Mapping the Hidden Secretome in Leishmania Parasites Using a Proteogenomics Approach

Introduction Leishmaniasis affects millions of people worldwide and is caused by obligate intracellular protozoan parasites transmitted through infected phlebotomine sand flies. Clinical outcomes range from self-healing cutaneous lesions to fatal visceral disease across twenty recognised human-pathogenic species. Although Leishmania genomes are remarkably conserved, substantial phenotypic diversity arises through post-transcriptional and post-translational regulatory mechanisms, highlighting the essential role of proteomic investigations. The Leishmania secretome contains virulence factors crucial for establishing infection, modulating host immunity, and promoting parasite survival within macrophages. Previous proteomic studies have characterised secreted proteins across multiple species, identifying a conserved core secretome predominantly released via extracellular vesicles through non-classical pathways. However, large portions of the predicted proteome remain experimentally unvalidated. Materials and Methods To uncover overlooked proteins, we conducted a comprehensive proteogenomic analysis using publicly available mass spectrometry datasets. Custom six-frame translated genome databases were generated for seven reference Leishmania species, enabling the identification of genome search-specific peptides. This approach helps to detect the novel secreted proteins that have been missed in previous secretome researches together with low-abundance features, hypothetical proteins and the proteins that are from the canonical translation procedures. Results This proteogenomic study identified 469 secreted proteins in the seven Leishmania species that were not reported previously. The proteins were significantly explored and validated in the Leishmania proteome and disclose a higher level of functional diversity. The visceralising species were abundant in ATP-binding proteins and oxidoreductases, while the cutaneous species were represented with proteasome-rich secretome profiles. Discussion The identification of these further secreted proteins highlights the boundaries of previous studies and emphasises the higher resolution provided by the proteogenomic approaches. The species-specific categorical and functional differences explored in this study can contribute to diverse tissue tropisms, host-pathogen interactions and the clinical indicators of leishmaniasis. The newly identified proteins in this study likely include previously unidentified virulence factors with potential significance for parasite variations, pathogenesis, and therapeutic interference. Conclusion This study reveals that the proteogenomic analysis is a prevailing approach for exploring the hidden secretome of Leishmania species. These findings advance the understanding of parasite evolution and biology by identifying 469 novel secreted proteins and detecting the significant species-specific functional diversity; also highlight potential indicators for future functional research and therapeutic interventions.