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Parasitic roundworms (nematodes) have lost genes involved in the de novo biosynthesis of haem, but have evolved the capacity to acquire and utilise exogenous haem from host animals. However, very little is known about the processes or mechanisms underlying haem acquisition and utilisation in parasites. Here, we reveal that HRG-1 is a conserved and unique haem transporter in a broad range of parasitic nematodes of socioeconomic importance, which enables haem uptake via intestinal cells, facilitates cellular haem utilisation through the endo-lysosomal system, and exhibits a conspicuous distribution at the basal laminae covering the alimentary tract, muscles and gonads. The broader tissue expression pattern of HRG-1 in Haemonchus contortus (barber's pole worm) compared with its orthologues in the free-living nematode Caenorhabditis elegans indicates critical involvement of this unique haem transporter in haem homeostasis in tissues and organs of the parasitic nematode. RNAi-mediated gene knockdown of hrg-1 resulted in sick and lethal phenotypes of infective larvae of H. contortus, which could only be rescued by supplementation of exogenous haem in the early developmental stage. Notably, the RNAi-treated infective larvae could not establish infection or survive in the mammalian host, suggesting an indispensable role of this haem transporter in the survival of this parasite. This study provides new insights into the haem biology of a parasitic nematode, demonstrates that haem acquisition by HRG-1 is essential for H. contortus survival and infection, and suggests that HRG-1 could be an intervention target candidate in a range of parasitic nematodes. Author summary: Parasitic nematodes cannot synthesise haem de novo and must exploit haem from the environment or host animals. Mechanisms securing haem acquisition and its systemic distribution in these pathogens remain to be elucidated. In the present study, we identify a unique haem transporter HRG-1 in a range of nematodes commonly found in animals, which shows haem binding activity in vitro, haem transporting activity in yeast, and an important role in haem homeostasis in the free-living model organism Caenorhabditis elegans. This transporter distributes in the basal laminae and interacts with a vacuolar ATPase in Haemonchus contortus (a paradigm and model to screen anthelmintic targets), enabling haem transport among tissues in this parasitic nematode. RNAi-mediated gene knockdown of hrg-1 results in decreased viability of the infective larvae of barber's pole worm, which cannot establish infection or survive in the host animal. The uniqueness and essentiality of HRG-1 in this parasitic nematode provide novel insights into the haem uptake and distribution in helminths and a target candidate for the control of nematode infection. [ABSTRACT FROM AUTHOR]

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]

Excretory/secretory proteins of Haemonchus contortus (HcESPs) intermingle comprehensively with host immune cells and modulate host immune responses. In this study, H contortus ES antigen named as elongation factor 1 alpha (HcEF‐1α) was cloned and expressed. The influences of recombinant HcEF‐1α on multiple functions of goat peripheral blood mononuclear cells (PBMCs) were observed in vitro. Immunoblot analysis revealed that rHcEF‐1α was recognized by the serum of goat infected with H contortus. Immunofluorescence analysis indicated that rHcEF‐1α was bound on surface of PBMCs. Moreover, the productions of IL‐4, TGF‐β1, IFN‐γ and IL‐17 of cells were significantly modulated by the incubation with rHcEF‐1α. The production of interleukin IL‐10 was decreased. Cell migration, cell proliferation and cell apoptosis were significantly increased; however, nitric oxide production (NO) was significantly decreased. The MHC II molecule expression of cells incubated with rHcEF‐1α was increased significantly, whereas MHC‐I was not changed as compared to the control groups (PBS control and pET32a). These findings indicated that rHcEF‐1α protein might play essential roles in functional regulations of HcESPs on goat PBMC and mediate the immune responses of the host during host‐parasite relationship. [ABSTRACT FROM AUTHOR]

Abstract: The nematode Haemonchus contortus (H. contortus) is one of the most pathogenic and economically important parasites of sheep. A 24kDa protein is one of the important components in H. contortus excretory/secretory (ES), which was shown to have important biological function. In our research, the cDNA of its open reading frame (ES24) was obtained and analyzed. Then the ES24 was sub-cloned into pET-30a expression vector. The recombinant vector that codes hexahistidyl peptide fusion protein (His-ES24) was transformed into Escherichia coli BL21 (DE3) strain. After induction, a high expression level of His-ES24 was found at 6h taking about 26% of the total bacterial protein analyzed by gel thin-layer scanning. The expressed His-ES24 was purified and then used in an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies in serum samples. The ELISA was able to differentiate between H. contortus-infected sheep serum and Fasciola hepatica-infected sheep serum or non-infected sheep serum. No cross-reaction was observed in sheep sera that have been experimentally infected with F. hepatica. A total of 153 field sheep serum samples conserved in our laboratory were examined using the His-ES24 ELISA, and 82 (53.6%) of them were found seropositive to H. contortus. Our results demonstrate that the prokaryotic-expressed His-ES24 might be a useful diagnostic reagent for epidemiological studies of H. contortus in sheep. [Copyright &y& Elsevier]

