Arginine kinase from Haemonchus contortus decreased the proliferation and increased the apoptosis of goat PBMCs in vitro

Abstract

BackgroundArginine 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.MethodsThe 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.ResultsThe 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-γ, nitric oxide (NO) production and cell apoptosis of goat PBMCs, whereas, TGF-β1 levels, cell proliferation and PBMCs migration were significantly decreased in a dose dependent manner.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2244-z) contains supplementary material, which is available to authorized users.

Tropomyosin: An Excretory/Secretory Protein from Haemonchus contortus Mediates the Immuno-Suppressive Potential of Goat Peripheral Blood Mononuclear Cells In Vitro

“…Nanjing (2005) strain H. contortus nematodes were isolated as detailed in a previous study [24] and maintained in one year old helminth-free goats. From eggs collection and culture to L3 stage larvae, experiments were carried out as described previously [25,26].…”

Y75B8A.8 (HC8) protein of Haemonchus contortus: A functional inhibitor of host IL‐2

“…Globin domain‐containing protein (HCGB ) (NCBI accession number CDJ94102.1) and Protein Y75B8A.8 ( HC8 ) (NCBI accession number CDJ95224.1) were identified as the binding proteins from the co‐immunoprecipitation. The extraction of RNA and composite of cDNA from H contortus were conducted as previously mentioned . Full‐length cDNA of HCGB and HC8 were amplified using the polymerase chain reaction (PCR) amplification (TAKARA, Japan), then cloned into Nub domain prey vector pPR3‐N.…”

“…The extraction of RNA and composite of cDNA from H contortus were conducted as previously mentioned. 37 Full-length cDNA of HCGB and HC8 were amplified using the polymerase chain reaction (PCR) amplification (TAKARA, Japan), then cloned into Nub domain prey vector pPR3-N.…”

Identification of a novel methyltransferase-type 12 protein from Haemonchus contortus and its effects on functions of goat PBMCs

“…The microscopical examination of fecal samples was performed on a weekly basis for the existence of parasite infection. The collection and preservation of parasite eggs, larvae, and adult worms were performed in accordance with the previously described methods [17].…”