Department of Parasitology, Medical College, Zhengzhou University, Zhengzhou, 450052, China.
Vet Res. 2021 Jan 7;52(1):6. doi: 10.1186/s13567-020-00877-8.
Inorganic pyrophosphatase (PPase) participates in energy cycle and plays a vital role in hydrolysis of inorganic pyrophosphate (PPi) into inorganic phosphate (Pi). The aim of this study was to investigate the biological properties of a Trichinella spiralis PPase (TsPPase) and its role in larval molting and developmental process. The predicted TsPPase consisted of 367 amino acids with a molecular mass of 41.48 kDa and a pI of 5.76. Amino acid sequence alignment and phylogenetic analysis showed that the TsPPase gene encodes a functional family I soluble PPase with the same characteristics as prokaryotic, plant and animal/fungal soluble PPase. The rTsPPase was expressed and purified, it has the activity to catalyze the hydrolysis of PPi to Pi, and the activity was dependent on Mg, pH and temperature. The enzymatic activity of rTsPPase was significantly inhibited after its metal binding sites mutation. TsPPase was transcribed and expressed in all T. spiralis phases, especially in muscle larvae (ML) and intestinal infective larvae (IIL). Immunofluorescence assay (IFA) revealed that TsPPase was mainly located in cuticle and stichosome. When the ML and IIL were treated with TsPPase-specific siRNA-279, TsPPase expression and enzymatic activity were obviously reduced, the larval molting and development were also impeded. Intestinal IIL as well as AW burden, IIL molting rates from mice infected with siRNA-treated ML were obviously suppressed. The results indicated that rTsPPase possesses the enzymatic activity of native inorganic pyrophosphatase, and TsPPase plays an important role in development and molting process of intestinal T. spiralis larval stages.
无机焦磷酸酶（PPase）参与能量循环，在无机焦磷酸（PPi）水解为无机磷酸盐（Pi）中起着至关重要的作用。本研究旨在研究旋毛虫 PPase（TsPPase）的生物学特性及其在幼虫蜕皮和发育过程中的作用。预测的 TsPPase 由 367 个氨基酸组成，分子量为 41.48 kDa，等电点为 5.76。氨基酸序列比对和系统发育分析表明，TsPPase 基因编码一种具有与原核生物、植物和动物/真菌可溶性 PPase 相同特征的功能性 I 族可溶性 PPase。rTsPPase 表达和纯化后，具有催化 PPi 水解为 Pi 的活性，该活性依赖于 Mg、pH 和温度。rTsPPase 的金属结合位点突变后，其酶活性显著受到抑制。TsPPase 在旋毛虫的所有阶段都有转录和表达，特别是在肌肉幼虫（ML）和肠感染幼虫（IIL）中。免疫荧光分析（IFA）显示 TsPPase 主要位于角质层和肌梭。当 ML 和 IIL 用 TsPPase 特异性 siRNA-279 处理时，TsPPase 的表达和酶活性明显降低，幼虫蜕皮和发育也受到阻碍。用 siRNA 处理的 ML 感染的小鼠的肠 IIL 以及 AW 负担、IIL 蜕皮率明显受到抑制。结果表明，rTsPPase 具有天然无机焦磷酸酶的酶活性，TsPPase 在肠道旋毛虫幼虫阶段的发育和蜕皮过程中发挥重要作用。
