Institute of Specific Prophylaxis and Tropical Medicine, Medical University Vienna, Kinderspitalgasse 15, A-1090 Vienna |
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MOLEKULARE PARASITOLOGIE Univ.-Doz. Mag. Dr. Julia Walochnik A d r e s s e: Kinderspitalgasse 15, A-1090 Wien Tel: +43-1-40 490-79446 Fax.: +43-1-40 490-79435 Email: julia.walochnik@meduniwien.ac.at
FORSCHUNG: Classification and phylogeny of free-living amoebae The free-living amoebae are ubiquitously occurring protozoans that feed on bacteria, algae and yeasts. They are widely distributed in terrestrial and aquatic habitats and have as a common feature that they are mitochondria bearing eukaryotic unicellular microorganisms that produce pseudopodia (literally “false feet”) protruding outward from the periphery of the cell serving feeding and locomotion. Locomotion is performed by directed cytoplasmic streaming and by rolling motion. Ingestion is linked very tightly to locomotion and represents a typical phagocytosis, which is generated by an exterior stimulus, cell-cell contact playing a major role. Most genera of the free-living amoebae are characterised by a biphasic life cycle consisting of a vegetative trophozoite stage and a persistant, physiologically static cyst stage enabling them to withstand adverse environmental conditions. Some genera show an additional flagellated dispersal form. The free-living amoebae are highly abundant in the natural environment and serve significant roles at the base of food webs. Moreover, they help to maintain the fertility of soils by making nutrients available to plants. Bacteria and fungi grazing amoebae are also of importance as biocontrol agents in both agriculture and forestry. In addition to their important ecological functions, various representatives of different genera of the free-living amoebae have been implicated in human disease, however identification of species confronts with veritable difficulties and the classification of the free-living amoebae is still unresolved. The classification of the free-living amoebae is a most contentious area and has been revised many times. The amoebae are asexually propagating organisms. This implies a general discrepancy in species determination, as the biological species is defined as a group of interbreeding individuals that are reproductively isolated from other organisms and is therefore not applicable on free-living amoebae. The free-living amoebae subsummized under the taxon Rhizopoda represent an eclectic assemblage of unicellular eukaryotes that use pseudopodia for locomotion and feed by phagocytosis. It is now generally recognized that the Rhizopoda are all but a monophylum and include highly diverse groups that have evolved along many different lines. The presence of an amoeboid habit undoubtedly represents a convergent evolution and is no longer considered to be a conservative feature. Because of the lack of fossil records in most protozoan groups including the free-living amoebae the reconstruction of phylogeny has to be achieved by comparing all available morphological, biochemical, and above all molecular biological data of recent organisms and by searching for homologies. Julia Walochnik Susanne Glöckl Jacek Pietrzak COOPERATIONS Dr. Rolf Michel, Ernst-Rodenwaldt-Institut, Koblenz, Germany Encystment and excystment in Acanthamoeba castellanii: RNA and protein expression in the differentiating cell Differentiation processes such as the encystment are common in numerous eukaryotic single cell organisms of which many are causative agents of human infections. The cyst enables these microorganisms to endure adverse environmental conditions and thus holds up the infectious cycle. In the ubiquitous genus Acanthamoeba, which comprises potential pathogens causing Acanthamoeba keratitis and granulomatous amoebic encephalitis, cysts are formed in response to unfavourable environmental conditions including also biocide treatment. Cysts surviving treatment lead to a more dramatic course of disease or to recurrent infection and are also important for the vector role of acanthamoebae. Although Acanthamoeba castellanii has, due to its rapid growth and easily inducible synchronous cell differentiation, been serving as a model organism for studies on a variety of problems in cell biology for many decades, the molecular mechanisms of Acanthamoeba encystment have not yet been elucidated. However, the ability to form cysts is not only crucial for the role of acanthamoebae as active and passive pathogens, but is of essential clinical and ecological importance also in other pathogenic protozoa. Thus, a deeper insight into this complex process is of extensive interest. It is the aim of our project to apply contemporary molecular biological methodology to