Investigation of the threonine metabolism ofEchinococcus multilocularis: the threonine dehydrogenase as a potential drug target in alveolar echinococcosis

Abstract

AbstractAlveolar echinococcosis (AE) is a severe zoonotic disease caused by the metacestode stage of the fox tapewormEchinococcus multilocularis. We recently showed thatE. multilocularismetacestode vesicles scavenge large amounts of L-threonine from the culture medium that were neither stored nor overused for protein synthesis. This motivated us to study the effect of L-threonine on the parasite and how it is metabolized. We established a novel metacestode vesicle growth assay with an automated readout, which showed that L-threonine treatment led to significantly increased parasite growth. In addition, L-threonine increased the formation of novel metacestode vesicles from primary parasite cell cultures in contrast to the non-proteinogenic threonine analog 3-hydroxynorvaline. Tracing of [U-13C]-L-threonine and metabolites in metacestode vesicles and culture medium resulted in the detection of [U-13C]-labeling in aminoacetone and glycine, indicating that L-threonine was metabolized by threonine dehydrogenase (TDH). In addition, the detection of [13C2]-glutathione, suggested thatE. multilocularismetacestode vesicles synthesize glutathione via L-threonine-derived glycine. EmTDH-mediated threonine metabolism in theE. multilocularismetacestode stage was further confirmed by quantitative real-time PCR, which demonstrated high expression ofemtdhinin vitrocultured metacestode vesicles and also in metacestode samples obtained from infected animals. EmTDH was enzymatically active in metacestode vesicle extracts. Thus, the drugs disulfiram, myricetin, quercetin, sanguinarine and seven quinazoline carboxamides were assessed for inhibition of recombinantly expressed EmTDH, and the most potent inhibitors disulfiram, myricetin and sanguinarine were further tested for activity againstE. multilocularismetacestode vesicles and primary parasite cells. Sanguinarine exhibited significantin vitroactivity and IC50-values for metacestode vesicles, primary parasite cells, as well as mammalian cells were determined. Our results suggest that sanguinarine treatment should be further assessedin vivoemploying suitable AE mouse models. Furthermore, the EmTDH assay could serve as high-throughput target-based discovery platform for novel anti-echinococcal compounds.