Over-Expression of 60s Ribosomal L23a Is Associated with Cellular Proliferation in SAG Resistant Clinical Isolates of Leishmania donovani

Background Sodium antimony gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism. Methodology and principal findings The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode. Conclusion/Significance This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism. Author Summary Visceral Leishmaniasis (VL) is the most fatal form in Indian subcontinent. Till last few years, the treatment of the disease was done with Sodium antimony gluconate (SAG), the first line drug against VL. This, however, was severely eroded by the resistance developed by the parasite against it. In order to understand the underlying mechanism, earlier a proteomic analysis of SAG sensitive as well as SAG resistant isolates of L.donovani (Ld) was done in which 60s ribosomal L23a (Ld60sRL23a) protein, one of the essential member of translational machinery, was found to be over-expressed. To examine its role in SAG resistance mechanism, which is hitherto not known, 60sRL23a was characterized and over-expressed in the sensitive isolate of L.donovani. The sensitivity of these transfectants, was found to be decreased towards SAG. The growth curve of transfectants clearly showed its proliferation potential in both promastigote and amastigote forms. Cell cycle analysis of the transfectants further assured its rapid progression towards the G2/M phase. The above studies, thus, indicate that 60s RL23a regulates proliferation of L.donovani parasites and represents a unique strategy to resist SAG. 60sRL23a could be further explored as a potential drug target to strengthen the chemotherapy strategy against L.donovani.