Department of Parasitology, Medical College, Zhengzhou University, Zhengzhou, People's Republic of China.
PLoS Negl Trop Dis. 2022 Oct 31;16(10):e0010881. doi: 10.1371/journal.pntd.0010881. eCollection 2022 Oct.
Pyruvate kinase widely exists in many parasites and plays an important role in the energy production for the parasites. Pyruvate kinase might be a potential drug target for killing the parasites. The aim of the present study was to evaluate the biological characteristics and roles of T. spiralis pyruvate kinase M (TsPKM) in sugar metabolism, larval molting and development of T. spiralis.
METHODOLOGY/PRINCIPAL FINDINGS: TsPKM has two functional domains of pyruvate kinase and the tertiary structure of TsPKM is tetramer which has the enzyme active site constituted by 8 amino-acid residues (Arg71, Asn73, Asp110, Phe241, Lys267, Glu269, Asp293 and Thr325). Recombinant TsPKM (rTsPKM) was expressed and purified. The rTsPKM had good immunogenicity. RT-PCR and Western blot showed that TsPKM was transcribed and expressed at various developmental stages in T. spiralis lifecycle. Immunofluorescence test showed that TsPKM was principally located in the cuticle, muscle, stichosome, intestine and the intrauterine embryos of female adults. rTsPKM catalyzed the reaction of phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) to produce pyruvic acid and adenosine triphosphate (ATP). TsPKM played an important role in the metabolism and energy production of T. spiralis. After silencing of TsPKM gene by specific dsRNA-TsPKM2, protein expression and enzyme activity of TsPKM decreased by 50.91 and 26.06%, respectively. After treatment with RNAi, natural TsPKM enzyme activity, larval molting, sugar metabolism, growth and development of T. spiralis were significantly reduced.
TsPKM participates in the larval molting, sugar metabolism, growth and development of T. spiralis and it might be a candidate target of therapeutic drug of trichinellosis.
丙酮酸激酶广泛存在于许多寄生虫中，在寄生虫的能量产生中起着重要作用。丙酮酸激酶可能是杀死寄生虫的潜在药物靶点。本研究旨在评估旋毛虫丙酮酸激酶 M（TsPKM）在糖代谢、幼虫蜕皮和旋毛虫发育中的生物学特性和作用。
方法/主要发现：TsPKM 具有丙酮酸激酶的两个功能域，TsPKM 的三级结构为四聚体，具有由 8 个氨基酸残基（Arg71、Asn73、Asp110、Phe241、Lys267、Glu269、Asp293 和 Thr325）组成的酶活性位点。重组 TsPKM（rTsPKM）被表达和纯化。rTsPKM 具有良好的免疫原性。RT-PCR 和 Western blot 显示 TsPKM 在旋毛虫生命周期的各个发育阶段都有转录和表达。免疫荧光试验显示 TsPKM 主要位于幼虫的角质层、肌肉、肌梭、肠和雌性成虫的子宫内胚胎中。rTsPKM 催化磷酸烯醇丙酮酸（PEP）和二磷酸腺苷（ADP）的反应，产生丙酮酸和三磷酸腺苷（ATP）。TsPKM 在旋毛虫的代谢和能量产生中起着重要作用。用特异性 dsRNA-TsPKM2 沉默 TsPKM 基因后，TsPKM 的蛋白表达和酶活性分别降低了 50.91%和 26.06%。用 RNAi 处理后，天然 TsPKM 酶活性、幼虫蜕皮、糖代谢、旋毛虫的生长和发育均显著降低。
TsPKM 参与旋毛虫的幼虫蜕皮、糖代谢、生长和发育，可能是旋毛虫病治疗药物的候选靶点。
