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Background: Nematodes have lost the ability to synthesise necessary lipids de novo and have complementally evolved the capacity to acquire fatty acids and their derivatives from a diet or host animal. Nematode-specific fatty acid- and retinol-binding protein (FAR) family is one approach that facilitates lipid acquisition, representing an Achilles heel and potential target against roundworms of socioeconomic significance. However, little is known about their detailed functional roles in either free-living or parasitic nematodes., Methods: A genome-wide identification and curation were performed to screen the FAR family members of Haemonchus contortus. Their transcription patterns in worms were also analysed to identify the targets. Ligand binding assay and molecular docking were conducted to verify the fatty acid binding activities of FAR proteins of interest. RNA interference (RNAi) and heterologous expression (rescuing) experiments were designed to explore the potential roles of the selected FAR protein in nematodes. Localisation of the protein was shown in sections of paraffin-embedded worms after an immunohistochemistry (IHC) assay., Results: Here, an orthologue of far-6 in the model organism Caenorhabditis elegans (Ce-far-6) was functionally characterised in a parasitic nematode, H. contortus (Hc-far-6). It is demonstrated that knockdown of Ce-far-6 gene did not affect worm fat content, reproduction, or lifespan, but decreased worm body length at an early life stage of C. elegans. In particular, the Ce-far-6 mutant associated phenotype was completely rescued by Hc-far-6, suggesting a conserved functional role. Surprisingly, there were distinct tissue expression patterns of FAR-6 in the free-living C. elegans and parasitic H. contortus. High transcriptional level of Hc-far-6 and dominant expression of FAR-6 in the intestine of the parasitic stage of H. contortus link this gene/protein to nematode parasitism., Conclusions: These findings substantially enhance our understanding of far genes and the associated lipid biology of this important parasitic nematode at a molecular level, and the approaches established are readily applicable to the studies of far genes in a broad range of parasites., (© 2023. The Author(s).)

Infective Haemonchus contortus larvae (L3s) are able to protect themselves from desiccation. To explore the molecular mechanisms of desiccation survival, mRNA differential display RT-PCR was used to screen differentially expressed genes in L3s upon desiccation, followed by RNAi experiments to define gene functions. In this, 58 differentially expressed transcripts were obtained. Among these, the BF-U01A and CH-U02A fragments represent genes with the highest identity percentage in bioinformatic analysis. They were named Hc-ubq and Hc-gst based on their respective homologous ubiquitin in Caenorhabditis elegans and glutathione S-transferase in H. contortus. Quantitive RT-PCR results indicated that they were both up-regulated in desiccated L3s. Hc-ubq and Hc-gst RNAi in H. contortus showed reduced survival rate of L3s, with unchanged locomotion behavior. Homologous Ce-ubq-2 and Ce-gst-7 RNAi in C. elegans also displayed higher larval death rate. These results suggest that ubq and gst may play important roles in nematode desiccation tolerance. Our study analyzed desiccation resistance related genes in H. contortus L3s, and revealed significant research implications on the mechanisms behind nematode desiccation survival., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Haemonchus contortus infections in small ruminants are of major economic importance worldwide. Heat shock proteins (HSPs) are a family of molecular chaperones that play important roles in the process of invasion and survival of nematodes. Although HSP70 has been identified in several parasitic nematodes, little is known of its distribution and function in Haemonchus contortus. The aims of this study were to characterize HSP70 from Haemonchus contortus (designed as Hc-hsp70), express Hc-hsp70 and analyse the promoter activity in Caenorhabditis elegans. Bioinformatic analysis revealed that the open reading frame of the Hc-hsp70 cDNA encodes a 646-amino acid peptide, which is highly conserved in comparison to HSP70 in other nematodes. Phylogenetic analysis indicated that H. contortus is closely related to Caenorhabditis. The 5'-flanking region promoted green fluorescence protein (GFP) expression in the intestine in all larval stages and adult with 2 expression patterns in C. elegans. Expression of Hc-hsp70 mRNA transcripts in C. elegans increased following 2, 4, 6 h of heat shock and peaked at 4 h. However, its expression induced down-regulation of hsp-1 of C. elegans. These results suggest that the H. contortus hsp70 might have a similar function to that of C. elegans hsp-1.

