Targeted and non-targeted proteomics to characterize the parasite proteins of Echinococcus multilocularis metacestodes

Abstract

The larval stage of the cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis. To investigate the biology of these stages and to test novel compounds, metacestode cultures represent a suitable in vitro model system. These metacestodes are vesicles surrounded by an envelope formed by the vesicle tissue (VT), which is formed by the laminated and germinal layer, and filled with vesicle fluid (VF). We analyzed the proteome of VF and VT by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified a total of 2,954 parasite proteins. The most abundant protein in VT was the expressed conserved protein encoded by EmuJ_000412500, followed by the antigen B subunit AgB8/3a encoded by EmuJ_000381500 and Endophilin B1 (protein p29). In VF, the pattern was different and dominated by AgB subunits. The most abundant protein was the AgB8/3a subunit followed by three other AgB subunits. In total, the AgB subunits detected in VF represented 62.1% of the parasite proteins. In culture media (CM), 63 E. multilocularis proteins were detected, of which AgB subunits made up 93.7% of the detected parasite proteins. All AgB subunits detected in VF (encoded by EmuJ_000381100–700, corresponding to AgB8/2, AgB8/1, AgB8/4, AgB8/3a, AgB8/3b, and AgB8/3c) were also found in CM, except the subunit encoded by EmuJ_000381800 (AgB8/5) that was very rare in VF and not detected in CM. The relative abundance of the AgB subunits in VF and CM followed the same pattern. In VT, only the subunits EmuJ_000381500 (AgB8/3a) and EmuJ_000381200 (AgB8/1) were detected among the 20 most abundant proteins. To see whether this pattern was specific to VF from in vitro cultured metacestodes, we analyzed the proteome of VF from metacestodes grown in a mouse model. Here, the AgB subunits encoded by EmuJ_000381100–700 constituted the most abundant proteins, namely, 81.9% of total protein, with the same order of abundance as in vitro. Immunofluorescence on metacestodes showed that AgB is co-localized to calcareous corpuscles of E. multilocularis. Using targeted proteomics with HA-tagged EmuJ_000381200 (AgB8/1) and EmuJ_000381100 (AgB8/2), we could show that uptake of AgB subunits from CM into VF occurs within hours.

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

“…Besides the results of the [U- 13 C]-L-threonine flux assay, we also measured significantly higher gene expression of emtdh and emkbl compared to emtd in metacestode vesicles, confirming previously reported results in metacestode vesicles in vitro and metacestodes in vivo (39,90). Furthermore, EmTDH and EmKBL, but not EmTD, were detected via non-targeted proteomics in VF and VT of in vitro cultured metacestode vesicles and also in VF of in vivo grown metacestodes obtained from experimentally infected mice (17).…”

“…This process also seemed to occur within E. multilocularis metacestode vesicles, as a combination of L-threonine and 3-HNV counteracted the negative effect of 3-HNV alone. We thus could show, that in a standardized in vitro setting (17), an active threonine metabolism is important for E. multilocularis. It is of note that we encountered variation in the number of metacestode vesicles formed from GL cell cultures, even in the control groups between different assays.…”

“…In a recent study, EmTDH and EmKBL were found via non-targeted proteomics samples of VF and VT of in vitro cultured metacestode vesicles, as well as in VF of in vivo grown metacestodes obtained from experimentally infected mice (17). Interestingly, EmTD was not detected in these samples.…”

“…In addition, the GL contains differentiated cell types such as muscle cells, nerve cells, glycogen storage cells and subtegumentary cytons (13,14). The inner fluid of metacestodes, also called vesicle fluid (VF) for in vitro grown metacestode vesicles (15), stores nutrients such as glucose, as well as various amino acids and proteins (17). The GL is further surrounded by a syncytial tegument and an outer acellular and carbohydrate-rich laminated layer (18,19).…”