Evolutionary duplication of the leishmanial adaptor protein α-SNAP plays a role in its pathogenicity.

Essential-gene duplication during evolution promotes specialized functions beyond the typical role. Our in silico study unveiled two α-SNAP paralogs in Leishmania, a crucial component that, along with NSF, triggers disassembly of the cis-SNARE complex, formed during vesicle fusion with target membranes. While multiple α-SNAPs are common in many flagellated protists, including the trypanosomatids, they are unusual among other eukaryotes. This study explores the evolutionary and functional relevance of α-SNAP gene duplication in Leishmania donovani, emphasizing both subfunctionalization and neofunctionalization. We discovered that L. donovani α-SNAP (Ldα-SNAP) genes are transcribed in promastigote and amastigote stages, indicating they are not pseudogenes. Although the two paralogs share essential residues and structural features, only Ldα-SNAP (Ldα-SNAP1) can effectively substitute the function of its yeast counterpart, while Ldα-SNAP (Ldα-SNAP2) cannot. This functional difference is attributed to a replacement of alanine with phosphorylatable-serine in Ldα-SNAP1 during evolution from the most common ancestral ortholog. This modification is rarely observed in corresponding orthologs of other trypanosomatids. Incidentally, Ldα-SNAP paralogs exhibit differential localization in the ER and flagellar pocket. However, both paralogs, either actively or passively, regulate the secretion of exosomes and PM blebs, containing the virulence protein GP63. This indicates functional division and their indirect participation in the host's macrophage inactivation. Moreover, a small fraction of Ldα-SNAP1's presence in the flagellum hints at a potential role in sensing environmental cues and aiding the parasite's attachment to the sandfly's hindgut. Our findings underscore that duplicated Ldα-SNAPs have retained ancestral functions through subfunctionalization, and subsequently, they acquired parasite-specific neofunction(s) through the accumulation of natural mutation(s).