Identification of enzymes potentially involved in the formation of the oocyst wall of coccidian parasites

The Coccidia are a group of single-celled, protozoan parasites within the phylum Apicomplexa. Adaptable and widely distributed, they are able to reproduce asexually and sexually within their host, causing serious diseases in poultry, livestock, pets, wildlife and people all over the world. One of the defining features of the group is the oocyst, which is the end-point of sexual reproduction and is pivotal for transmission of these parasites. Oocysts are surrounded by a wall, which has a chemical structure that enables them to persist for long periods in the environment until ingested by new hosts. The resilience of the oocyst wall has been attributed to the formation of dityrosine cross-links between tyrosinerich proteins that originate in the sexual stages (gametocytes) of the parasite. These tyrosine-rich proteins are processed into smaller proteins, then undergo oxidation to form the dityrosine bonds as they are incorporated into the oocyt wall matrix. The hypotheses investigated in this study were that: processing of tyrosine-rich proteins is via proteases; and catalysis of dityrosine crosslinking is via oxioreductases. The aim of the thesis was to identify proteases and oxioreductases that are expressed specifically in gametocytes as they transform into oocysts. The poultry coccidian, Eimeria tenella, was chosen as the model for this study because of the availability of a genome database and reliable methods to purify gametocytes and oocysts of this parasite. Bioinformatic analyses identified 45 potential proteases in the E. tenella genome. However, expression of only three subtilisin-like serine proteases was up-regulated in gametocytes, as revealed by qualitative and quantitative RT-PCR. Expression of one, EtSUB2, was specific to the sexual phase of development and localised within the cytoplasm of macrogametocytes, adjacent to the wall forming bodies that store tyrosine-rich proteins prior to their incorporation into the oocyst wall. Data mining of the E. tenella database also identified five genes ...