Identification and Characterization of a Cytosolic Malate Dehydrogenase as a Potential Drug Target in Echinococcus multilocularis

Abstract Alveolar echinococcosis, caused by the larval stage (metacestode) of Echinococcus multilocularis, is an emerging zoonotic disease with limited treatment options, highlighting the need for new therapeutic targets. Like other helminths, E. multilocularis relies on anaerobic metabolism via malate dismutation, a poorly characterized pathway. In this study, cytosolic malate dehydrogenase (cMDH), a key enzyme in this pathway, was investigated as a potential drug target. Bioinformatic analyses identified three cMDH homologues in E. multilocularis, all exhibiting canonical domain architecture and clustering with metazoan orthologs. Transcriptomic profiling revealed that EmuJ_000417100 (EmcMDH) is highly expressed in both in vitro-cultured metacestode vesicles and parasite lesions in mice, whereas other paralogs show minimal expression. Recombinant EmcMDH catalyzed the reversible conversion of malate and oxaloacetate, displaying typical cMDH kinetics with higher catalytic efficiency and lower Km values for oxaloacetate (16.4 ± 3.2 μM) and NADH (11.5 ± 3.9 μM) compared to malate (0.21 ± 0.10 mM) and NAD+ (0.43 ± 0.11 mM), indicating a preference for oxaloacetate reduction. RNAi-mediated knockdown of EmcMDH impaired metacestode vesicle development, demonstrating its important role in parasite growth. The natural compound gossypol potently inhibited EmcMDH (IC50 = 0.25 ± 0.04 μM) with ∼80-fold selectivity over murine cMDH. As proof-of-concept, gossypol also reduced parasite viability (IC50 = 6.43 ± 0.66 μM), though cytotoxicity was observed in mammalian cells. While the known promiscuity of gossypol precludes attributing these effects solely to EmcMDH inhibition, these findings hint at EmcMDH as a functionally relevant metabolic enzyme and a potential drug target, providing a foundation for further validation and development of selective cMDH-targeted therapies.