To construct the recombinant vector Pact-EGFP, the Act-1 core promoter region was amplified from the pUCm-T/Act-1 and subcloned into pEGFP-4.1 vector (derived from pEGFP-N1 with the removal of human cytomegalovirus immediate early promoter), by restriction enzymes Bgl II and Hind III. Transfection of Pact-EGFP vector into Vero cell by liposome indicated that Act-1 core promoter regulated the expression of EGFP gene in lower level in Vero cells. After Pact-EGFP microinjection into the gonad of Caenorhabditis elegans with pRF4 as a gene marker, green fluorescence was detected in the cortex, vice cortex and the pharyngeal of C. elegans. According to the locations, two different transgene lines were separated. The expression level of EGFP expressed in C. elegans was more than that in Vero cell. Some unique motifs might exist in Act-1 core promoter region of C. elegans, which was closely related to the expression level of EGFP. These results lay the foundation for the further research on gene function of parasitic nematodes using C. elegans.

Hypobiosis (facultative developmental arrest) is the most important life-cycle adaptation ensuring survival of parasitic nematodes under adverse conditions. Little is known about such survival mechanisms, although ascarosides (ascarylose with fatty acid-derived side chains) have been reported to mediate the formation of dauer larvae in the free-living nematode Caenorhabditis elegans. Here, we investigated the role of a key gene acox-1, in the larval development of Haemonchus contortus, one of the most important parasitic nematodes that employ hypobiosis as a routine survival mechanism. In this parasite, acox-1 encodes three proteins (ACOXs) that all show a fatty acid oxidation activity in vitro and in vivo, and interact with a peroxin PEX-5 in peroxisomes. In particular, a peroxisomal targeting signal type1 (PTS1) sequence is required for ACOX-1 to be recognised by PEX-5. Analyses on developmental transcription and tissue expression show that acox-1 is predominantly expressed in the intestine and hypodermis of H. contortus, particularly in the early larval stages in the environment and the arrested fourth larval stage within host animals. Knockdown of acox-1 and pex-5 in parasitic H. contortus shows that these genes play essential roles in the post-embryonic larval development and likely in the facultative arrest of this species. A comprehensive understanding of these genes and the associated β-oxidation cycle of fatty acids should provide novel insights into the developmental regulation of parasitic nematodes, and into the discovery of novel interventions for species of socioeconomic importance. Author summary: Haemonchus contortus is one of the most pathogenic nematodes causing substantial economic losses worldwide that undergo facultative arrest (diapause) to survive harsh environmental and host conditions. However, the molecular basis (e.g., fatty acid oxidation and dauer pheromone synthesis) underlying such survival mechanisms has not yet been elucidated in parasitic nematodes. Here, we show that an acyl-CoA oxidase ACOX-1 interacts with a peroxin PEX-5 and functions in fatty acid oxidation in peroxisomes. Further, knockdown of ACOX-1 and PEX-5 results in a lethal phenotype of the infective stage and likely developmental parasitic larval stage within host animals. This data demonstrates an essential role of ACOX-1-PEX-5 and potentially ascarosides signalling in the facultative arrested larval development of parasitic nematodes, and suggests possible intervention targets. [ABSTRACT FROM AUTHOR]

