Experimental Resistance to Drug Combinations in Leishmania donovani: Metabolic and Phenotypic Adaptations

Abstract

f Together with vector control, chemotherapy is an essential tool for the control of visceral leishmaniasis (VL), but its efficacy is jeopardized by growing resistance and treatment failure against first-line drugs. To delay the emergence of resistance, the use of drug combinations of existing antileishmanial agents has been tested systematically in clinical trials for the treatment of visceral leishmaniasis (VL). In vitro, Leishmania donovani promastigotes are able to develop experimental resistance to several combinations of different antileishmanial drugs after 10 weeks of drug pressure. Using an untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics approach, we identified metabolic changes in lines that were experimentally resistant to drug combinations and their respective single-resistant lines. This highlighted both collective metabolic changes (found in all combination therapy-resistant [CTR] lines) and specific ones (found in certain CTR lines). We demonstrated that single-resistant and CTR parasite cell lines show distinct metabolic adaptations, which all converge on the same defensive mechanisms that were experimentally validated: protection against drug-induced and external oxidative stress and changes in membrane fluidity. The membrane fluidity changes were accompanied by changes in drug uptake only in the lines that were resistant against drug combinations with antimonials, and surprisingly, drug accumulation was higher in these lines. Together, these results highlight the importance and the central role of protection against oxidative stress in the different resistant lines. Ultimately, these phenotypic changes might interfere with the mode of action of all drugs that are currently used for the treatment of VL and should be taken into account in drug development.

Genomic Appraisal of the Multifactorial Basis forIn VitroAcquisition of Miltefosine Resistance in Leishmania donovani

“…It remains to be established whether this accumulation is a consequence o (i) a lower rate of proline metabolism, (ii) a lack of correlation of RNA and protein expression levels, or (iii) its connection with arginine levels. Interestingly, higher proline levels were associated with playing a protective role against antimonialinduced, but not HePC-induced, oxidative stress (57). Metacyclogenesis accounts for the transformation of poorly infective procyclic promastigotes into highly infective metacyclic promastigotes.…”

Effect of clinically approved HDAC inhibitors on Plasmodium, Leishmania and Schistosoma parasite growth

“…On an annual basis 1-2 million and ∼250 million people are reported to be infected with Leishmania (World Health Organization, 2008, Pigott et al., 2014) and Schistosoma parasites, respectively (Colley et al., 2014). Although drugs for each of these parasitic infections are available, prevention and treatment is often difficult due to side-effects (Sundar and Chakravarty, 2015) or ineffective due to drug-resistant parasites (Croft et al., 2006, Dondorp et al., 2009, Dondorp and Ringwald, 2013, Berg et al., 2015, Takala-Harrison et al., 2015). There is no vaccine that is clinically available or widely effective for any of the human parasitic diseases.…”

A mitochondrial HSP70 (HSPA9B) is linked to miltefosine resistance and stress response in Leishmania donovani

“…S-adenosylmethionine syntethase is downregulated (0.461-fold change) in transgenic lines and from this we may infer that the levels of its substrate, AdoMet, part of the trans-sulfuration pathway, may also be affected. Changes in AdoMet production have been identified in drug resistant Leishmania and a correlation with oxidative stress has been established [49]. The second protein identified in our comparative proteomics analysis (Fig.…”

“…Several studies have pinpointed mitochondrial-dependent apoptosis and generation of ROS as major effectors in the mechanism of action of HePC [48, 49]. We measured superoxide accumulation with the fluorescent dye MitoSox (indicative of ROS levels in the mitochondria) under low pH and high temperature, and showed that transgenic parasites overexpressing HSPA9B are more susceptible to oxidative stress damage (Fig.…”

“…This result may support one of the potential functions of HSPA9B: the underexpression of HSPA9B in the HePC resistant lines generated in our laboratory partially protects the cells against drug-induced oxidative stress. Furthermore, it is well known that L. donovani promastigotes resistant to HePC usually exhibit different metabolic adaptations leading to protection against oxidative stress [49]. In this regard we performed 2D-DIGE analysis of enriched mitochondrial proteins from HSPA9B overexpressing promastigotes and control cells (Fig.…”