Gene expression modulation and the molecular mechanisms involved in N elfinavir resistance in L eishmania donovani axenic amastigotes

Summary Drug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging L eishmania / HIV ‐1 co‐infections. We have delineated the mechanism of cell death induced by the HIV ‐1 protease inhibitor, N elfinavir, in the L eishmania parasite. In order to further study N elfinavir– L eishmania interactions, we selected N elfinavir‐resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations of closely related L eishmania species were used as a screening tool to compare N elfinavir‐resistant and ‐sensitive parasites in order to identify candidate genes involved in drug resistance. Microarray analyses of N elfinavir‐resistant and ‐sensitive L eishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters. Transporter assays using radiolabelled N elfinavir suggest a greater drug accumulation in the resistant parasites and in a time‐dependent manner. Furthermore, high‐resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in N elfinavir‐resistant parasites. Together these results suggest that N elfinavir is rapidly and dramatically sequestered in drug‐induced intracellular vesicles.