The proteome of the coccidian oocyst wall
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NO FULL TEXT AVAILABLE. This thesis contains 3rd party copyright material. ----- The Coccidia comprise pathogens including Eimeria, which causes losses of one billion dollars annually to the poultry meat industry, and the human pathogen, Toxoplasma, causing abortion, congenital defects and encephalitis. Their pathogenesis is enhanced by the highly resilient dityrosine-crosslinked oocyst lifecycle stage, which remains infective in the environment for months. Despite the importance of the oocyst wall and its effectiveness as a vaccine target in Eimeria, little is known about its composition. The aim of this proteomics study was to use Eimeria tenella to gain an understanding of the coccidian oocyst wall. Oocyst walls were isolated to high purity as assessed by electron microscopy. Solubilisation for SDS-PAGE separation was difficult, but optimised with lauryldimethylamine-oxide in 6M guanadinium with heat after reduction and alkylation. MALDI-MS and ESI-MS were compared before a final, successful approach was identified. An SDS-PAGE gel was dissected before ESI-MS analysis using an LTQ ion trap instrument. Preliminary E. tenella predicted protein databases were used to identify 610 proteins, 50% of which were determined to be non-wall contaminants by PSI-BLAST analysis. To reduce contaminants and improve identification of potential oocyst wall proteins, a detergent wash, subtractive analysis was performed where the purified oocyst walls were washed with various detergents before solubilisation and ESI-IT analysis. Four samples were analysed, a no-wash (610 proteins identified) Triton X-100 was (557 proteins idenitified), 0.01% SDS wash (335 proteins identified) and 0.1% SDS wash (581 proteins identified). A total of 1098 different proteins were identified. Comparison of proteins identified from this subtractive anlaysis led to the identification of potential oocyst wall proteins including a high proportion without homology to any known proteins. To help identify these, a database containing known wall and structural protein motifs and amino acid characteristics was created and searched. Initial immunolocalisation studies were performed on likely oocyst wall proteins. Three proteins E. tenella cysteine modular repeat protein (EtCMRP), collagen and ALSC rich protein, were localised to the oocyst wall. This validates the study and along with the other coccidian conserved, proline and leucine rich proteins identified increase understanding of the nature of the oocyst wall. This study was able to construct effective approaches for oocyst wall purification, solubilisation and MS analysis despite the limited sample, highly resilient dityrosine crosslinking and very preliminary databases available and went on to identify novel oocyst wall proteins highly conserved across the Coccidia.
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