The characterisation of selected molecules expressed exclusively in the sexual stages of Eimeria tenella and Eimeria maxima
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NO FULL TEXT AVAILABLE. This thesis contains 3rd party copyright material. ----- The highly infectious nature of avian coccidiosis is attributed primarily to the durability of the excreted oocyst from the causative parasite, Eimeria. Despite the known importance of this life-cycle stage, little is known about the molecular basis of sexual stage development, a process that precedes oocyst formation. It was the aim of this study to identify and characterise genes and/ or proteins that are expressed in sexual stages of Eimeria tenella and Eimeria maxima (two of the most economically important species) with an underlying goal of identifying novel transmission blocking targets for controlling coccidiosis. The first strategy focussed on identifying eimerian proteins showing homology to known gametocyte proteins of related, cyst-forming Apicomplexa. The Cryptosporidium oocyst wall proteins (COWPs) are a group of cysteine-rich, oocyst wall proteins initially expressed in gametocyte stages. COWP6 was shown to have a putative orthologue in E. tenella (EtOWP6) and a more divergent homologue in E. maxima (EmOWPx). In addition to observed sequence similarities to COWP6, EtOWP6 and EmOWPx both displayed similar sexual stage-exclusive gene and protein expression. EtOWP6, in particular, showed strong gametocyte-exclusivity and localised to wall-forming bodies (putatively, type I), organelles responsible for the development of the outer oocyst wall in Eimeria. In the second strategy, a high-throughput BLAST analysis was performed on proteins identified exclusively in either male gametes or female gametes of Plasmodium berghei (Khan et al., 2005) in order to identify microgamete- and macro gamete-exclusive genes/proteins in E. tenella. Although a large number of putative microgamete- and macrogamete-exclusive E. tenella genes were identified, only a small proportion of these genes displayed gametocyte-biased transcription. Further analysis of protein expression in a number of promising candidates lead to the identification of EtGFAT, a macrogamete-exclusive glucosamine: fructose 6-phosphate aminotransferase. This protein is the first enzyme in the glycosylation pathway and was localised to E. tenella macrogametes. Furthermore, E. tenella genes coding for other enzymes in the glycosylation pathway also displayed gametocyte-exclusive transcription. The apparent upregulation of glycosylation in gametocytes correlated with increased levels of N acetylglucosamine and, in particular, N-acetylgalactosamine, the two potential products of glycosylation. The identification and partial characterisation of a gametocyteexclusive glycosylation pathway in this study is consistent with the known role of gametocyte-exclusive glycoproteins, Gam56 and Gam82, in the formation of the oocyst wall in E. maxima. The findings of this study have expanded the knowledge of Eimeria sexual stage development. Future work should be directed to (1) characterising the exact function of EtOWP6 and related cysteine-rich proteins, particularly in relation to oocyst wall formation and (2) defining the mechanisms and triggers of gametocyte-exclusive glycosylation in Eimeria.
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