Nuclear hormone receptors (NHRs) are emerging target candidates against nematode infection and resistance. However, there is a lack of comprehensive information on NHR-coding genes in parasitic nematodes. In this study, we curated the nhr gene family for 60 major parasitic nematodes from humans and animals. Compared with the free-living model organism Caenorhabditis elegans , a remarkable contraction of the nhr family was revealed in parasitic species, with genetic diversification and conservation unveiled among nematode Clades I (10–13), III (16–42), IV (33–35) and V (25–64). Using an in vitro biosystem, we demonstrated that 40 nhr genes in a blood-feeding nematode Haemonchus contortus (clade V; barber's pole worm) were responsive to host serum and one nhr gene (i.e. , nhr-64) was consistently stimulated by anthelmintics (i.e. , ivermectin, thiabendazole and levamisole); Using a high-throughput RNA interference platform, we knocked down 43 nhr genes of H. contortus and identified at least two genes that are required for the viability (i.e. , nhr-105) and development (i.e. , nhr-17) of the infective larvae of this parasitic nematode in vitro. Harnessing this preliminary functional atlas of nhr genes for H. contortus will prime the biological studies of this gene family in nematode genetics, infection, and anthelmintic metabolism within host animals, as well as the promising discovery of novel intervention targets. [Display omitted] • The first genetic landscape and functional atlas for the nhr gene family in parasitic nematodes. • nhr-64 is consistently stimulated by ivermectin, thiabendazole and levamisole. • nhr-17 and nhr-105 are required for larval viability in Haemonchus contortus. [ABSTRACT FROM AUTHOR]

[Display omitted] • Haemonchus contortus protein Hc -SPI-I8A functions as an inhibitor of blood clotting contributed by the TIL domain. • Hc -SPI-I8 is highly expressed in the parasitic stages and at the hypodermis, intestine and gonads of H. contortus. • Hc -SPI-I8 was in the same clade as the homologues, which could inhibit coagulation by interacting with clotting factors. • Hc -SPI-I8A is most likely disturbing the extrinsic coagulation cascade by interacting with Oa TSP1CP through its TIL domain. • Hc -SPI-I8A might also interfering in the intrinsic coagulation cascade by an unknown mechanism. Haemonchus contortus , a blood-feeding nematode, inhibits blood coagulation at the site of infection to facilitate blood-sucking and digesting for successful parasitism. However, the mechanism underlying anti-coagulation at the host-parasite interface is largely unknown. In the current study, Hc-spi-i8 , which has two greatly different transcripts named Hc-spi-i8a and Hc-spi-i8b , respectively, was described. Hc -SPI-I8A was a serine protease inhibitor containing a trypsin inhibitor-like cysteine rich (TIL) domain, while Hc -SPI-I8B was not. Hc -SPI-I8A/B were primarily expressed in the hypodermis, intestines and gonads in the parasitic stages of H. contortus. Hc -SPI-I8A interacted with Ovis aries TSP1-containing protein (Oa TSP1CP), which was determined by yeast two-hybrid, co-immunoprecipitation (Co-IP), pull down and co-localization experiments. The blood clotting time contributed by the TIL domain was prolonged by Hc -SPI-I8A. Hc -SPI-I8A is most likely interfering in the extrinsic coagulation cascade by interacting with Oa TSP1CP through its TIL domain and intrinsic coagulation cascade by an unknown mechanism. These findings depict a crucial point in the host-parasite interaction during H. contortus colonization, which should contribute to drug discovery and vaccine development in fighting against this important parasite worldwide. [ABSTRACT FROM AUTHOR]

Protease inhibitors are major components of excretory/secretory products released by parasitic nematodes and have been proposed to play roles in host-parasite interactions. Haemonchus contortus (the barber's pole worm) encodes for several serine protease inhibitors, and in a previous study we identified a trypsin inhibitor-like serine protease inhibitor of this blood-feeding nematode, SPI-I8, as necessary for anticoagulation. Here, we demonstrated that a bovine pancreatic trypsin inhibitor/Kunitz-type serine protease inhibitor (BPTI/Kunitz) domain-containing protein highly expressed in parasitic stages, HCON_00133150, is involved in suppressing proinflammatory cytokine production in mammalian cells. Fluorescent labelling of HCON_00133150 revealed a punctate localisation at the inner hypodermal membrane of H. contortus , an organ closely related to the excretory column. Yeast two-hybrid screening and immunoprecipitation-mass spectrometry identified that the recombinant HCON_00133150 physically interacted with a range of host proteins including the G protein subunit beta 1 of sheep (Ovis aries ; Oa GNB1), a negative regulator of NLRP3 inflammasome activation. Interestingly, heterologous expression of HCON_00133150 enhanced the inhibitory effect of Oa GNB1 on NLRP3 inflammasome and the maturation of proinflammatory cytokines IL-1β and IL-18 in transfected cells. 1-to-1 orthologues (n = 33) of BPTI/Kunitz inhibitor domain-containing proteins were predicted in clades III, IV and V (but not clade I) parasitic nematodes. Structural (tandem BPTI/Kunitz inhibitor domains inverted into the globular reticulation) and functional (a GNB1 enhancer) characterisation of HCON_00133150 and its orthologues elucidated that these molecules might contribute to immune suppression by parasitic nematodes in animals and humans. • HCON_00133150 encodes a collagen-like BPTI/Kunitz domain-containing protein. • More than 700 proteins of sheep PBMCs and abomasum interact with HCON_00133150. • HCON_00133150 inhibits IL-1β/IL-18 maturation in vitro by interacting with GNB1. [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]

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. [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]