daf–22 in vivo, transformations of N2 and daf–22 (ok693) strains with cp–Hc–daf–22::gfp were performed by micro-injection. The detection of GFP revealed that the recombinant Hc-DAF-22 could be expressed in the intestine region in a similar pattern to Ce–daf–22 in C. elegans [18]. Besides, Hc-DAF-22 could partially restore the phenotype of the daf–22 mutant (ok693) as the fat granules were significantly reduced or even completely absent (Fig. 8). The subsequent analysis of the post-embryonic development showed that Hc-DAF-22 also partially rescued the body size and brood size of the daf–22 (ok693) mutants (Fig. 9a, c, d), although no significant change was seen in growth rates. Similar results were observed by other researchers when expressing Hc–hsp–90 in C. elegans daf–21 mutant. Hc-HSP-90 could only partially rescue the phenotype of the mutant [31]. Nevertheless, other studies showed a full restore of transgenetic expression of parasitic genes in mutant strains of C. elegans. The expression of A. caninum slo–1 and C. oncophora slo–1 genes in C. elegans slo–1(js379) mutant was able to rescue the phenotype of worm locomotion and phenotypic behavior [32]. In another study, the glutamate-gated chloride channel (GluCl) subunit of H. contortus was expressed in a C. elegans mutant (DA1316), which was able to rescue the ivermectin sensitivity of mutant C. elegans [33]. These data indicate that the expression of parasitic genes in mutants of C. elegans could provide a first step in understanding gene functions in vivo, some genes could fully rescue the functions of C. elegans mutant while some could not. The similarity of parasitic genes with the homologues in C. elegans may play a key role in the successful rate of rescue. In our case, the partial rescue of daf–22 (ok693) mutant with Hc–daf–22 may be caused by the difference of gene structure between Hc–daf–22 and Ce–daf–22 as described above.

RNA interference (RNAi), inducing gene silencing, has been applied successfully to C. elegans and provides a functional genomic platform in a range of organisms including parasitic nematodes. Although RNAi experiments in parasitic nematodes such as in Ostertagia ostertagi [34], Heligmosomoides polygyrus [35], H. contortus [36] have been successful, there is still a limited number of genes which can be successfully silenced with RNAi due to many complicated reasons, including the lack of appropriate methods of dsRNA delivery and in vitro culture systems for parasitic nematodes, differences in RNAi effector protein functionality and in the complement of RNAi effectors between nematodes [33, 35, 37, 38]. In this study, we used RNAi to silence the Ce–daf–22 of C. elegans with Hc–daf–22 by feeding. Based on our observation, the daf–22 (RNAi) phenotype showed more fat storage and larger fat granules in a manner similar to daf–22 (ok693) mutant strain. Furthermore, the relative quantification of mRNA levels showed that Hc–daf–22 could successfully partially silence the Ce–daf–22, which further confirmed the similarity of gene structure and possible functions between these two genes.

Based on the expression pattern in different stages of H. contortus, it was noteworthy that Hc–daf–22 reached its peak in L3 and L4 stages. L3 stage worms have sealed mouth and live without feeding, so it is very important for them to take full advantage of the inner sources of energy like fat to survive, while L4 worms are just starting to feed on blood; this gene may play important roles in non-feeding stages of H. contortus.

In this study, we demonstrated that Hc–daf–22 has similar gene structure with the SCPx protein genes, which all act as peroximal ?-oxidation enzymes associated in the fatty acid metabolism. We also confirmed that Hc–daf–22 could partially recued the function of Ce–daf–22 in daf–22 mutant (ok693). It is safe to infer that Hc–daf–22 may play a similar role in the same pathway in H. contortus. Further studies are needed to confirm its enzyme activity as thiolase, its position in the peroximal ?-oxidation pathway and its relationship with diapause formation in H. contortus in developmental level. The results of our study primary demonstrated the possible role of this gene and showed a promising future in the research of parasitic diapause formation mechanisms.