The goal of this thesis is to shed more light on the oocyst stage of Toxoplasma gondii regarding several key aspects such as possible new molecules in the oocyst wall that can be harnessed for ligand-based methods for detection, molecules inside the oocyst that contribute to the increased stress resistance and molecules that can be used in serological detection methods. To this end, the project is split into three parts. The first part aims to lay the groundwork for establishment of new methods for oocyst enrichment in environmental samples by identifying new molecules binding to the oocyst wall. As a basis for further experiments, a method for robust immunofluorescence experiments using only small numbers of oocysts is developed. Using this method, molecules binding to the oocyst wall can be identified and their binding characteristics further analyzed. The generation of nanobodies targeting the oocyst wall is explored. Also, members from the Dectin-1 CLR cluster are investigated with special focus on their binding to the oocyst wall. Results from these experiments will also contribute to new insights into the molecular composition of the oocyst wall. In the second part, factors contributing to the oocysts resilience to environmental stress are investigated. Oocysts are known to stay viable in the environment under varying conditions, relatively untouched by environmental factors. This is often attributed to the rigidity of the bilayered walls that protect the oocyst and the sporocysts. However, the presence of so-called LEA proteins inside the oocyst that contribute to protection against damage induced by certain stress factors has been hypothesized. These LEA proteins are known to confer stress resistance in plants and invertebrates through their disordered structure. Through in silico, in vitro and in vivo characterization of the TgLEAs, more in depth knowledge will be acquired, collecting predictions and findings regarding their IDP characteristics, their biochemical properties as well as their effect on bacterial growth upon several stresses. The in vitro analyses will investigate the LEA protein’s potential to protect an important T. gondii pathogenesis factor from stress induced damage. The findings from these experiments will lead to a better understanding of oocyst physiology as well as opening up more possibilities to develop methods to eradicate oocysts from the environment in a targeted and more efficient manner and prevent parasite spread. Lastly, this thesis analyzes the oocyst specific TgLEAs regarding their putative role as antigens that can elicit a specific antibody response, serving as infection marker. Such a serological tool would be highly beneficial in early identification of infection clusters and efficient mounting of counter measures to prevent further spread. First, antibodies against two most promising LEA proteins will be generated by immunization of two rabbits to serve as additional controls in future experiments as well as proof of basic antigenicity of TgLEAs. Subsequently, the Luminex technology will be shortly introduced and adapted to allow feasible analyses of large numbers of chicken sera for T. gondii infections. ELISA tests will assess the TgLEAs’ antigenic potential by analysis of sera from experimentally infected chickens. Lastly, the Luminex technology will be employed to corroborate the ELISA findings and investigate on a large-scale different serum dilutions to confirm if TgLEAs are suitable antigens to differentiate different T. gondii infection routes, i.e. tachyzoite, bradyzoite or oocyst. In summary, this project will expand the knowledge on molecules (1) binding to the oocyst wall, (2) contributing to stress tolerance inside the oocyst and (3) as antigens for identification of oocyst-mediated infections.
