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.
Highlights
H. contortus is an important pathogenic nematode causing huge economic losses worldwide [1] and is able to coexist with its host under harsh conditions that escape digestion by stomach acid and pepsin
The PCR product of Hc-TPS amplification was attained from H. contortus by using cDNA with a precise set of primers, and the right segment size of 1,629 bp (Figure 1A) of the 543 proteinencoding amino acids was detected
The Hc-TPS gene was effectively cloned into the pET-28a expression vector, and 1,629 bp was verified by enzymatic digestion via online BLAST analysis (Figure 1B)
Summary
H. contortus is an important pathogenic nematode causing huge economic losses worldwide [1] and is able to coexist with its host under harsh conditions that escape digestion by stomach acid and pepsin. An in-depth study of key molecules involved in the mechanism of H. contortus interaction with the host is expected to identify new drug targets for controlling H. contortus infection in the future. H. contortus is one of the most important parasitic nematodes of considerable economic importance in small ruminants such as sheep and goats [6]. In the host–parasite relationship, the parasite excretes and secretes a large number of molecules into the host to regulate the host’s immune function. A variety of molecules derived from H. contortus nematodes have been identified to modulate the function of PBMCs, such as rHc-STP-1 [8] and rHc-TpMy [9], which have unique immunosuppressive effects on goat PBMCs and may be one of the mechanisms that promote immune evasion
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