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Investigations using TEM confirmed these findings. Sections through control metacestodes showed an intact germinal layer, composed of numerous cell types that form the parasite tissue Figure 3A, B. The most distal part of the parasite tissue is composed of the tegument, a syncytial layer that surrounds the entire metacestode, with numerous microtriches protruding into the laminated layer. The adjacent metacestode tissue is composed of undifferentiated cells with a large nucleus and nucleolus also described as germinative or stem cells , and differentiated cell types such as glycogen storage cells, muscle cells, nerve cells and connective tissue Figure 3A, B.

However, treatment with 0. Overall, BTZ treatment was followed by a distinct degeneration of the tegumental structure and in particular of the microtriches Figure 4A, C. In many instances, cells were harboring large cytoplasmic vacuoles filled with membranous material that structurally resembled autophagosomes Figure 4B, E. Black arrows point towards microtriches that originate at the tegument and protrude into the LL. In parasites treated with 0. Undifferentiated cells and mitochondria exhibit a similar morphology as the controls, microtriches black arrows are still intact.

C shows a higher magnification view of microtriches. A shows an overview of a section through the metacestode wall after treatment with BTZ at 0. Profound changes occurred at the interface between tegument Te and laminated layer LL , with evident microtriches distortion black arrows, A, C. The cytoplasm of germinal layer cells is filled with electron dense deposits marked by white arrow in A and designated as ed in B and D , and often vacuoles containing membrane stacks ms resembling authophagosomes are visible B, E and insert in E.

Nuclei nuc appear still largely intact A, B. PSMB5 sequence alignments from E. Thus the described molecular target of BTZ in humans is also present in E. The amino acids of the active core of PSMB5 are highlighted in light grey, amino acids involved in BTZ binding are highlighted in dark grey based on [22] , [23]. The alignment was performed in ClustalW2. BTZ was developed as an anti-cancer drug that would target the proteasome, and thus induces apoptosis of tumor cells [20]. While TEM suggested that BTZ treatment of metacestodes resulted in features resembling apoptotic cells, we investigated whether BTZ also induced accumulation of ubiquitinated proteins, which represents another hallmark of proteasome inhibition.

To rule out that this was due to an increased activity of ubiquitinating enzymes, the proteolytic activity of the proteasome was assessed. Upon addition of BTZ to E. In order to block other proteases that show chymotrypsin-like activity, Halt protease-inhibitor cocktail, that could also inhibit parts of the proteasome [30] , was added.

Treatment with equal amounts of DMSO served as a negative control. Semiquantitative analysis of ubiquitinated proteins was performed in Image J and relative protein abundance shown in relation to the untreated controls C. This experiment was performed twice and lead to similar results of 3. Chymotrypsin-like activity of the E.

Addition of BTZ led to dose-dependent inhibition of this activity. A, in solution proteasome assay with and without Halt protease inhibitor to inhibit other proteases. This experiment was performed in technical quadruplicates and was repeated three times, which led to similar results each time. B, in gel assay to detect chymotrypsin-like activity in E.

Bovine chymotrypsin was used as positive control C, 5 ng per lane , E. C, Western blot was performed to detect the E. Note that this protein migrates at the same apparent molecular mass as the active band in B that could be inhibited by BTZ. Experiments B and C were repeated both twice leading to the same results.

One clear chymotryptic active band was visible at around 23 kDa Figure 7B. Western blot analysis revealed that EmPSMB5 had the same apparent molecular mass as the chymotryptic active band from the parasite extract Figure 7C. In conclusion, BTZ has a strong impact on the functionality of the proteasome of E.

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The effects of BTZ treatment by i. In general, mild to heavy diarrhea was observed for all mice treated i. In addition, BTZ-treated mice showed neurological symptoms such as slight ataxia and reduced fur care eventually. One mouse of the BTZ-treated group died during the course of the experiment. Due to these adverse effects of BTZ, treatment was adjusted to a dosage of 0. After another 3 weeks of treatment at lower BTZ concentrations, all mice were euthanized, carefully examined and total parasite weight determined.

In the control group, proliferating metacestodes with many small vesicles and surrounding blood vessels were found. In contrast, BTZ treatment led to whitish and unilocular cysts, some of them being calcified Figure 8A. Thus, in vivo treatment with BTZ has a clear effect on the morphological appearance of recovered E. After euthanization parasite material was resected A and weighed B. A, macroscopical assessment showed proliferating metacestode masses with many big and small vesicles see black arrows in the control group. All treated groups showed less metacestodes, especially less proliferating ones and more white and encapsulated, small cysts see white arrows.

