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Recombinant elongation factor 1 alpha of Haemonchus contortus affects the functions of goat PBMCs.

ABSTRACT: 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-1?) was cloned and expressed. The influences of recombinant HcEF-1? on multiple functions of goat peripheral blood mononuclear cells (PBMCs) were observed in vitro. Immunoblot analysis revealed that rHcEF-1? was recognized by the serum of goat infected with H contortus. Immunofluorescence analysis indicated that rHcEF-1? was bound on surface of PBMCs. Moreover, the productions of IL-4, TGF-?1, IFN-? and IL-17 of cells were significantly modulated by the incubation with rHcEF-1?. 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-1? was increased significantly, whereas MHC-I was not changed as compared to the control groups (PBS control and pET32a). These findings indicated that rHcEF-1? 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.

SUBMITTER: Ehsan M

PROVIDER: S-EPMC7187238 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Recombinant elongation factor 1 alpha of Haemonchus contortus affects the functions of goat PBMCs.

Ehsan Muhammad M Gadahi Javaid Ali JA Lu MingMin M Yan RuoFeng R Xu LiXin L Song XiaoKai X Zhu Xing-Quan XQ Du AiFang A Hu Min M Li XiangRui X

Parasite immunology 20200228 5

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. I ...[more]

PMID: 32043596

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Project description:BackgroundMethyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals. MTFs proteins have been associated with various biological/cellular processes including transcriptional regulation, subcellular protein and RNA localization, signal transduction and DNA-damage repair. However, the role of MTFs in immune mechanism during host-parasite interaction has not been addressed yet.ResultsAn open reading frame (764 bp) of methyltransferase-type 12 gene of H. contortus denoted as HcMTF-12, was successfully cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by prokaryotic expression in Escherichia coli BL21 (DE3 strain). The recombinant HcMTF-12 protein (rHcMTF-12) was about 47 kDa along with a fusion vector protein of 18 kDa. Immunoblot results identified the native protein MTF-12 with antibodies produced in rats against rHcMT-12, whereas rHcMTF-12 protein was recognized with sera of goat experimentally infected with H. contortus. Immunohistochemical analysis revealed that the native MTF-12 protein was mainly located in the periphery (cuticle) of parasite sections as well as within the pharynx and intestinal region. An immunofluorescence assay validated that rHcMTF-12 attached to the surface of goat PBMCs. Furthermore, the cytokines transcription of IL-2, IFN-γ and IL-4 transcripts of PBMCs incubated with rHcMTF-12 were enhanced in a dose-dependent manner. The secretion of TGF-β1 and IL-10 was significantly decreased. However, IL-6 production was not significantly different as compared to the control groups. Moreover, the migration activity and nitric oxide (NO) production by PBMCs were induced considerably, whereas the proliferation of PBMCs cells was negatively affected when incubated with the rHcMTF-12 protein.ConclusionsOur findings suggest that HcMTF-12 significantly mediated the functions of PBMCs, and it might be a potential candidate for therapeutic interventions against haemonchosis.

Project description:Arginine kinase (AK), an important member of phosphagen kinase family has been extensively studied in various vertebrates and invertebrates. Immunologically, AKs are important constituents of different body parts, involved in various biological and cellular functions, and considered as immune-modulator and effector for pro-inflammatory cytokines. However, immunoregulatory changes of host cells triggered by AK protein of Haemonchus contortus, a parasitic nematode of ruminants, are still unknown. The current study was focused on cloning and characterisation of Hc-AK, and its regulatory effects on cytokines level, cell migration, cell proliferation, nitric oxide production and apoptosis of goat peripheral blood mononuclear cells (PBMCs) were observed.The full-length sequence of the Hc-AK gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sub-cloned into the prokaryotic expression vector pET-32a. The biochemical characteristics of recombinant protein Hc-AK, which was purified by affinity chromatography, were performed based on the enzymatic assay. Binding of rHc-AK with PBMCs was confirmed by immunofluorescence assay (IFA). Immunohistochemical analysis was used to detect localisation of Hc-AK within adult worms sections. The immunoregulatory effects of rHc-AK on cytokine secretions, cell proliferation, cell migration, nitric oxide production and apoptosis were determined by co-incubation of rHc-AK with goat PBMCs.The full-length ORF (1080 bp) of the Hc-AK gene was successfully cloned, and His-tagged AK protein was expressed in the Escherichia coli strain BL21. The recombinant protein of Hc-AK (rHc-AK) was about 58.5 kDa together with the fused vector protein of 18 kDa. The biochemical assay showed that the protein encoded by the Hc-ak exhibited enzymatic activity. Western blot analysis confirmed that the rHc-AK was recognised by the sera from rat (rat-antiHc-AK). The IFA results showed that rHc-AK could bind on the surface of goat PBMCs. Immunohistochemically, Hc-AK was localised at the inner and outer membrane as well as in the gut region of adult worms. The binding of rHc-AK to host cells increased the levels of IL-4, IL-10, IL-17, IFN-?, nitric oxide (NO) production and cell apoptosis of goat PBMCs, whereas, TGF-?1 levels, cell proliferation and PBMCs migration were significantly decreased in a dose dependent manner.Our findings suggested that rHc-AK is an important excretory and secretory (ES) protein involved in host immune responses and exhibit distinct immunomodulatory properties during interaction with goat PBMCs.

Dataset Information

Recombinant elongation factor 1 alpha of Haemonchus contortus affects the functions of goat PBMCs.

Publications

Recombinant elongation factor 1 alpha of Haemonchus contortus affects the functions of goat PBMCs.

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