TY - JOUR A1 - Brehm, Klaus A1 - Koziol, Uriel A1 - Rauschendorfer, Theresa A1 - Rodríguez, Luis Zanon A1 - Krohne, Georg T1 - The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis N2 - Background It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal. Results We show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells. Conclusions In E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae. KW - Cestoda KW - Echinococcus KW - Neoblast KW - Germinative cell KW - Stem cell KW - Nanos KW - Argonaute KW - Mucin KW - Alkaline phosphatase Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110315 ER - TY - THES A1 - Zavala Góngora, Ricardo T1 - Isolierung, Charakterisierung und Funktionsanalyse von TGF-Beta-Signaltransduktionskomponenten des Fuchsbandwurms Echinococcus multilocularis T1 - Structural and functional characterization of TGFß signaling systems in Echinococcus multilocularis N2 - Die molekularen Mechanismen der Wirt-Parasit-Interaktion bei der durch den Zestoden Echinococcus multilocularis ausgelösten Erkrankung der alveolären Echinokokkose sind bislang ungeklärt. Zudem liegen keine Daten über Entwicklungs- und Differenzierungsmechanismen dieses Parasiten vor, die für die Entwicklung neuer Antiparasitika genutzt werden könnten. Ein bei der Evolution der Metazoen bereits frühzeitig entstandener Signaltransduktionsmechanismus zur Steuerung von Entwicklungsvorgängen ist das TGFβ/BMP-System, das aus strukturell verwandten Zytokinen der TGFβ (transforming growth factor β) bzw. BMP (bone morphogenetic protein)-Familie, oberflächenständigen Rezeptoren der TGFβ-Rezeptorfamilie (Typ I und Typ II) und intrazellulären Signaltransduktoren der Smad-Familie besteht. Außer an Entwicklungsvorgängen tierischer Organismen könnte diesem System eine wichtige Rolle bei der Wirt-Helminth-Kommunikation während Infektionsprozessen zukommen, wie in vorherigen Studien am Nematoden Brugia malayi und am Trematoden Schistosoma mansoni gezeigt werden konnte. Erste, wichtige Schritte zur Charakterisierung von TGFβ und BMP-Signalsystemen in Zestoden wurden in der vorliegenden Arbeit getan. Aufbauend auf einem vorherigen Bericht zu einem Transmembranrezeptor (EmRSK1) und einem Smad-Homologen (EmSmadA) aus Echinococcus multilocularis wurde die Liste der TGFβ/BMP Signaltransduktionsfaktoren in E. multilocularis in dieser Arbeit deutlich erweitert und erstmals umfangreiche funktionelle Studien durchgeführt. Die hier charakterisierten Faktoren umfassen zwei weitere Serin/Threonin-Kinasen der TGFβ/BMP-Rezeptorfamilie (EmRSK2, EmRSK3) sowie intrazelluläre Transduktoren der R-Smad-Subfamilie (EmSmadB, EmSmadC) und ein Homologes zur MAP-kinase-kinase-kinase TAK1 (TGFβ activated kinase 1), genannt EmTAK1. Zudem konnte erstmals für einen parasitären Helminthen ein Zytokin der BMP-Subfamilie, EmBMP, auf molekularer Ebene charakterisiert werden. Strukturelle und funktionelle Untersuchungen legen nahe, dass E. multilocularis sowohl ein TGFβ wie auch ein BMP-Signalsystem exprimiert. Ersteres wird sehr wahrscheinlich durch die Kinase EmRSK2 und den Smad-Faktor EmSmadC gebildet, letzteres durch EmRSK1 und EmSmadB. EmSmadA nimmt eine Sonderstellung ein, da es sowohl durch TGFβ- wie auch durch BMP-Rezeptoren aktiviert werden kann. Die genaue Rolle von EmRSK1 und EmTAK1 wäre durch weitere Untersuchungen zu klären. Signifikante funktionelle Homologien zwischen den TGFβ/BMP-Signalsystemen des Parasiten und Säugern konnten nachgewiesen werden, die sich u.a. darin äußern, dass die Echinococcus Smad-Proteine durch entsprechende Rezeptoren des Menschen aktiviert werden können. Darüber hinaus konnten jedoch auch einige deutliche Unterschiede zwischen den Systemen aus Parasit und Wirt nachgewiesen werden, die sich als Angriffspunkte zur Entwicklung von Chemotherapeutika eignen könnten. So fehlt den Smad-Faktoren EmSmadA und EmSmadC eine MH1-Domäne, die sonst unter allen R-Smads hoch konserviert ist. Zudem sind einige bislang noch nie beschriebene, strukturelle Besonderheiten der Echinococcus TGFβ/BMP-Rezeptoren zu verzeichnen. Auch die Regulation dieser Faktoren und die Kreuz-Interaktion mit weiteren intrazellulären Signalwegen (z.B. der MAP Kinase Kaskade) scheint in