“To be or not to be” the importance and applications of EmPGI in alveolar echinococcosis

The parasite Echinococcus multilocularis, grows as a small tapeworm in final hosts (foxes mainly). Parasite eggs, released into the environment within faeces of final hosts, can infect intermediate hosts, when taken up orally. Consequently, multivesicular and infiltrative metacestodes can grow and cause the disease alveolar echinococcosis (AE). Apart from natural intermediate hosts, such as small rodents, also humans can accidentally ingest infectious eggs and acquire AE. Overall, human AE is a rare disease, but there are several highly endemic focuses described and incidences have been increasing drastically recently. Therefore, human AE is regarded as an emerging disease. In addition, the consequences of acquiring AE are very serious: if untreated, the disease is fatal in > 75 % of cases. To be… The manifestation of AE takes more than 10-15 years, meaning that - in order “to be” - the parasite can reside, grow and spread within its intermediate host without being actively noticed by the patient – similar to a malignant tumor. Thus, the parasite is thought to apply survival-strategies and to modulate the host’s physiology and immune response. Two parts of E. multilocularis metacestodes are thought to play major roles in this modulation of the host: laminated layer (LL) and excretory / secretory (E/S) products, two components that are in direct contact with the host environment. Only little is known concerning composition or detailed function of LL and E/S products. Within this thesis, attention has been given to E/S products, and especially to LL, with regards to their role in host-parasite interaction. The LL is a heavily glycosylated, acellular layer that surrounds the entire metacestode like a glycocalyx. It builds up the direct host-parasite interface and some fractions of the LL were described to be important in the host-parasite interaction. Almost nothing is known with regards to protein composition of the LL. The only described isolation method for LL proteins does not result in satisfying protein resolution by SDS-PAGE. To get a better view on LL proteins, several new methods for their isolation, basically based on removal of carbohydrates from metacestode tissue, are described within this thesis. One of the proteins identified to be associated with purified LL, is the enzyme phosphoglucose isomerase (PGI). PGI, well-known to be an enzyme of glycolysis, exhibits several extracellular, moonlighting functions in mammalian cells. It is described as a cytokine and growth factor and suggested to play, among others, roles in tumor invasion, metastasis formation and angiogenesis induction. Due to parallels in the growth characteristics of tumors and E. multilocularis metacestodes and due to the extracellular localization of E. multilocularis PGI (EmPGI), particular attention was given to this protein with respect to a potential role of a moonlighting protein in the host-parasite interplay of AE. 2 ABSTRACT EmPGI was cloned, sequenced and functionally expressed in E. coli. It was localized in vesicle fluid, different LL extracts and E. multilocularis cells. Experimentally infected mice showed a clear humoral immune response against recEmPGI. However, release of EmPGI into the metacestode environment in vitro was shown for damaged vesicles only. Investigation of host-modulating roles of proteins from LL and E/S products revealed that the parasite vesicle fluid (VF) induces a proliferative response in vitro in bovine adrenal cortex endothelial cells (ACEs) and Reuber rat hepatocytes, as well as in germinal layer cells (forming the living parasite vesicle tissue). PGI, as part of VF and LL of the E. multilocularis metacestode and as a moonlighting protein of mammalian cells, was shown to enhance selectively the proliferation of ACEs and, to a lesser extent, also of germinal layer cells. For these reasons, EmPGI is discussed as a potential moonlighting protein in AE that, as a pathogenicity factor, could influence the host-parasite interplay in favor of the parasite. …or not to be The same enzyme shown to be enzymatically active within E. multilocularis metacestodes and possibly to be involved in the host-parasite interaction in AE, can be exploited as a marker for drug screening against AE. Despite fatal consequences of AE, no chemotherapeutical treatment option acting parasitocidal - and thereby making the parasite “not to be” - is available. To date there has been no reliable, cheap and easy-to-perform in vitro drug assay, which would have allowed the efficient discovery of new potential drug candidates. Within this thesis, a combination of the recently described in vitro large-scale cultivation of metacestode vesicles and the detection of EmPGI activity released from damaged vesicles in vitro is applied as a new screening option. By the assay, several compounds and their derivatives, namely thiazolides and the antimalarials artemisinin, pentamidine and mefloquine, were tested and compared concerning their in vitro efficiency against metacestodes. For thiazolides, a more detailed analysis of the structure-function relationship was performed. It was shown that, in contrary to activity against bacteria or Giardia lamblia, not only nitro-thiazolides, but also halogenated molecules were strong inducers of EmPGI-release and that tri-bromothiazolides, such as Rm4841, in particular, exhibited a very strong activity against metacestodes. Among the anti-malarials, especially mefloquine, due to its approval and low toxicity to host cells, was proposed as a potential new drug against AE