The spatial proteome of Eimeria tenella provides a high-resolution view of proteins localised to key invasion organelles

Eimeria tenella is one of the most prevalent species of Eimeria that infects chickens causing coccidiosis, an enteric disease estimated to cost the poultry industry over £10.4 billion globally per year. The E. tenella genome contains ∼7,268 protein-coding genes, almost half of which are annotated as hypothetical with no known function. In this study we investigated the localisation of E. tenella sporozite proteins using Hyperplexed Localisation of Organelle Proteins by Isotope Tagging (hyperLOPIT) combined with Bayesian machine learning. We assigned 722 identified proteins with previously unknown localisation to one of 12 subcellular niches at ≥0.99 confidence, substantially expanding the known proteome of this invasive parasite stage. These included 49 and 70 proteins assigned to the invasion-related microneme and rhoptry organelles respectively. Importantly, we observed distinct separation of previously described surface antigen (SAG) A and B proteins into separate subcellular niches including one (SAGB) within the micronemes, suggestive of functional divergence between these two SAG sub-families and highlighting a potential mechanism contributing to virulence. Furthermore, our spatial map provides vital functional context for hypothetical proteins, notably assigning a highly conserved ortholog of the T. gondii SPATR (secreted protein with an altered thrombospondin repeat) to the microneme, supporting its shared role in apicomplexan host cell invasion. This spatial proteome provides a valuable resource to the research community, revealing complex evolutionary divergences of key protein subfamilies. These create opportunities to further our understanding of parasite pathogenicity, identify vaccine candidates and enable comparative evolutionary and biological analyses across diverse apicomplexan parasites that impact human, livestock and wider animal welfare.