study the encystment in A. castellanii and establish a model for protozoan cell differentiation. Proteome analysis with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and subsequent genetic analysis will be applied to elucidate the genetic and physiological key players involved in the encystment and excystment processes of this organism. First, proteins that are strongly expressed during differentiation will be isolated from 2D-gels, identified and checked for regulatory sequences on the genetic level. With regulatory sequences at hand, the A. castellanii genome will be searched for other differentiation specific genes, including low copy proteins such as transcriptional activators and repressors. This reverse genetic approach will provide us with data on a large number of structural and regulatory genes, thereby allowing the elucidation of A. castellanii differentiation. The Acanthamoeba model will include the temporal and hierarchical order of expressed genes that range from the induced early cyst to the mature cyst and will be the basis for comparative studies including other cyst forming protozoan pathogens as Entamoeba histolytica and Giardia lamblia. Moreover, the influence of bacterial endocytobionts on Acanthamoeba castellanii encystment will be assessed by RNA-profiling experiments based on the knowledge of the genes involved in differentiation. Julia Walochnik Martina Köhsler David Leitsch COOPERATIONS Univ.-Prof. Dr. Michael Duchêne, Medical University of Vienna: Department of Specific Prophylaxis and Tropical Medicine Univ.-Prof. Dr. Günther Allmaier, Technical University of Vienna: Department of Analytical Chemistry Univ.-Prof. Dr. Matthias Horn, University of Vienna: Department of Microbial Ecology this project is supported by the FWF Identification of pathogenicity related proteins in Acanthamoeba spp. Acanthamoebae are highly abundant in natural as well as in man-made habitats, however, clinical manifestations of Acanthamoeba infections are rare. Although Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE) are highly divergent with respect to their immunobiologies, it has been shown that in both infections host-parasite interactions play a crucial role for the establishment of an infection. Yet the immunobiology of Acanthamoeba infections is still poorly understood. In the current study we are focussing on proteins involved in the establishment of an Acanthamoeba infection, comparing virulent and non-virulent strains on one hand and AK and GAE causing strains on the other. Julia Walochnik Wilawan Pumidonming this project is supported by the ÖAD Anti-Protozoal and Anti-Fungal Activity of Plant Derived Substances Protozoans are the cause of widespread morbidity and mortality in many countries of the world, including diseases such as malaria, sleeping sickness, Chagas disease, amoebosis and leishmaniosis. Malaria alone causes more than a million deaths and several hundred million cases of severe disease each year. Unlike in several other diseases there are still no vaccines on the market for any disease caused by protozoans, and very often anti-protozoal agents are rather old drugs associated with severe side-effects and/or problems of resistance. This project aims to find new antiprotozoal natural compounds derived from higher plants, focussing on bioactive substances occuring in tropical plant families. Five different bioactive substances will be isolated and purified and tested in vitro against different pathogenic protozoa and fungi. All five substances have already shown anti-protozoal activity in preliminary tests against Plasmodium falciparum, the causative agent of malaria, and Entamoeba histolytica, the causative agent of amoebic dysentery or liver abscess. Moreover, preliminary experiments have been performed against plant-pathogenic species like Pyricularia grisea, Fusarium spp., Botrytis cinerea, Pestalotiopsis spp. and Colletotrichum gloeosporioides. In the first instance toxicity assays will be performed and substances will be screened for potential genotoxicity. Subsequently, in vitro assays in 96-well plates will be developed in order to perform high throughput analysis and substances will be tested against Entamoeba histolytica, Giardia intestinalis, Leishmania donovani, Leishmania infantum, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trichomonas vaginalis and Trypanosoma cruzi. Julia Walochnik Florian Astelbauer COOPERATIONS Univ.-Prof. Dr. Michael Duchêne, Medical University of Vienna: Department of Specific Prophylaxis and Tropical Medicine Univ.-Prof. Dr. Harald Greger, University of Vienna: Department of Phytochemistry Dr. Andreas Obwaller, Orphanidis Research GmbH this project is supported by the FFG |
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