Host Proteins in Echinococcus multilocularis Metacestodes

Abstract

Metacestodes of Echinococcus multilocularis are the causative agents of alveolar echinococcosis, a neglected, life-threatening, zoonotic disease. To study these metacestodes in vitro, a model system using a culture medium conditioned by rat hepatoma cells is available. A key question is how the parasite interacts with the host and, in particular, which host-derived compounds are taken up. In this study, we focus on the uptake of host-derived proteins. Studies with artificially labeled proteins suggest that this uptake may occur independently of protein size or charge. Closer investigation using proteomics draws, however, a different picture. Of 1170 host (i.e., rat or bovine) proteins as identified by LC-MS/MS-based proteomics present in the culture medium, only 225 are found in metacestode vesicle tissue or fluid. Moreover, their relative abundances differ. Serum albumin, the most abundant culture medium host protein, is only the third most abundant protein in vesicle fluid, where Alpha-2-HS-glycoprotein becomes the most abundant protein. In vesicle fluid obtained ex vivo from experimentally infected mice, the situation is again different, with histone isoforms as the most abundant proteins. This suggests that while maintaining their internal milieu constant, metacestodes may adjust the spectrum of host proteins taken up. Potential uptake mechanisms and functions are discussed.

Keywords: helminth proteomics; homeostasis; host-parasite interaction; model system; systems biology.

Figure 1

Protein concentrations in vesicle fluid obtained from single or pooled metacestode vesicles of different sizes. The protein concentrations were determined using the bichinolic acid (BCA) assay. The in vitro vesicles were incubated in a culture medium (CM) containing 5% fetal bovine serum (FBS). After three days, The vesicles were washed with phosphate buffered saline (PBS), and vesicle fluid (VF) was harvested with a syringe. In the case of pooled vesicles, mean values and standard deviations correspond to three independent pools.

Figure 2

Uptake of fluorescence-labelled proteins into metacestode vesicles. (A), Intact metacestode vesicles were incubated in PBS buffer containing 200 nM Cy3-labelled chymotrypsin A (Chy A), bovine serum albumin (BSA), catalase or thyreoglobulin (Thyreo). (B), Intact metacestode vesicles were incubated in PBS buffer containing FITC-labelled mouse monoclonal antibodies (mAbg11, IFA), goat-anti-rabbit (GaR), or goat-anti-mouse (GaM) IgG (10 mg/L). After 3 h of incubation at 37 °C, the metacestode vesicles were thoroughly washed with PBS, and protein concentrations and label fluorescences of vesicle fluid (VF) and vesicle tissue (VT) were determined. Mean values ± standard deviation are given for three replicates per condition.

Figure 3

Ranking of relative abundances of host proteins identified both in culture medium and in vesicle fluid. The relative abundances were calculated based on the IBAQ values of the respective proteins and ranked from 1 (the most abundant protein within a class) to n (the least abundant protein). The values are listed in Tables S1 and S2. The ideal correlation (r = 1) is presented by a continuous black line. The real correlation is presented as a dotted line. CM, culture medium; VF, vesicle fluid.

Figure 4

Ranking of relative abundances of proteins identified both in vesicle tissue and in vesicle fluid. The relative abundances were calculated based on the IBAQ values of the respective proteins and ranked from 1 (the most abundant protein within a class) to n (the least abundant protein). The values are listed in Table S2. The ideal correlation (r = 1) is presented by a continuous black line. The real correlation is presented as a dotted line. A, group of proteins overrepresented in vesicle fluid (VF). B, group of proteins overrepresented in vesicle tissue (VT).

Figure 5

The seven most abundant proteins of group (A) (overrepresented in vesicle fluid) and group (B) (proteins overrepresented in vesicle tissue) as depicted in Figure 3 and listed in Table S2. Mean IBaq values ± standard deviations are given for five biological replicates. VF, vesicle fluid; VT, vesicle tissue.

Figure 6

Schematic representation of potential import routes and roles of host proteins within E. multilocularis metacestode vesicles. CM, culture medium; HP, host protein (pink); VF, vesicle fluid (light blue); VT, vesicle tissue, including GL, germinal layer (green); LL, laminated layer (red); T, tegument (brown). Invaginations from the GL represent the formation of brood capsules. Figure adapted from reference [63].