ONT-based comparative transcriptomic analysis reveal pyruvate kinase plays an important role in the differentiation of segments of Spirometra mansoni by regulating the glycolysis/gluconeogenesis pathway
Background: The differentiation and maturation of proglottids are the basis for the growth and development of tapeworms. However, we still know little about the molecular mechanisms underlying the differentiation of proglottids of Spirometra mansoni. Methods: Here, the Oxford Nanopore Technologies (ONT) method was used to perform full-length transcriptomic analysis of 3 types of proglottid of S. mansoni. Comparative transcriptomic analysis revealed pyruvate kinase (PK) is a key gene affecting segmental differentiation. Then, the PK family members of S. mansoni (SmPKs) were screened and systematically analyzed. Meanwhile, a representative member, SmPK1, was chosen for cloning expression and functional characterization. Finally, the functional mechanism of SmPK1 in segmental differentiation was explored through in vitro inhibition experiments. Results: A total of 4,486 differentially expressed genes (DEGs) were identified across 3 proglottid types. GO analysis showed that DEGs were mostly enriched in metabolism-related terms. KEGG and Gene Set Enrichment Analysis (GSEA) further revealed that the enrichment degree of glycolysis/gluconeogenesis pathway gradually increases as the segments develop and mature. Protein-protein interaction (PPI) analysis confirmed that pyruvate kinase occupies a central position among energy metabolism-related genes, and playing key roles in glycolysis/gluconeogenesis. Based on the omics data, 4 members of SmPKs were identified. Phylogenetic analysis revealed that SmPKs have varying degrees of evolution and exhibit high diversity. Both immune and infected mouse serum can specifically recognize the recombinant SmPK1 (rSmPK1). The optimal reaction conditions for rSmPK1 were 37 degrees Celsius and pH 8.0, and the addition of K+ ang Mg2+ significantly enhanced its catalytic activity. Tannic acid can significantly inhibit the activity of SmPK1 in vitro, block the production of pyruvate, and force the organism to compensate for energy supply through rapid lipolysis and delayed glycogen depletion, thereby affecting tapeworm energy metabolism. Conclusion: This study provided the first comprehensive characterization of gene expression profiles across different proglottids of S. mansoni. It was found that PK plays a pivotal role in proglottid differentiation, laying the foundation for further exploration the differentiation mechanism of segments in tapeworms.