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

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.

Ascorbate peroxidase overexpression protects Leishmania braziliensis against trivalent antimony effects

Ascorbate peroxidase (APX) is a redox enzyme of the trypanothione pathway that converts hydrogen peroxide (H2O2) into water molecules. In the present study, the APX gene was overexpressed in Leishmania braziliensis to investigate its contribution to the trivalent antimony (SbIII)-resistance phenotype. Western blot results demonstrated that APX-overexpressing parasites had higher APX protein levels in comparison with the wild-type line (LbWTS). APX-overexpressing clones showed an 8-fold increase in the antimony-resistance index over the parental line. In addition, our results indicated that these clones were approximately 1.8-fold more tolerant to H2O2 than the LbWTS line, suggesting that the APX enzyme plays an important role in the defence against oxidative stress. Susceptibility tests revealed that APX-overexpressing L. braziliensis lines were more resistant to isoniazid, an antibacterial agent that interacts with APX. Interestingly, this compound enhanced the anti-leishmanial SbIII effect, indicating that this combination represents a good strategy for leishmaniasis chemotherapy. Our data demonstrate that APX enzyme is involved in the development of L. braziliensis antimony-resistance phenotype and may be an attractive therapeutic target in the design of new strategies for leishmaniasis treatment.

Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates

The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime(R)) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.