In vitro characterization of Haemonchus contortus trehalose-6-phosphate phosphatase and its immunomodulatory effects on peripheral blood mononuclear cells (PBMCs)

Abstract

Background Trehalose-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). Methods The 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. Results The 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. Conclusions These 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 Abstract

Histidine acid phosphatase domain-containing protein from Haemonchus contortus is a stimulatory antigen for the Th1 immune response of goat PBMCs

“…The PBMCs were washed 3 times with sterile phosphate-buffered solution (PBS), and the cell density was adjusted to 1 × 10 6 cells/ml. Then, the cell viability of isolated PBMCs was detected with trypan blue dye [ 12 ].…”

“…The relative protein expression of rHc-HAP was detected by western blot, as described in our previous studies [ 12 , 20 ]. Briefly, the proteins (30 µg) were transferred onto polyvinyl difluoride (PVDF) membranes [GE Healthcare Life Science (China) Co., Beijing, China] after 12% SDS-PAGE and the membranes incubated with 5% bovine serum albumin (BSA) at 37 °C for 1 h. After washing the membranes 5 times with TBST (Tris-buffered saline containing 0.05% Tween-20), the membranes were incubated overnight at 4 °C with goat serum artificially infected with H. contortus .…”

“…Gadahi et al discovered that the excretory secretions of H. contortus inhibit the function of goat peripheral blood mononuclear cells (PBMCs) [ 11 ]. Moreover, we have recently demonstrated that trehalose-6-phosphate phosphatase (HcGOB) and trehalose phosphate synthase structural domain protein (Hc-TPS) in H. contortus inhibit PBMC function by activating the interleukin (IL)-10/signal transducer and activator of transcription (STAT) 3 (IL-10/STAT3) pathway [ 12 , 13 ].…”

MSC-ACE2 Ameliorates Streptococcus uberis-Induced Inflammatory Injury in Mammary Epithelial Cells by Upregulating the IL-10/STAT3/SOCS3 Pathway

“…The relative transcript levels of IL-6, IL-10, IL-18, TNF-a, IL-Iβ, Apoptosis-associated speck-like protein containing CARD (ASC), Suppressor of cytokine signaling 3 (SOCS3), ZO-1, Claudin-3, and Occludin in EpH4-Ev cells were analyzed by qRT-PCR assay. The methods of obtaining cDNA and PCR and extracting and obtaining total RNA are based on a previous study ( 22 , 23 ). There is a list of primer sequences in Table 1 ( Additional File 1 ).…”

“…Western blot assays were performed with reference to the description of previous studies ( 23 ). After co-culture for 24 h, the culture medium was discarded and washed twice with cold PBS.…”