Transforming growth factor-β signalling regulates protoscolex formation in the Echinococcus multilocularis metacestode

Abstract

The lethal zoonosis alveolar echinococcosis (AE) is caused by tumor-like, infiltrative growth of the metacestode larval stage of the tapeworm Echinococcus multilocularis. We previously showed that the metacestode is composed of posteriorized tissue and that the production of the subsequent larval stage, the protoscolex, depends on re-establishment of anterior identities within the metacestode germinative layer. It is, however, unclear so far how protoscolex differentiation in Echinococcus is regulated. We herein characterized the full complement of E. multilocularis TGFβ/BMP receptors, which is composed of one type II and three type I receptor serine/threonine kinases. Functional analyzes showed that all Echinococcus TGFβ/BMP receptors are enzymatically active and respond to host derived TGFβ/BMP ligands for activating downstream Smad transcription factors. In situ hybridization experiments demonstrated that the Echinococcus TGFβ/BMP receptors are mainly expressed by nerve and muscle cells within the germinative layer and in developing brood capsules. Interestingly, the production of brood capsules, which later give rise to protoscoleces, was strongly suppressed in the presence of inhibitors directed against TGFβ/BMP receptors, whereas protoscolex differentiation was accelerated in response to host BMP2 and TGFβ. Apart from being responsive to host TGFβ/BMP ligands, protoscolex production also correlated with the expression of a parasite-derived TGFβ-like ligand, EmACT, which is expressed in early brood capsules and which is strongly expressed in anterior domains during protoscolex development. Taken together, these data indicate an important role of TGFβ/BMP signalling in Echinococcus anterior pole formation and protoscolex development. Since TGFβ is accumulating around metacestode lesions at later stages of the infection, the host immune response could thus serve as a signal by which the parasite senses the time point at which protoscoleces must be produced. Overall, our data shed new light on molecular mechanisms of host-parasite interaction during AE and are relevant for the development of novel treatment strategies.

Genome-wide transcriptome analysis of Echinococcus multilocularis larvae and germinative cell cultures reveals genes involved in parasite stem cell function

“…(2019) , are among the GC associated genes of our study. In contrast, numerous genes which we had identified to be predominantly expressed in muscle and nerve cells, encoding Wnt ligands ( Koziol et al., 2016a ) or TGFβ signalling components ( Kaethner et al., 2023 ), are not part of the list.…”

“…Amplification primers for all probes used in this study are listed in Table S1 . After hybridization, fluorescent specimens were processed and analyzed essentially as described recently ( Koike et al., 2022 ; Kaethner et al., 2023 ). Control experiments using labeled sense probes were always negative.…”

“…Control experiments using labeled sense probes were always negative. In vitro staining of S-phase stem cells was carried out as described ( Koziol et al., 2014 ) using 50 µM 5-ethynyl-2’-deoxyuridine (EdU; Life Technologies, Darmstadt, Germany) for a 5 h pulse after vesicle isolation, followed by fluorescent detection with Alexa Fluor 555 azide as described previously ( Koike et al., 2022 ; Kaethner et al., 2023 ). For in situ hybridization on primary cell preparations, the established protocol for metacestode vesicles ( Koziol et al., 2016a ) was slightly modified by including additional sedimentation steps during washing to avoid material loss.…”

“…Enhanced GC proliferation has, for example, been demonstrated in response to exogenously added mammalian insulin ( Hemer et al., 2014 ), epidermal growth factor (EGF; Cheng et al., 2017b ), fibroblast growth factor (FGF; Förster et al., 2019 ), or serotonin ( Herz and Brehm, 2021 ). Very recently it has also been shown that the GC dependent differentiation of E. multilocularis towards the protoscolex is responsive to host and parasite cytokines of the transforming growth factor-β family ( Kaethner et al., 2023 ). It is, however, unknown so far how these exogenous signals are integrated into gene regulatory networks within GC to affect differentiation of progeny cells.…”

“…As in the case of the somatic stem cells of the related but free-living planarians, GC are morphologically undistinguishable but heterogenous regarding the expression of conserved regulators of pluripotency, indicating that sub-populations exist which differ in self-renewal and differentiation potential ( Koziol et al., 2014 ). Several signalling components have been shown to be expressed in GC, but also in post-mitotic cells ( Hemer et al., 2014 ; Förster et al., 2019 ; Kaethner et al., 2023 ). Furthermore, only few markers have been identified that are strongly associated with GC.…”