Unconventional role of Rab4 in the secretory pathway in Leishmania.

ABSTRACT

Leishmania donovani is an auxotroph for heme. Parasite acquires heme by clathrin-mediated endocytosis of hemoglobin by specific receptor. However, the regulation of receptor recycling pathway is not known in Leishmania. Here, we have cloned, expressed and characterized the Rab4 homologue from L. donovani. We have found that LdRab4 localizes in both early endosomes and Golgi in L. donovani. To understand the role of LdRab4 in L. donovani, we have generated transgenic parasites overexpressing GFP-LdRab4WT, GFP-LdRab4Q67L, and GFP-LdRab4S22N. Our results have shown that overexpression of GFP-LdRab4Q67L or GFP-LdRab4S22N does not alter the cell surface localization of hemoglobin receptor in L. donovani. Surprisingly, we have found that overexpression of GFP-LdRab4S22N significantly blocks the transport of Ldgp63 to the cell surface whereas the trafficking of Ldgp63 is induced to the cell surface in GFP-LdRab4WT and GFP-LdRab4Q67L overexpressing parasites. Consequently, we have found significant inhibition of gp63 secretion by GFP-LdRab4S22N overexpressing parasites whereas secretion of Ldgp63 is enhanced in GFP-LdRab4WT and GFP-LdRab4Q67L overexpressing parasites in comparison to untransfected control parasites. Moreover, we have found that survival of transgenic parasites overexpressing GFP-LdRab4S22N is severely compromised in macrophages in comparison to GFP-LdRab4WT and GFP-LdRab4Q67L expressing parasites. These results demonstrated that LdRab4 unconventionally regulates the secretory pathway in L. donovani.