Structural studies of surface antigen proteins of Eimeria tenella and related Apicomplexan parasites

Apicomplexa comprises a diverse group of parasitic organisms responsible for severe diseases in humans and livestock. Toxoplasmosis, caused by Toxoplasma gondii, poses risks to pregnant women, a problem with animals such as sheep and immunocompromised individuals and Malaria, caused by Plasmodium spp., remains a global health challenge with complex transmission dynamics and widespread drug resistance. Chicken coccidiosis, caused by Eimeria spp., leads to substantial economic losses in the poultry industry. Eimeria parasites exhibit distinct preferences for infecting specific sections of the chicken gut, mediated by proteins from micronemes protein, rhoptries proteins and surface antigens (SAGs) proteins of the CAP-like superfamily that are involved in host-parasite interactions and pathogenicity. Surface antigen proteins that belong to the separate SRS family in Toxoplasma and Plasmodium parasites play similar critical roles in invasion, immune evasion, and virulence. Understanding the diversity and functions of these surface antigen proteins may pave the way for novel intervention strategies against apicomplexan diseases.

This thesis presents the design and evaluation of a construct for the efficient production of large quantities of soluble CAP-like SAG proteins, based on a cleavable thioredoxin solubility tag, optimised codon usage and structure based identification of the core SAG domain boundaries, resulting in the successful expression of twenty-two CAP-like SAG proteins. The structures of six representative SAG proteins from the three Eimeria SAG subfamilies were determined by X-ray crystallography, with each having the same core single domain  sandwich structure, containing two or three disulphide bonds and a conserved buried arginine in an NxxR motif. Despite the similarity in secondary structure, clear differences in the length, position and sequence of the connecting loops results in a wide diversity of surface shape, charge and functional groups, indicating that this family of CAP-like SAG proteins are unlikely to share a common binding partner. Genomic analysis revealed that CAP-like SAG genes are present in other apicomplexan parasite genomes that contain multiple SRS-like SAG proteins such as Toxoplasma gondii, Neospora caninum, Besnoitia besnoiti and Cystoisospora suis, with Plasmodium spp., Babesia bovis and Theileria orientalis having just a single CAP-like SAG gene. The structure of the P. vivax CAP-like SAG was determined revealing the same core, single domain structure, with sequence analysis across these parasites indicating that some parasites have examples of genes with multiple CAP-like SAG domains. Transcriptome analysis showed no consistent expression of this protein family in different parasite life stages, possibly suggesting that the primary function of some, or perhaps all, of these proteins is more likely to be associated with the stimulation of the immune system as a response to parasite invasion. In conclusion, this thesis contributes to our understanding of the structural and functional diversity of CAP-like SAGs across apicomplexan parasites, paving the way for future studies aimed at elucidating their roles in parasite biology and exploring their potential as therapeutic targets or vaccine candidates.