Identifying Rab2 Protein as a Key Interactor of Centrin1 Essential for Leishmania donovani Growth

Previously, we have demonstrated that deletion of a growth-regulating gene (LdCen1) in the Leishmania donovani parasite (LdCen1^-/-) attenuated the parasite's intracellular amastigote growth but not the growth of extracellular promastigotes. LdCen1^-/- parasites were found to be safe and efficacious against homologous and heterologous Leishmania species as a vaccine candidate in animal models. The reason for the differential growth of LdCen1^-/- between the two stages of the parasite needed investigation. Here, we report that LdCen1 interacts with a novel Ras-associated binding protein in L. donovani (LdRab2) to compensate for the growth of LdCen1^-/- promastigotes. LdRab2 was isolated by protein pull-down from the parasite lysate, followed by nano-LC-MS/MS identification. The RAB domain sequence and the functional binding partners of the LdRab2 protein were predicted via Search Tool for the Retrieval of Interacting Proteins (STRING) analysis. The closeness of the LdRab2 protein to other reported centrin-binding proteins with different functions in other organisms was analyzed via phylogenetic analysis. Furthermore, in vitro and in silico analyses revealed that LdRab2 also interacts with other L. donovani centrins 3-5. Since centrin is a calcium-binding protein, we further investigated calcium-based interactions and found that the binding of LdRab2 to LdCen1 and LdCen4 is calcium-independent, whereas the interactions with LdCen3 and LdCen5 are calcium-dependent. The colocalization of LdCen1 and LdRab2 at the cellular basal-body region by immunofluorescence supports their possible functional association. The elevated expression of the LdRab2 protein in the mutant promastigotes suggested a probable role in compensating for the promastigote growth of this mutant strain, probably in association with other parasite centrins.