The trypsin inhibitor-like domain is required for a serine protease inhibitor of Haemonchus contortus to inhibit host coagulation

Wu, Fei; Zhang, Hui; Zhou, Jingru; Wu, Jie; Tong, Danni; Chen, Xueqiu; Huang, Yan; Shi, Huiping; Yang, Yi; Ma, Guangxu; Yao, Chaoqun; Du, Aifang

doi:10.1016/j.ijpara.2021.05.002

Cited by 7 publications

(7 citation statements)

References 62 publications

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“…Coimmunoprecipitation (Co-IP) was performed as described previously ( 64 ). LCMT1-HA, PME-1-HA, or the parental vector plasmid was cotransfected with PP2Acα-FLAG into HEK 293T, respectively.…”

Section: Methodsmentioning

confidence: 99%

PP2Acα-B′/PR61 Holoenzyme of Toxoplasma gondii Is Required for the Amylopectin Metabolism and Proliferation of Tachyzoites

et al. 2023

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Section: Methodsmentioning

confidence: 99%

PP2Acα-B′/PR61 Holoenzyme of Toxoplasma gondii Is Required for the Amylopectin Metabolism and Proliferation of Tachyzoites

et al. 2023

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“…The free-living nematode C. elegans (wild-type N2 strain and mutant-type RB1515 strain) was obtained from the Caenorhabditis Genetics Center (CGC) and maintained following the manufacturer’s instructions. Parasitic nematode H. contortus (ZJ isolate) was experimentally maintained in Hu sheep and the eggs, first- (L1s), second- (L2s), third- (L3s), and fourth-stage larvae (L4s), and the adult worms were collected as described previously [ 33 , 34 ]. The human embryonic kidney 293 T (HEK 293 T) cell line (Beyotime, Shanghai) was maintained following the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

“…Polyclonal antibodies against the recombinant HCON_00092800 protein were produced in Kunming mice ( n = 5) using a well-established protocol [ 33 , 54 ]. In brief, the purified recombinant protein (50 µg per mouse) was mixed with Freund’s complete adjuvant (1:1) and used for the initial immunisation of mice by subcutaneous injection.…”

Section: Methodsmentioning

confidence: 99%

Fatty acid- and retinol-binding protein 6 does not control worm fatty acid content in Caenorhabditis elegans but might play a role in Haemonchus contortus parasitism

Wei

Chen

et al. 2023

Parasites Vectors

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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. Graphical Abstract

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“…The recombinant plasmid (pET28a (+) ligated with Hc-TPS) was again cultured into LB (Luria-Bertani) media used with kanamycin sulfate (optical density at OD600), and the expression was induced into E. coli (BL21, DE3) via isopropylb-D-thiogalactopyranoside (IPTG; working concentration: 1 mM) for 5 h at 37°C (29). The rHc-TPS protein was attached with a histidine-tag and obtained from bacterial lysis via His-Trap HP columns (GE Healthcare, Piscataway, NJ, USA), and dialysis was performed by renaturation buffer (20 mmol/l Tris-HCl, 500 mmol/l NaCl, 1 mmol/l GSH, 0.1 mmol/l GSSG, pH 8.0) containing different urea concentrations (8,6,4,2, and 0 M) and using PBS (pH 8.0). The rHc-TPS protein was determined (size and purity) on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).…”

Section: Cloning Of Hc-tps and Expression Of Rhc-tpsmentioning

confidence: 99%

“…H. contortus is one of the most important parasitic nematodes of considerable economic importance in small ruminants such as sheep and goats ( 6 ). In the host–parasite relationship, the parasite excretes and secretes a large number of molecules into the host to regulate the host’s immune function.…”

Section: Introductionmentioning

confidence: 99%

The GT1-TPS Structural Domain Protein From Haemonchus contortus Could Be Suppressive Antigen of Goat PBMCs

et al. 2022

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Trehalose phosphate synthase (TPS), a key enzyme in trehalose synthesis, is not present in mammals but critical to the viability of a wide range of lower organisms. However, almost nothing is known about the function of Hc-TPS (GT1-TPS structural domain protein from Haemonchus contortus). In this study, Hc-TPS gene was cloned and the recombinant protein (rHc-TPS) was expressed and purified. The quantitative real-time PCR (qPCR) results showed that Hc-TPS was transcribed at different stages of H. contortus, with higher levels of transcription at the molting and embryo stages. Immunofluorescence analysis showed that Hc-TPS was widely distributed in adults, but the expression was mainly localized on the mucosal surface of the intestine as well as in the embryos of female worms. The impacts of rHc-TPS on peripheral blood mononuclear cell (PBMC) proliferation, nitric oxide (NO) generation, transcriptional expression of cytokines, and related pathways were examined by co-incubating rHc-TPS with goat PBMCs. The results showed that rHc-TPS significantly inhibited PBMC proliferation and NO secretion in a dose-dependent manner. We also found that rHc-TPS activated the interleukin (IL)-10/signal transducer and activator of transcription 3/suppressor of cytokine signaling 3 (IL-10/STAT3/SOCS3) axis and significantly promoted SOCS3 expression, while inhibiting interferon-gamma (INF-γ), IL-4, IL-9, and IL-2 pathways. Our findings may contribute to understanding the immune evasion mechanism for the parasite during host–parasite interactions and also help to provide ideas for discovering new drug targets.

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The trypsin inhibitor-like domain is required for a serine protease inhibitor of Haemonchus contortus to inhibit host coagulation

Cited by 7 publications

References 62 publications

PP2Acα-B′/PR61 Holoenzyme of Toxoplasma gondii Is Required for the Amylopectin Metabolism and Proliferation of Tachyzoites

PP2Acα-B′/PR61 Holoenzyme of Toxoplasma gondii Is Required for the Amylopectin Metabolism and Proliferation of Tachyzoites

Fatty acid- and retinol-binding protein 6 does not control worm fatty acid content in Caenorhabditis elegans but might play a role in Haemonchus contortus parasitism

The GT1-TPS Structural Domain Protein From Haemonchus contortus Could Be Suppressive Antigen of Goat PBMCs

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