Enhancing the copy number of Ldrab6 gene in Leishmania donovani parasites mediates drug resistance through drug - thiol conjugate dependent multidrug resistance protein A (MRPA)

Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan Leishmania donovani parasite which may be fatal if left untreated. While drug-sensitive parasites are able to live and multiply within the host macrophages, they develop resistance to drugs used against them for survival and multiplication in the infected patients undergoing routine treatment. Development of new agents devoid of such drug resistance potential is achievable by identifying new drug targets in the parasite. One such target is the key regulator of intracellular vesicular trafficking protein, RabGTPase which belongs to the Ras GTPase superfamily. We recently elucidated whole genome sequence (WGS) of L. donovani (clinical Indian isolate; BHU 1220, GenBank: AVPQ00000000.1) and identified Ldrab6 gene. We now provide experimental evidence for this gene's ability to impart drug-resistant phenotype to wild-type (sensitive) Leishmania upon transfection. trans-Dibenzalacetone (DBA), a synthetic analog of curcumin, was used to determine its antileishmanial activity in wild-type parasites and parasites transfected with Ldrab6 gene. Dose-response study showed that DBA had no effect on transfected parasites at 20 µg/mL dose, whereas wild-type promastigotes showed 50% inhibition (IC50) at the same dose. This indicates the development of resistant mechanism in the transfected parasites due to enhancement of the copy number of Ldrab6 gene in L. donovani parasites. Flow cytometric analysis revealed elevated level of thiols in transfectants when compared to wild-type parasites treated with DBA. To assess the functional activity of multidrug resistance-associated protein (MRP) pump in transfectants, the accumulation of calcein, a known MRP pump substrate and probenecid, a known MRP pump regulator, were analyzed. The results indicate that Ldrab6 gene in Leishmania conferred resistance by the well-established mechanism of drug-thiol conjugation and sequestration by ABC transporter multidrug resistance-protein A (MRPA). Accordingly, Leishmania parasites transfected with Ldrab6 gene can be used as an experimental cell line for the screening of new lead molecules for their propensity to develop drug resistance.