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.

Keywords: cestodes; antigen B; echinococcosis; model system; targeted proteomics; transport; untargeted proteomics.

Figure 1

Antigen B (AgB) subunits in E multilocularis. (A) Alignments of polypeptide sequences of subunits encoded by the ORFs EmuJ_000381100, 200, 400, 500, 700, and 800 without signal peptides. Conserved amino acids are shown in dark gray; similar ones are shown in light gray. Annotations to AgB subunits AgB8/1–AgB8/5 are given in parentheses. (B) Relative abundances of these subunits in vesicle fluid (VF in vitro), vesicle tissue (VT), and culture medium (CM) of cultured metacestodes, as well as VF from metacestodes ex vivo (VF ex vivo). Note that EmuJ_000381600 and EmuJ_000381700 cannot be distinguished. Mean values ± standard deviations of five biological replicates are shown.

Figure 2

Whole-mount immunofluorescence with polyclonal antibodies against AgB8/1 polypeptide. With all other AgB antisera, similar results were obtained ( Figure S2 ), and representative pictures of AgB8/1 labelings are shown. No signal was detected with preimmune sera ( Figure S3 ). (A) E. multilocularis metacestode grown in vitro labeled with anti-AgB8/1 (green) and rhodamine-phalloidin (magenta). The upper panel shows maximum intensity projection and lower panels show transverse sections. (B) Maximum intensity projection with higher magnification showing a calcareous corpuscle stained with anti-AgB8/1 and nuclei (DAPI, cyan) in the upper panel, with DIC (differential interference contrast, gray) in the lower panels (note that not all calcareous corpuscles were stained). Scale bars: 30 µm.

Figure 3

Uptake of HA-tagged AgB subunits into E multilocularis metacestodes. (A) Polypeptide sequence of HA-tagged AgB8/1 encoded by EmuJ_000381200. (B) Polypeptide sequence HA-tagged AgB8/2 encoded by EmuJ_000381100. HA-tag is underlined. (C) Pre-study uptake with HA-tagged AgB8/1. (D) Uptake study with HA-tagged AgB8/2. Mean values ± standard errors correspond to three replicates. The calibration curves of the corresponding HA-tagged polypeptides are presented as inlets. Culture medium was spiked with HA-tagged polypeptide, vesicle fluid was harvested, and samples were processed as described in Materials and Methods. The full dataset is presented in Tables S5 , S6 .