Finding the hidden genes in Leishmania guyanensis genome: Application of proteogenomics approach to improve the genome annotation in the kinetoplastid parasite.

The kinetoplastid protozoan parasite Leishmania guyanensis a member of the Leishmania (Viannia) species complex is the causative agent of American tegumentary leishmaniasis (ATL). L. guyanensis infections typically result in cutaneous lesions, but in some cases can progress to mucocutaneous leishmaniasis, particularly when co-infected with Leishmania RNA virus 1 (LRV1), thus leading to exacerbated inflammation, treatment failure, and relapse. Despite the availability of its reference genome, limitations in gene annotation persist due to the presence of hypothetical proteins, pseudogenes, and unrecognised coding regions. In this study, we employed a proteogenomic approach integrating publicly available high-resolution mass spectrometry data with a custom six-frame translated genome database to refine the genome annotation of L. guyanensis strain MHOM/BR/75/M4147. Utilising stringent database-dependent searches with a 1% false discovery rate, we identified numerous unique peptides, of which 653 were genome search-specific peptides (GSSPs) mapping exclusively to unannotated genomic regions. These GSSPs facilitated the discovery of 65 novel protein-coding genes and the correction of 62 existing gene models, including N- and C-terminal extensions. Our findings demonstrate the power of proteogenomics to uncover cryptic protein-coding regions and improve genome annotations beyond conventional predictions. This refined annotation enhances our understanding of L. guyanensis biology, providing a more accurate proteomic landscape that can inform studies on parasite virulence, host interaction, and potential therapeutic targets. The study underscores the importance of integrating proteomic evidence with genomic data to capture the full coding potential of kinetoplastid parasites, paving the way for improved diagnostics and interventions against leishmaniasis.