Characterization of a novel pyruvate kinase from Trichinella spiralis and its participation in sugar metabolism, larval molting and development.

Background: 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. Conclusions: 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. Author summary: Pyruvate kinases belong to transferases and can transfer the high-energy phosphate bond of phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP) to produce pyruvic acid and adenosine triphosphate (ATP). Pyruvate kinases play a significant biological role in the parasite survival in hosts. Our results revealed that TsPKM was expressed at various T. spiralis developmental stages, and principally located in the cuticle, 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. Protein expression and enzyme activity of TsPKM were decreased by 50.91 and 26.06% respectively through silencing of TsPKM gene using specific dsRNA-TsPKM2. After treatment with RNAi and inhibitor tannin, natural TsPKM activity, larval molting, sugar metabolism, growth and development of T. spiralis were obviously inhibited. Our results showed that TsPKM participates in T. spiralis molting, sugar metabolism and development, and it might be a candidate target for anti-Trichinella drugs. [ABSTRACT FROM AUTHOR]