Functional characterization of monomeric GTPase Rab1 in the secretory pathway of Leishmania [Microbiology]

October 23rd, 2015 by Bahl, S., Parashar, S., Malhotra, H., Raje, M., Mukhopadhyay, A.

Leishmania secretes large number of their effectors to the extracellular milieu. However, regulation of secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression and characterization of Rab1 homologue from Leishmania. We have found that Ld-Rab1 localizes in Golgi in Leishmania. To understand the role of Ld-Rab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing Ld-GFP-Rab1:WT, Ld-GFP-Rab1:Q67L, a GTPase deficient dominant-positive mutant of Rab1 and Ld-GFP-Rab1:S22N, a GDP locked dominant-negative mutant of Rab1. Surprisingly, our results have shown that overexpression of Ld-GFP-Rab1:Q67L or Ld-GFP-Rab1:S22N does not disrupt the trafficking and localization of HbR in Leishmania. To determine whether Rab1 dependent secretory pathway is conserved in parasites, we have analyzed the role of Ld-Rab1 in the secretion of secretory acid phosphatase (SAP) and Ld-gp63 in Leishmania. Our results have shown that overexpression of Ld-GFP-Rab1:Q67L or Ld-GFP-Rab1:S22N significantly inhibits the secretion of SAP by Leishmania. We have also found that overexpression of Ld-GFP-Rab1:Q67L or Ld-GFP-Rab1:S22N retains Ld-RFP-gp63 in Golgi and blocks the secretion of Ld-gp63 whereas the trafficking of Ld-RFP-gp63 in Ld-GFP-Rab1:WT expressed cells is unaltered in comparison to control cells. Taken together, our results have shown that Rab1 regulated secretory pathway is well conserved and HbR trafficking follows Rab1 independent secretory pathway in Leishmania.