Abstract
Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL) and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr) strain of L. donovani. Stable isotope labeling of amino acids in cell culture (SILAC) followed by quantitative mass spectrometry of the wild type AG83 strain and the paromomycin resistant (PRr) strain identified a total of 226 proteins at ≥95% confidence. Data analysis revealed upregulation of 29 proteins and down-regulation of 21 proteins in the PRr strain. Comparative proteomic analysis of the wild type and the paromomycin resistant strains showed upregulation of the ribosomal proteins in the resistant strain indicating role in translation. Elevated levels of glycolytic enzymes and stress proteins were also observed in the PRr strain. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival and vesicular trafficking in the PRr strain. Furthermore, ultra-structural analysis by electron microscopy demonstrated increased number of vesicular vacuoles in PRr strain when compared to the wild-type strain. Drug affinity pull-down assay followed by mass spectrometery identified proteins in L. donovani wild type strain that were specifically and covalently bound to paromomycin. These results provide the first comprehensive insight into the mode of action and underlying mechanism of resistance to paromomycin in Leishmania donovani.
Highlights
Paromomycin is an aminoglycosidic antibiotic which was initially used against bacterial infection [1]
In order to understand the mode of action and possible mechanism of resistance of this antibiotic at the molecular level, we have investigated the protein expression profile of genetically related pair of paromomycin susceptible/-resistant strains
Identification of Proteins Differentially Expressed in Wild Type (WT) and the Paromomycin Resistant (PRr) Strain
Summary
Paromomycin is an aminoglycosidic antibiotic which was initially used against bacterial infection [1]. It is used against giardiasis, amoebaiasis [2] and cryptosporidiosis [3]. The mechanism of action of paromomycin has been well studied in E. coli. It is known to inhibit protein synthesis by interacting with the ribosomal subunits [1]. It has been shown to inhibit the anti-association activity of initiation factor 3 and promote association of the ribosomal subunits [4]. It is known to bind to the major groove in the A-site of 16S rRNA in E. coli and induces misreading of mRNA [5]
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