Identification of specific reversal agents for Leishmania ABCI4-mediated antimony resistance by flavonoid and trolox derivative screening

Manzano, José Ignacio; Lecerf-Schmidt, Florine; Lespinasse, Marie-Ange; Pietro, Attilio Di; Castanys, Santiago; Boumendjel, A.; Gamarro, Francisco

doi:10.1093/jac/dkt407

Cited by 15 publications

(17 citation statements)

References 36 publications

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“…The reduction in ISM accumulation does not appear to be due to an increase in efflux of the compound, however, since after washing out ISM from the trypanosomes’ culture medium the rate by which the intracellular ISM concentration diminished was roughly the same between resistant and sensitive cell lines. Also, the application of inhibitors of ABC transporters (increased expression of which is implicated in multi-drug resistance in many different systems including the related kinetoplastid Leishmania [ 58 ]) did not enhance accumulation of ISM as one would expect if they were responsible for ISM efflux. However, only a limited panel of ABC transporter inhibitors was tested, as active extrusion was clearly not the main resistance mechanism.…”

Section: Discussionmentioning

confidence: 99%

Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo-ATPase

et al. 2016

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BackgroundIsometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine.Methodology/Principal FindingsTwo isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15), being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA) and both lines carried a mutation in the nuclearly encoded γ subunit gene of F1 ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance.Conclusions/SignificanceSub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial mutation is in the F1 ATPase γ subunit, which allows loss of kDNA and results in a reduction of the mitochondrial membrane potential.

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Section: Discussionmentioning

confidence: 99%

Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo-ATPase

et al. 2016

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“…To analyze the relationship between thiol levels and susceptibility to Sb III , parasites were previously grown in M199 culture medium supplemented with 10% hiFBS plus 3 mM BSO (a ␥-glutamylcysteine synthetase inhibitor) for 48 h at 28°C. For assays of susceptibility of intracellular Leishmania amastigotes to Sb III and Sb V , stationary-phase promastigotes were used to infect macrophage-differentiated THP-1 cells at a macrophage/parasite ratio of 1:10, as described previously (17). After overnight infection at 35°C with 5% CO 2 in RPMI 1640 medium plus 5% hiFBS, extracellular parasites were removed by washing with serum-free medium.…”

Section: Methodsmentioning

confidence: 99%

“…Infected macrophages were incubated at 37°C with different concentrations of Sb III and Sb V with 5% CO 2 in RPMI 1640 medium plus 10% hiFBS for 72 and 120 h, respectively. Following incubation, the cultures were fixed and analyzed as described previously (17).…”

Section: Methodsmentioning

confidence: 99%

The LABCG2 Transporter from the Protozoan Parasite Leishmania Is Involved in Antimony Resistance

Perea

Manzano

Castanys

et al. 2016

Antimicrob Agents Chemother

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Treatment for leishmaniasis, which is caused by Leishmania protozoan parasites, currently relies on a reduced arsenal of drugs. However, the significant increase in the incidence of drug therapeutic failure and the growing resistance to first-line drugs like antimonials in some areas of Northern India and Nepal limit the control of this parasitic disease. Understanding the molecular mechanisms of resistance in Leishmania is now a matter of urgency to optimize drugs used and to identify novel drug targets to block or reverse resistant mechanisms. Some members of the family of ATP-binding cassette (ABC) transporters in Leishmania have been associated with drug resistance. In this study, we have focused our interest to characterize LABCG2's involvement in drug resistance in Leishmania. Leishmania major parasites overexpressing the ABC protein transporter LABCG2 were generated in order to assess how LABCG2 is involved in drug resistance. Assays of susceptibility to different leishmanicidal agents were carried out. Analysis of the drug resistance profile revealed that Leishmania parasites overexpressing LABCG2 were resistant to antimony, as they demonstrated a reduced accumulation of Sb III due to an increase in drug efflux. Additionally, LABCG2 was able to transport thiols in the presence of Sb III . Biotinylation assays using parasites expressing LABCG2 fused with an N-terminal green fluorescent protein tag revealed that LABCG2 is partially localized in the plasma membrane; this supports data from previous studies which suggested that LABCG2 is localized in intracellular vesicles that fuse with the plasma membrane during exocytosis. In conclusion, Leishmania LABCG2 probably confers antimony resistance by sequestering metal-thiol conjugates within vesicles and through further exocytosis by means of the parasite's flagellar pocket.

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“…, 10 , 11 The same phenotype is reached when Sb is a substrate of the ABC transporters and can be pumped out or transported into intracellular vesicles, followed by extrusion. Several transporter proteins were associated with Sb resistance in different Leishmania species, from both field isolates and laboratory-selected resistant mutants such as, multidrug resistant protein A (MRPA)/ABCC3 (also known as PGPA), 12 , 13 ABCG2, 14 ABCI4, 15 and antimony resistance marker (ARM)56/ARM58 16 . However, the roles of such resistance genes are still unknown for L. braziliensis isolates derived from AT lesions.…”

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confidence: 99%

Antimony resistance in Leishmania (Viannia) braziliensis clinical isolates from atypical lesions associates with increased ARM56/ARM58 transcripts and reduced drug uptake

Rugani

Gontijo

Frézard

et al. 2019

Mem. Inst. Oswaldo Cruz

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BACKGROUND In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.

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Identification of specific reversal agents for Leishmania ABCI4-mediated antimony resistance by flavonoid and trolox derivative screening

Cited by 15 publications

References 36 publications

Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo-ATPase

Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo-ATPase

The LABCG2 Transporter from the Protozoan Parasite Leishmania Is Involved in Antimony Resistance

Antimony resistance in Leishmania (Viannia) braziliensis clinical isolates from atypical lesions associates with increased ARM56/ARM58 transcripts and reduced drug uptake

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