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Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains
C D Shaw1, H Imamura2, T Downing3
1Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom.
Abstract:
Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMMr) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC50) values for PMMr parasites varied between 104 and 481 μM at the promastigote stage and 32 and 195 μM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage (P < 0.05). This effect was most marked in the Sb-resistant (Sbr) PMMr clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type (P < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMMr strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMMr) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMMr and Sbr PMMr). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMMr In addition, we found that PMMr is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.
Insights
Developing paromomycin resistance in Leishmania donovani involves increased tolerance to nitric oxide and altered amino acid metabolism. This drug resistance is linked to aneuploidy, potentially accelerating genetic adaptation.
Area of Science:
- Molecular parasitology
- Drug resistance mechanisms
- Leishmania donovani pathogenesis
Background:
- Drug resistance in Leishmania donovani poses a significant challenge for treating leishmaniasis.
- Understanding resistance mechanisms is crucial for developing effective therapeutic strategies.
- Paromomycin (PMM) is a key drug used in leishmaniasis treatment, but resistance can emerge.
Purpose of the Study:
- To investigate the mechanisms underlying paromomycin resistance (PMMr) in clinical strains of Leishmania donovani.
- To explore the association between PMM resistance and resistance to other stressors like nitric oxide.
- To identify metabolic and genetic changes associated with PMM resistance.
Main Methods:
- Stepwise induction of PMM resistance in three Nepalese clinical Leishmania donovani strains.
- Measurement of PMM 50% inhibitory concentration (IC50) at promastigote and intracellular amastigote stages.
- Assessment of cross-resistance to nitric oxide, analysis of glutathione and trypanothione levels, and detection of metabolic alterations.
- Evaluation of aneuploidy in PMM-selected parasite populations.
Main Results:
- Induced PMM resistance resulted in mixed parasite populations with varying IC50 values.
- PMM resistance correlated with increased nitric oxide resistance in amastigotes, particularly in antimony-resistant strains.
- Upregulation of glutathione was observed in resistant strains, alongside alterations in amino acid metabolism (isoleucine, arginine, tyrosine derivatives).
- A significant increase in aneuploidy was detected during PMM selection across all strains.
Conclusions:
- Paromomycin resistance in Leishmania donovani is a complex phenotype associated with cross-resistance to nitric oxide and specific metabolic shifts.
- Upregulation of glutathione and altered amino acid metabolism are key features of PMM resistance.
- Increased aneuploidy during PMM selection may facilitate the rapid acquisition of resistance-conferring genetic changes.
