Leishmania donovani is the causative agent of visceral leishmaniasis, a tropical disease affecting millions worldwide. While proteomic studies of Leishmania species have been conducted, the organization of protein-protein interaction (PPI) networks in L. donovani remains largely unexplored. Here, we present a protein interaction network for L. donovani generated through size-exclusion chromatography coupled with mass spectrometry (SEC-MS) and computational analysis. We quantified 3,468 proteins with high confidence, of which approximately 70% are conserved across the Tritryps (Trypanosoma brucei, T. cruzi, and L. donovani).

The resulting network contains 1,509 nodes and 16,095 interactions, exhibiting scale-free topology and covering key cellular machineries such as the proteasome, ribosome, translation initiation complexes, and BBSome. Remarkably, most annotated Leishmania complexes remained intact within the network, highlighting its high quality. Paralogs within L. donovani frequently interacted with each other, a phenomenon observed at a higher rate than reported in different organisms. Beyond structural organization, the network also provided interaction-based evidence that functionally contextualizes previously uncharacterized or poorly annotated proteins. Complexes involved in mRNA metabolism and flagellar assembly revealed novel components supported by conserved interaction patterns, underscoring the biological utility of the network for functional inference. Our study provides the first experimentally derived, large-scale interaction network specific to L. donovani, offering critical insights into the parasite’s molecular architecture. All interaction data are available through our dedicated database at https://2025.trypsnetdb.org.