Improving Genome Annotation of the Animal Trypanosomiasis (surra) Pathogen Trypanosoma evansi Using the Proteogenomics Approach.

Abstract

BACKGROUND: Trypanosoma evansi, the causative agent of surra, remains a significant threat to livestock productivity across Africa, Asia, and Latin America. Despite the availability of a reference genome sequence since 2015, annotation of this pathogen remains incomplete, particularly in repetitive subtelomeric regions containing variant surface glycoprotein (VSG) genes and expression site-associated genes (ESAGs). METHODS: To address these limitations, we utilised an integrated proteogenomic approach combining publicly available high-resolution mass spectrometry (LC-MS/MS) data with a custom six-frame translated genome database for the T. evansi STIB 805 strain. Analysis of three independent proteomics datasets yielded 39,025 unique peptides, of which 2008 mapped exclusively to the translated genome database rather than the annotated proteome. RESULTS: These genome-search-specific peptides (GSSPs) provided experimental evidence for previously uncharacterized genomic features and identified 24 novel protein-coding genes located in intergenic regions, including a transferrin-binding protein ortholog essential for nutrient acquisition and a ribosomal protein S14p critical for translation. We discovered 30N-terminal extensions that refine existing gene models, including the purine salvage enzyme inosine monophosphate dehydrogenase, a promising drug target. Most significantly, we provided the first biochemical evidence for translation of 182 regions previously classified as pseudogenes, including VSG proteins that contribute to antigenic variation and immune evasion. CONCLUSIONS: These findings demonstrate that the T. evansi pseudogene archive represents a dynamic, functional component of the parasite's proteome rather than evolutionary relics. This genome annotation improves our understanding of T. evansi biology and provides a valuable resource and empirical evidence to guide future studies aimed at developing improved diagnostics and potential therapeutics against surra.

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