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Mitochondrial Proteomics of Antimony and Miltefosine Resistant Leishmania infantum.

ABSTRACT: Antimony (SbIII) and miltefosine (MIL) are important drugs for the treatment of Leishmania parasite infections. The mitochondrion is likely to play a central role in SbIII and MIL induced cell death in this parasite. Enriched mitochondrial samples from Leishmania promastigotes selected step by step for in vitro resistance to SbIII and MIL were subjected to differential proteomic analysis. A shared decrease in both mutants in the levels of pyruvate dehydrogenase, dihydrolipoamide dehydrogenase, and isocitrate dehydrogenase was observed, as well as a differential abundance in two calcium-binding proteins and the unique dynamin-1-like protein of the parasite. Both mutants presented a shared increase in the succinyl-CoA:3-ketoacid-coenzyme A transferase and the abundance of numerous hypothetical proteins was also altered in both mutants. In general, the proteomic changes observed in the MIL mutant were less pronounced than in the SbIII mutant, probably due to the early appearance of a mutation in the miltefosine transporter abrogating the need for a strong mitochondrial adaptation. This study is the first analysis of the Leishmania mitochondrial proteome and offers powerful insights into the adaptations to this organelle during SbIII and MIL drug resistance.

SUBMITTER: Vincent IM

PROVIDER: S-EPMC5217391 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Mitochondrial Proteomics of Antimony and Miltefosine Resistant Leishmania infantum.

Vincent Isabel M IM Racine Gina G Légaré Danielle D Ouellette Marc M

Proteomes 20151021 4

Antimony (SbIII) and miltefosine (MIL) are important drugs for the treatment of Leishmania parasite infections. The mitochondrion is likely to play a central role in SbIII and MIL induced cell death in this parasite. Enriched mitochondrial samples from Leishmania promastigotes selected step by step for in vitro resistance to SbIII and MIL were subjected to differential proteomic analysis. A shared decrease in both mutants in the levels of pyruvate dehydrogenase, dihydrolipoa ...[more]

PMID: 28248274

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Project description:BackgroundSuperoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this enzyme is a promising molecular target for drug development against trypanosomatids.ResultsWe obtained Leishmania infantum mutant clones with lower FeSOD-A expression and investigated their phenotypes. Our attempts to delete this enzyme-coding gene using three different methodologies (conventional allelic replacement or two different CRISPR/methods) failed, as FeSOD-A gene copies were probably retained by aneuploidy or gene amplification. Promastigote forms of WT and mutant parasites were used in quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and western blot analyses, and these parasite forms were also used to assess drug susceptibility. RT-qPCR and western blot analyses revealed that FeSOD-A transcript and protein levels were lower in FeSOD-A-/-/+ L. infantum mutant clones than in the wild-type (WT) parasite. The decrease in FeSOD-A expression in L. infantum did not interfere with the parasite growth or susceptibility to amphotericin B. Surprisingly, FeSOD-A-/-/+ L. infantum mutant clones were 1.5- to 2.0-fold more resistant to trivalent antimony and 2.4- to 2.7-fold more resistant to miltefosine. To investigate whether the decrease in FeSOD-A expression was compensated by other enzymes, the transcript levels of five FeSODs and six enzymes from the antioxidant defence system were assessed by RT-qPCR. The transcript level of the enzyme ascorbate peroxidase increased in both the FeSOD-A-/-/+ mutants tested. The FeSOD-A-/-/+ mutant parasites were 1.4- to 1.75-fold less tolerant to oxidative stress generated by menadione. Infection analysis using THP-1 macrophages showed that 72 h post-infection, the number of infected macrophages and their intracellular multiplication rate were lower in the FeSOD-A-/-/+ mutant clones than in the WT parasite.ConclusionsThe unsuccessful attempts to delete FeSOD-A suggest that this gene is essential in L. infantum. This enzyme plays an important role in the defence against oxidative stress and infectivity in THP-1 macrophages. FeSOD-A-deficient L. infantum parasites deregulate their metabolic pathways related to antimony and miltefosine resistance.

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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Mitochondrial Proteomics of Antimony and Miltefosine Resistant Leishmania infantum.

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Mitochondrial Proteomics of Antimony and Miltefosine Resistant <i>Leishmania infantum</i>.

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