Paromomycin Affects Translation and Vesicle-Mediated Trafficking as Revealed by Proteomics of Paromomycin –Susceptible –Resistant Leishmania donovani

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.

New trends in the use of aminoglycosides

“…PAR was also found to affect translation and vesicle-mediated trafficking in studies using PAR-susceptible and PAR-resistant L. donovani . 133 …”

Multi-analytical platform metabolomic approach to study miltefosine mechanism of action and resistance in Leishmania

“…Proteomics partially overcomes this hurdle: it has been extensively employed to decipher mechanisms of drug resistance [47][48][49], but this still overlooks some metabolic subtleties that are inherent to the complexity of the intracellular environment. A metabolome database for Leishmania has been developed [50], and a number comprehensive reviews have been published [51][52][53].…”

Drug Resistance in Leishmania

“…This suggests that PM could affect vesicle-mediated trafficking. The quantitative proteomic analysis also revealed an upregulation of ribosomal proteins, suggesting that PM also affects translation by targeting these ribosomal proteins (Chawla et al 2011) (Fig. 3c), although the exact molecular mechanistic remains to be elucidated (Fernandez et al 2011).…”

“…Since there is a concern on the development of clinical resistance, the longevity of PM would be better preserved in combination regimens than as a single agent therapy. Recently, a quantitative SILAC-based proteomic analysis of an in vitro-generated PM-resistant strain has brought some light on the mode of action of PM and the underlying resistance mechanisms (Chawla et al 2011). The uptake of PM is thought to proceed by endocytosis, a process facilitated by the binding of PM to a number of parasite surface proteins (Fig.…”