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MRPA-independent mechanisms of antimony resistance in Leishmania infantum.

ABSTRACT: Control of both human and canine leishmaniasis is based on a very short list of chemotherapeutic agents, headed by antimonial derivatives (Sb). The utility of these molecules is severely threatened by high rates of drug resistance. The ABC transporter MRPA is one of the few key Sb resistance proteins described to date, whose role in detoxification has been thoroughly studied in Leishmania parasites. Nonetheless, its rapid amplification during drug selection complicates the discovery of other mechanisms potentially involved in Sb resistance. In this study, stepwise drug-resistance selection and next-generation sequencing were combined in the search for novel Sb-resistance mechanisms deployed by parasites when MRPA is abolished by targeted gene disruption. The gene mrpA is not essential in L. infantum, and its disruption leads to an Sb hypersensitive phenotype in both promastigotes and amastigotes. Five independent mrpA^-/- mutants were selected for antimony resistance. These mutants displayed major changes in their ploidy, as well as extrachromosomal linear amplifications of the subtelomeric region of chromosome 23, which includes the genes coding for ABCC1 and ABCC2. Overexpression of ABCC2, but not of ABCC1, resulted in increased Sb tolerance in the mrpA^-/- mutant. SNP analyses revealed three different heterozygous mutations in the gene coding for a serine acetyltransferase (SAT) involved in de novo cysteine synthesis in Leishmania. Overexpression of sat^Q390K, sat^G321R and sat^G325R variants led to a 2-3.2 -fold increase in Sb resistance in mrpA^-/- parasites. Only sat^G321R and sat^G325R induced increased Sb resistance in wild-type parasites. These results reinforce and expand knowledge on the complex nature of Sb resistance in Leishmania parasites.

SUBMITTER: Douanne N

PROVIDER: S-EPMC7225602 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOne of the major challenges to leishmaniasis treatment is the emergence of parasites resistant to antimony. To study differentially expressed genes associated with drug resistance, we performed a comparative transcriptomic analysis between wild-type and potassium antimonyl tartrate (SbIII)-resistant Leishmania infantum lines using high-throughput RNA sequencing.MethodsAll the cDNA libraries were constructed from promastigote forms of each line, sequenced and analyzed using STAR for mapping the reads against the reference genome (L. infantum JPCM5) and DESeq2 for differential expression statistical analyses. All the genes were functionally annotated using sequence similarity search.ResultsThe analytical pipeline considering an adjusted p-value < 0.05 and fold change > 2.0 identified 933 transcripts differentially expressed (DE) between wild-type and SbIII-resistant L. infantum lines. Out of 933 DE transcripts, 504 presented functional annotation and 429 were assigned as hypothetical proteins. A total of 837 transcripts were upregulated and 96 were downregulated in the SbIII-resistant L. infantum line. Using this DE dataset, the proteins were further grouped in functional classes according to the gene ontology database. The functional enrichment analysis for biological processes showed that the upregulated transcripts in the SbIII-resistant line are associated with protein phosphorylation, microtubule-based movement, ubiquitination, host-parasite interaction, cellular process and other categories. The downregulated transcripts in the SbIII-resistant line are assigned in the GO categories: ribonucleoprotein complex, ribosome biogenesis, rRNA processing, nucleosome assembly and translation.ConclusionsThe transcriptomic profile of L. infantum showed a robust set of genes from different metabolic pathways associated with the antimony resistance phenotype in this parasite. Our results address the complex and multifactorial antimony resistance mechanisms in Leishmania, identifying several candidate genes that may be further evaluated as molecular targets for chemotherapy of leishmaniasis.

Project description:BackgroundTryparedoxin peroxidase (TXNPx) participates in defence against oxidative stress as an antioxidant by metabolizing hydrogen peroxide into water molecules. Reports suggest that drug-resistant parasites may increase the levels of TXNPx and other enzymes, thereby protecting them against oxidative stress.MethodsIn this study, the gene encoding cytosolic TXNPx (cTXNPx) was characterized in lines of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum that are susceptible and resistant to potassium antimony tartrate (Sb(III)). We investigated the levels of mRNA and genomic organization of the cTXNPx gene. In addition, we transfected the Leishmania lines with the cTXNPx gene and analysed the susceptibility of transfected parasites to Sb(III) and to hydrogen peroxide (H2O2).ResultsNorthern blot and real-time reverse transcriptase polymerase chain reaction analyses revealed that the level of TXNPx mRNA was approximately 2.5-fold higher in the Sb(III)-resistant L. braziliensis line than in the parental line. In contrast, no significant difference in cTXNPx mRNA levels between the L. infantum lines was observed. Southern blot analyses revealed that the cTXNPx gene is not amplified in the genome of the Sb(III)-resistant Leishmania lines analysed. Functional analysis of cTXNPx was performed to determine whether overexpression of the enzyme in L. braziliensis and L. infantum lines would change their susceptibility to Sb(III). Western blotting analysis showed that the level of cTXNPx was 2 to 4-fold higher in transfected clones compared to non-transfected cells. Antimony susceptibility test (EC50 assay) revealed that L. braziliensis lines overexpressing cTXNPx had a 2-fold increase in resistance to Sb(III) when compared to the untransfected parental line. In addition, these clones are more tolerant to exogenous H2O2 than the untransfected parental line. In contrast, no difference in Sb(III) susceptibility and a moderate index of resistance to H2O2 was observed in L. infantum clones overexpressing cTXNPx.ConclusionOur functional analysis revealed that cTXNPx is involved in the antimony-resistance phenotype in L. braziliensis.

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MRPA-independent mechanisms of antimony resistance in Leishmania infantum.

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