Abstract
BackgroundTrehalose-6-phosphate phosphatase (TPP6) is a key enzyme in the trehalose biosynthesis pathway. The accumulation of TPP6 inside the body is harmful to the pathogen, but almost nothing is currently known about the function of TPP6 from Haemonchus contortus (CRE-GOB-1).MethodsThe H. contortus CRE-GOB-1 (HcGOB) gene was cloned and recombinant protein of GOB (rHcGOB) was expressed; transcription of the HcGOB gene at different developmental stages of H. contortus was then studied. The spatial expression pattern of the HcGOB gene in adult female and male worms was determined by both quantitative real-time PCR (qPCR) and immunofluorescence. The binding of the rHcGOB protein to goat PBMCs was assessed by immunofluorescence assay. The immunomodulatory impacts of rHcGOB on cell proliferation, nitric oxide generation and cytokine secretion were assessed by co-culture of rHcGOB protein with goat PBMCs.ResultsThe HcGOB protein was transcribed in eggs, infective third-stage larvae (iL3s) and adults of H. contortus, with the highest transcript levels found in the egg stage. The transcript levels were significantly elevated in iL3s after manual desheathing. HcGOB was widely distributed in adult worms where it was mainly localized in the gut and gonads. rHcGOB was observed to bind to PBMCs and also to be recognized by sera collected from a goat infected with H. contortus. rHcGOB significantly activated the interleukin-10/transforming growth factor β/signal transducer and activator of transcription 3 (IL-10/TGF-β/STAT3) pathway in PBMCs while suppressing the transcription and expression of IL-4 and IL-17.ConclusionsThese results suggest that the HcGOB gene plays an important role in the development, parasitism and reproduction of H. contortus. The rHcGOB protein affected the immunomodulatory function of PBMCs in the in vitro study, suggesting that this protein would be a promising vaccine target.Graphical
Highlights
Trehalose-6-phosphate phosphatase (TPP6) is a key enzyme in the trehalose biosynthesis pathway
Previous studies have shown that the excreted secretory proteins (ESPs) of H. contortus can inhibit the functions of peripheral blood mononuclear cells (PBMCs) in vitro, thereby inhibiting cell proliferation and nitric oxide (NO) secretion [12]
Expression and purification of H. contortus CRE-GOB-1 (HcGOB) Using the Complementary DNA (cDNA) of adult H. contortus worms as a template, the HcGOB gene was amplified using primers GOB-F and GOB-R, and the size of the amplified band was about 1299 bp (Fig. 1a)
Summary
Trehalose-6-phosphate phosphatase (TPP6) is a key enzyme in the trehalose biosynthesis pathway. Haemonchus contortus is an important gastrointestinal nematode of ruminants in tropical and subtropical regions of the world. It is a major health burden in sheep and causes significant economic losses to farmers [1, 2]. Vaccines offer an attractive alternative strategy for the control of haemonchosis [1], but both the extensive genetic diversity and the complicated immunomodulatory properties of H. contortus hinder the development of a suitable vaccine [1, 4]. Understanding how the mechanisms of H. contortus regulate the host immune response by promoting coexistence with the host may help towards the development of novel preventive strategies against haemonchosis
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