E. multilocularis anders zu verlaufen als bislang für Vertebraten, Insekten oder Nematoden beschrieben. Schließlich konnte, als sehr wichtiger Befund, auch nachgewiesen werden dass mindestens ein Rezeptor des Parasiten, EmRSK1, mit einem Zytokin des Wirts (BMP2) in vitro funktionell interagiert. Da BMP2 in Zellkultursystemen, die das Wachstum des Parasiten am befallenen Wirtsorgan nachstellen, einen deutlichen Effekt auf E. multilocularis ausübt, könnte die hier beschriebene EmRSK1/BMP2 – Interaktion von entscheidender Bedeutung für die Wirt-Parasit-Interaktion bei der alveolären Echinokokkose sein. N2 - Up to now, the molecular mechanisms of the interactions between host and parasite in the disease of alveolar echinococcosis, caused by the cestode Echinococcus multilocularis, are not understood. Furthermore there are not data available about the mechanisms of development and differentiation in this parasite that could be used for the design of novel antiinfectives. One of the signaling systems which emerged very early in metazoan evolution and which presumably controls developmental processes in all animals is the TGFβ signal transduction system. This system consists of various factors: structurally related cytokines of the TGFβ (transforming growth factor β) and the BMP (bone morphogenetic protein) family, surface associated receptors of the TGFβ receptor family (type I and type II) and intracellular signal transduction factors of the Smad family. In addition to their crucial role in animal development, TGFβ/BMP systems could also play an important role in the communication between host and helminths during an infection, as has been shown previous studies on the nematode Brugia malayi and the trematode Schistosoma mansoni. In this study, the initial steps towards a characterization of TGFβ/BMP signaling in the third large group of parasitic helminths, the cestodes, have been made. Adding to a previous report on a transmembrane receptor (EmRSK1) and a Smad homologue (EmSmadA) from E. multilocularis, this work significantly extends the list of known TGFβ/BMP signaling factors from Echinococcus and provides, for the first time, functional studies on these systems. The newly characterized factors comprise two further serin/threonin kinases of the TGFβ/BMP receptor family (EmRSK2, EmRSK3), two further intracellular transducing factors belonging to the subfamiliy of R-smads (EmSmadB, EmSmadC) and one homologue of the MAP-kinase-kinase-kinase TAK1 (TGFβ activated kinase 1), which was designated EmTAK1. Furthermore, and for the first time in a parasitic helminth, a cytokine of the BMP subfamily was characterized on the molecular level. Structural and functional studies suggested that E. multilocularis expresses both a TGFβ and a BMP signaling system. The kinase EmRSK2 and the Smad factor EmSmadC are most probably components of the first, EmRSK1 and EmSmadB parts of the latter system. Surprisingly, EmSmadA seems to constitute an unusual Smad since it can be activated by both TGFβ and the BMP receptors upon expression in mammalian cells. The precise roles of EmRSK3 and EmTAK1 have to be determined in future studies. In the present work, significant structural and functional homologies between the TGFβ/BMP systems of E. multilocularis and its mammalian hosts have been detected. Upon expression in human cells, the Echinococcus Smad proteins were, for example, able to functionally interact with the corresponding receptors from Homo sapiens. On the other hand, the E. multilocularis TGFβ/BMP signaling factors also displayed several biochemical differences to those of the host, which could be exploited for the development of antiparasitic drugs. One of these differences is the lack of a usually conserved MH1 domain in EmSmadA and EmSmadC. Moreover, the Echinococcus TGFβ/BMP receptors display several structural features which have not yet been detected in other members of the protein superfamily. Likewise, the regulation of TGFβ/BMP pathways in Echinococcus as well as their cross-interaction with other signaling pathways (e.g. the MAP kinase cascade) seems to differ from the situation in vertebrates, insects and nematodes. Finally, this work also provides