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

Abstract

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.

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

“…A 25-µl aliquot of the reaction system [1 µl of each primer, 12.5 µl of 2× Phanta Master Mix (Vazyme Biotech Co., Ltd.), 1 µl of cDNA and 9. 5 µl of ddH 2 O] was prepared in a PCR tube and amplified by following the PCR procedure described by Wen et al [ 13 ]. The gene products were cleaned using a Gel Extraction Kit (Vazyme Biotech Co., Ltd.) and ligated to the pET32a(+) prokaryotic expression vector.…”

“…The rHc-HAP protein was expressed and purified as previously described [ 13 ]. The pET32a(+)/Hc-HAP plasmid was transformed into Escherichia coli BL21(DE3) and induced to express rHc-HAP protein using isopropyl- D -thiogalactoside (IPTG).…”

“…The rat anti-Hc-HAP polyclonal antibody was prepared as described previously [ 13 ]. Briefly, rHc-HAP protein (300 μg) was mixed with adjuvant (Freund's complete adjuvant; Sigma-Aldrich, St. Louis, MO, USA) and immunized subcutaneously in Wistar rats.…”

“…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 ).…”