B, parasite weight visualized by boxplot. Chemotherapeutical treatment of AE relies on the application of the two benzimidazoles ABZ and MBZ, which have been shown to interact with beta-tubulin, and thus lead to distortion of the cytoskeletal organization and associated functions within E. In this study, we characterized the in vitro and in vivo activity of the proteasome inhibitor BTZ and defined the proteasome as a drug target in this parasite. This could have important implications also with regard to other clinically relevant cestodes. This is the first time that such an extended screening with hundreds of compounds against E.

We hereby employed an established assay, which demonstrates the damaging impact of compounds by detection of PGI activity, an enzyme that is abundant in the vesicle fluid and is released upon distortion of the physical integrity of the parasite cyst. Further concentration series of the most active drugs were performed and showed that in particular 3 drugs were also very highly active at lower concentrations: BTZ, nitazoxanide and crystal violet.

The issue of false negative compounds staying undetected by employing the PGI assay has arisen before [31]. A likely explanation for the missing activity in vitro is that in vivo ABZ gets further metabolized to its active derivatives albendazolsulfoxide and albendazolsulfone. In addition, the impact of host immunity cannot be simulated through in vitro testing.

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The pronounced impact of BTZ on the structural integrity of the germinal layer of E. At a concentration of 0. Even though a minor morphological effect on stem cells was observed by electron microscopy, BTZ is expected to be able to efficiently inhibit the growth and survival of germinal layer cells as they are actively metabolizing cells. This is crucial for a new treatment option against AE, since stem cells are the central target to prevent recurrence after drug treatment [32] , [33]. BTZ had been developed as a proteasome-inhibitor for the treatment of multiple myeloma and mantle cell lymphoma [20] , [21].

In protozoan parasites such as Plasmodium spp. In addition, the anti-malarial activity of BTZ and one of its derivatives was assessed earlier [51]. In helminths, however, the proteasome largely received attention within the nematode species Caenorhabditis elegans regarding developmental studies and in Schistosoma mansoni regarding studies on development and stress response [52]. As a drug target BTZ has not been experimentally addressed so far. However, it is worth mentioning that within the framework of the recently completed Echinococcus and Taenia genome sequencing project, the proteasome has been implicated as a potential drug target [7].

The proteasome of eukaryotes is built of a core particle 20S and one or two regulatory particles 19S that together form the 26S proteasome [53]. The 26S proteasome degrades proteins that are conjugated to ubiquitin in an ATP-dependent manner. In addition the 20S proteasome degrades unfolded, damaged, mutated and short-lived proteins also in an ubiquitin- and ATP-independent manner [54]. Thereby the proteasome takes over a critical role in regulating cell survival and cell cycle control [55].

In cancer cells, the inhibition of the proteasome leads to accumulation of proteasome-regulated proteins such as cell cycle-regulatory proteins, cyclin-dependent kinases, tumor suppressors and transcription factors [55] , [56]. Upon treatment of cancer cells with BTZ, reduced degradation of proteins of cell cycle control leads to apoptosis [55] , [56].

Induction of apoptosis has been previously postulated as a strategy to target cestode metacestodes [7] and certain hallmarks of apoptosis have been observed in protoscoleces of E. Overall, inhibition of the proteasome causes multiple effects, as this leads to functional impairment of multiple proteasome-regulated proteins, especially in actively dividing cells such as the E. This makes the proteasome a very attractive drug target. Therefore we further assessed the mode of action of BTZ in the cestode E. Exposure of in vitro-cultured E.

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In yeast, BTZ was described to inhibit the chymotryptic activity of the proteasome by binding through its boronic acid residue to the beta 5 subunit of the core particle, PSMB5 [23]. To confirm that BTZ also inhibited chymotrypsin-like activity of the E. Since BTZ inhibited the substrate cleavage activity only to a certain degree, Halt protease inhibitor mix was added. The effects of Halt protease inhibitor revealed that other, non-BTZ sensitive proteases were cleaving the substrate in use. It can, however, not be completely excluded that Halt also affects the chymotrypsin-like activity of the proteasome, as it has been shown by Yabe et al that one component Aprotinin has inhibiting activity on all three active subunits of the proteasome [30].

Nevertheless, we included Halt protease inhibitor, because only by this a stable DMSO control could be achieved. Thus, in-gel assays were performed using the same substrate, which revealed that only one band of chymotryptic activity was visible that could be partially inhibited by BTZ. Since BTZ inhibits the chymotryptic activity of the proteasome [23] , an ortholog to the beta 5 subunit was searched for in the E.

The proteasome inhibitor bortezomib is effective in hematologic malignancies such as multiple myeloma but has little activity against solid tumors, acts covalently, and is associated with undesired side effects.

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