• Haemonchus contortus protein Hc-DHS-28 has 3-hydroxyacyl CoA dehydrogenase activity. • Hc-dhs-28 is highly transcribed in the diapause worm and located in the peroxisome. • Hc-dhs-28 shares high similarity in gene function with Caenorhabditis elegans (Ce) -dhs-28. • Hc-dhs-28 might regulate the formation of diapause in H. contortus. • Immunisation with recombinant Hc-DHS-28 could protect the host after H. contortus infection. Haemonchus contortus could enter the diapause stage to avoid hostile conditions, however the inducing mechanism still remains poorly understood. A similar dauer strategy exists in Caenorhabditis elegans , and dauer phenomones, which are produced through a four step cycle of peroxisomal fatty acid β-oxidation, are essential in this stage. In this study, a novel gene, Hc-dhs-28 , was identified and characterised. Hc-DHS-28 was the homologue of Ce-DHS-28, a key enzyme in the oxidation cycle, and the protein contained a short chain dehydrogenase domain and a peroxisomal targeting signal 1. The expression pattern of Hc-DHS-28 detected by quantitative real-time PCR and indirect immunofluorescence assay revealed that this protein was mainly expressed in the intestine and subdermal regions of larvae at diapause and in free-living stages. Enzyme activity analysis confirmed its 3-hydroxyacyl CoA dehydrogenase activity with 121, 149, 162 and 166 as key functional sites; meanwhile co-localization in human embryonic kidney 293 cells indicated that Hc-DHS-28 was targeted to the peroxisome of cytoplasm under the guide of peroxisomal targeting signal 1, which was consistent with the functional domain prediction of Hc-dhs-28. Overexpression, rescue and RNA interference experiments were carried out to explore the function of Hc-dhs-28. Our results showed that Hc-dhs-28 was very similar to Ce-dhs-28 and partially rescued its function in C. elegans. RNAi with Hc-dhs-28 in C. elegans led to decreased transcription of genes in the peroxisomal fatty acid β-oxidation cycle, considerable fat accumulation and dauer formation defects. Furthermore, immunisation with recombinant Hc-DHS-28 protein in sheep was able to maintain the body weight of the host after infection and reduce the worm burden. In conclusion, Hc-DHS-28 is most likely involved in the peroxisome fatty acid β-oxidation as the third 3-hydroxyacyl CoA dehydrogenase to regulate the production of diapause-related pheromones, and then influence the formation of diapause in H. contortus. [ABSTRACT FROM AUTHOR]

Diapause induced in the early fourth stage of Haemonchus contortus is a strategy to adapt this nematode to hostile environmental conditions. In this study, we identified a new gene, Hc-fau , a homologue of human fau and Caenorhabditis elegans Ce-rps30 . Hc-fau encodes two proteins through alternative RNA splicing, Hc -FAUA and Hc -FAUB, consisting of 130 and 107 amino acids, respectively. Hc -FAU possesses a diverged ubiquitin-like (UBiL) protein domain and a conserved ribosome protein S30 domain. The protein is ubiquitously expressed, except in the gonad. However Hc-fau transcripts decrease significantly in diapausing L4s of H. contortus . In C. elegans , knockdown of Ce-rps30 confers an extended lifespan, increased lipid storage in the intestine and shortened body length. These morphological characteristics are comparable with dauer larvae of C. elegans , in which the gonad is condensed considerably. In contrast, a shortened lifespan is observed in C. elegans over-expressing Hc-faua , and especially Hc-faub , with hatching failure detected. The genes of insulin/IGF-1 signalling (IIS), TGF-β, cGMP, dafachronic acid (DA), apoptosis (AP) and fatty acids (FA) metabolism are all down-regulated in Ce-rps30 RNAi (RNA interference) worms, except for akt-1 and daf-16 . However, daf-16 up-regulation is inconsistent with its target gene down-regulation and the result from a heat stress assay in these worms. Daf-16 RNAi conducted in Ce-rps30 (tm6034/nt1) mutants failed to rescue the worms. The S30 domain stays in the nucleus, while UBiL accumulates in the cytoplasm. Compared with Hc -FAUA, results of UBiL domain and S30 domain over-expression indicate synergism between UBiL and S30 in regulating lifespan and reproduction. These results suggest the potential functions of Hc-fau in regulating larval diapause in H. contortus . [ABSTRACT FROM AUTHOR]