evidence that at least one host cytokine, BMP-2, can functionally interact with a receptor of the parasite, EmRSK1. This interaction could be highly relevant for host-parasite interaction mechanisms in alveolar echinococcosis since BMP-2 also exerts clear effects on Echinococcus growth and differentiation in an in vitro cultivation system that mimicks the situation at the affected organ during an infection. KW - Fuchsbandwurm KW - Ontogenie KW - Signaltransduktion KW - Echinococcus KW - TGFß KW - BMP KW - Smad KW - Zestode KW - Echinococcus KW - TGFß KW - BMP KW - Smad KW - cestode Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-17755 ER - TY - JOUR A1 - Brehm, Klaus A1 - Hemer, Sarah A1 - Konrad, Christian A1 - Spiliotis, Markus A1 - Koziol, Uriel A1 - Schaack, Dominik A1 - Förster, Sabine A1 - Gelmedin, Verena A1 - Stadelmann, Britta A1 - Dandekar, Thomas A1 - Hemphill, Andrew T1 - Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development N2 - Background The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host’s liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood. Results Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite’s glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin. Conclusions Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs. KW - Cestode KW - Tapeworm KW - Echinococcus KW - Echinococcosis KW - Insulin KW - Receptor kinase KW - Kinase inhibitor KW - Host-parasite interaction Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110357 ER - TY - JOUR A1 - Brehm, Klaus A1 - Koziol, Uriel A1 - Krohne, Georg T1 - Anatomy and development of the larval nervous system in Echinococcus multilocularis JF - Frontiers in Zoology N2 - Background The metacestode larva of Echinococcus multilocularis (Cestoda: Taeniidae) develops in the liver of intermediate hosts (typically rodents, or accidentally in humans) as a labyrinth of interconnected cysts that infiltrate the host tissue, causing the disease alveolar echinococcosis. Within the cysts, protoscoleces (the infective stage for the definitive canid host) arise by asexual multiplication. These consist of a scolex similar to that of the adult, invaginated within a small posterior body. Despite the importance of alveolar echinococcosis for human health, relatively little is known about the basic biology, anatomy and development of E. multilocularis larvae, particularly with regard to their nervous system. Results We describe the existence of a subtegumental nerve net in the metacestode cysts, which is immunoreactive for acetylated tubulin-α and contains small populations of nerve cells that are labeled by antibodies raised against several invertebrate neuropeptides. However, no evidence was found for the existence of cholinergic or serotoninergic elements in the cyst wall. Muscle fibers occur without any specific arrangement in the subtegumental layer, and accumulate during the invaginations of the cyst wall that form brood capsules, where protoscoleces develop. The nervous system of the protoscolex develops independently of that of the metacestode cyst, with an antero-posterior developmental gradient. The combination of antibodies against several nervous system markers resulted in a detailed description of the protoscolex nervous system, which is remarkably complex and already similar to that of the adult worm. Conclusions We provide evidence for the first time of the existence of a nervous system in the metacestode cyst wall, which is remarkable given the lack of motility of this larval stage, and the lack of serotoninergic and cholinergic elements. We propose that it could function as a neuroendocrine system, derived from the nervous system present in the bladder tissue of other taeniids. The detailed description of the development and anatomy of the protoscolex neuromuscular system is a necessary first step toward the understanding of the developmental mechanisms operating in these peculiar larval stages. KW - Echinococcus KW - Metacestode KW - Protoscolex KW - Nervous system KW - Neuropeptide KW - Serotonin KW - Acetylated tubulin Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96504 UR - http://www.frontiersinzoology.com/content/10/1/24 ER -