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

Sharman, Philippa Anne (2013) Identification of enzymes potentially involved in the formation of the oocyst wall of coccidian parasites. PhD thesis, James Cook University.

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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 coding for oxioreductases. Quantitative RT-PCR revealed that the relative transcript abundance of three of these, amine oxidase 2, glutathione peroxidase and an oxioreductase, was increased dramatically in gametocytes. All three proteins could be detected specifically in macrogametocytes within infected caeca of chickens, displaying a punctate distribution in the cytoplasm of the parasite. Thus, this thesis identifies candidates for two putative enzymatic steps in the generation of chemical bonds that are crucial for the formation of oocyst walls and, therefore, the completion of the lifecycle of coccidian parasites. These enzymes are potential targets for novel parasite control strategies.

Item ID: 40602
Item Type: Thesis (PhD)
Keywords: Apicomplexa; coccidia; coccidian; dityrosine; dityrosine crosslinking; Eimeria; gametocyte; infectious agents; infectious diseases; macrogamete; oocyst; oocyst wall; oxioreductases; parasites; protease; protease inhibitors; proteins; proteolytic enzymes; protozoan; serine proteinases; toxoplasma; tyrosine; vaccines; veterinary parasitology; wall-forming bodies
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For this thesis, Philippa Sharman received the Dean's Award for Excellence 2014.

Publications arising from this thesis are available from the Related URLs field. The publications are:

Chapter 1: Sharman, P.A., Smith, N.C., Wallach, M.G., and Katrib, M. (2010) Chasing the golden egg: vaccination against poultry coccidiosis. Parasite Immunology, 32 (8). pp. 590-598. (This paper constitutes a large proportion (60%) of Chapter 1 of this thesis.)

Katrib, Marilyn, Ikin, Rowan J., Brossier, Fabien, Robinson, Michelle, Slapetova, Iveta, Sharman, Philippa A., Walker, Robert A., Belli, Sabina I., Tomley, Fiona M., and Smith, Nicholas C. (2012) Stage-specific expression of protease genes in the apicomplexan parasite, Eimeria tenella. BMC Genomics, 13 (685). pp. 1-18.

Mai, Kelly, Sharman, Philippa A., Walker, Robert A., Katrib, Marilyn, De Souza, David, McConville, Malcolm J., Wallach, Michael G., Belli , Sabina I., Ferguson, David J.P., and Smith, Nicholas C. (2009) Oocyst wall formation and composition in coccidian parasites. Memorias do Instituto Oswaldo Cruz , 104 (2). pp. 281-289.

Walker, Robert A., Sharman, Philippa A., Miller, Catherine M., Lippuner, Christoph, Okoniewski, Michal, Eichenberger, Ramon M., Ramakrishnan, Chandra, Brossier, Fabien, Deplazes, Peter, Hehl, Adrian B., and Smith, Nicholas C. (2015) RNA Seq analysis of the Eimeria tenella gametocyte transcriptome reveals clues about the molecular basis for sexual reproduction and oocyst biogenesis. BMC Genomics, 16. pp. 1-20.

Date Deposited: 01 Oct 2015 02:49
FoR Codes: 07 AGRICULTURAL AND VETERINARY SCIENCES > 0707 Veterinary Sciences > 070708 Veterinary Parasitology @ 50%
06 BIOLOGICAL SCIENCES > 0605 Microbiology > 060502 Infectious Agents @ 50%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 50%
92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920109 Infectious Diseases @ 50%
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