Deep-sequencing revealing mutation dynamics in the miltefosine transporter gene in Leishmania infantum selected for miltefosine resistance.

Miltefosine is the first oral drug used in chemotherapy against leishmaniasis. In vitro studies found that resistance to miltefosine in Leishmania is often associated with the acquisition of point mutations in the miltefosine transporter, leading to a decrease in drug uptake. In this study, the dynamics of mutations upon miltefosine selection was studied by deep-sequencing of the miltefosine transporter gene. Deep-sequencing data revealed that no mutation was detected in the miltefosine transporter at sub-inhibitory concentrations of miltefosine. We show that the prevalence of mutated alleles was increasing when the drug pressure heightened, that more mutations were observed in highly resistant mutants, and that most mutations remained when parasites were cultured for a few passages in the absence of miltefosine.

Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism.

The parasite Leishmania often relies on gene rearrangements to survive stressful environments. However, safeguarding a minimum level of genome integrity is important for cell survival. We hypothesized that maintenance of genomic integrity in Leishmania would imply a leading role of the MRE11 and RAD50 proteins considering their role in DNA repair, chromosomal organization and protection of chromosomes ends in other organisms. Attempts to generate RAD50 null mutants in a wild-type background failed and we provide evidence that this gene is essential. Remarkably, inactivation of RAD50 was possible in a MRE11 null mutant that we had previously generated, providing good evidence that RAD50 may be dispensable in the absence of MRE11. Inactivation of the MRE11 and RAD50 genes led to a decreased frequency of homologous recombination and analysis of the null mutants by whole genome sequencing revealed several chromosomal translocations. Sequencing of the junction between translocated chromosomes highlighted microhomology sequences at the level of breakpoint regions. Sequencing data also showed a decreased coverage at subtelomeric locations in many chromosomes in the MRE11-/-RAD50-/- parasites. This study demonstrates an MRE11-independent microhomology-mediated end-joining mechanism and a prominent role for MRE11 and RAD50 in the maintenance of genomic integrity. Moreover, we suggest the possible involvement of RAD50 in subtelomeric regions stability.

Mitochondrial Proteomics of Antimony and Miltefosine Resistant Leishmania infantum.

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.

Formation of linear amplicons with inverted duplications in Leishmania requires the MRE11 nuclease.

Extrachromosomal DNA amplification is frequent in the protozoan parasite Leishmania selected for drug resistance. The extrachromosomal amplified DNA is either circular or linear, and is formed at the level of direct or inverted homologous repeated sequences that abound in the Leishmania genome. The RAD51 recombinase plays an important role in circular amplicons formation, but the mechanism by which linear amplicons are formed is unknown. We hypothesized that the Leishmania infantum DNA repair protein MRE11 is required for linear amplicons following rearrangements at the level of inverted repeats. The purified LiMRE11 protein showed both DNA binding and exonuclease activities. Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents. The MRE11(-/-) parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity. These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment.

Quantitative proteomic analysis of amphotericin B resistance in Leishmania infantum.

Amphotericin B (AmB) in its liposomal form is now considered as either first- or second-line treatment against Leishmania infections in different part of the world. Few cases of AmB resistance have been reported and resistance mechanisms toward AmB are still poorly understood. This paper reports a large-scale comparative proteomic study in the context of AmB resistance. Quantitative proteomics using stable isotope labeling of amino acids in cell culture (SILAC) was used to better characterize cytoplasmic and membrane-enriched (ME) proteomes of the in vitro generated Leishmania infantum AmB resistant mutant AmB1000.1. In total, 97 individual proteins were found as differentially expressed between the mutant and its parental sensitive strain (WT). More than half of these proteins were either metabolic enzymes or involved in transcription or translation processes. Key energetic pathways such as glycolysis and TCA cycle were up-regulated in the mutant. Interestingly, many proteins involved in reactive oxygen species (ROS) scavenging and heat-shock proteins were also up-regulated in the resistant mutant. This work provides a basis for further investigations to understand the roles of proteins differentially expressed in relation with AmB resistance.

Proteomic and genomic analyses of antimony resistant Leishmania infantum mutant.

BACKGROUND: Antimonials remain the primary antileishmanial drugs in most developing countries. However, drug resistance to these compounds is increasing and our understanding of resistance mechanisms is partial. METHODS/PRINCIPAL FINDINGS: In the present study, quantitative proteomics using stable isotope labelling of amino acids in cell culture (SILAC) and genome next generation sequencing were used in order to better characterize in vitro generated Leishmania infantum antimony resistant mutant (Sb2000.1). Using the proteomic method, 58 proteins were found to be differentially regulated in Sb2000.1. The ABC transporter MRPA (ABCC3), a known marker of antimony resistance, was observed for the first time in a proteomic screen. Furthermore, transfection of its gene conferred antimony resistance in wild-type cells. Next generation sequencing revealed aneuploidy for 8 chromosomes in Sb2000.1. Moreover, specific amplified regions derived from chromosomes 17 and 23 were observed in Sb2000.1 and a single nucleotide polymorphism (SNP) was detected in a protein kinase (LinJ.33.1810-E629K). CONCLUSION/SIGNIFICANCE: Our results suggest that differentially expressed proteins, chromosome number variations (CNVs), specific gene amplification and SNPs are important features of antimony resistance in Leishmania.

Gene amplification and point mutations in pyrimidine metabolic genes in 5-fluorouracil resistant Leishmania infantum.

BACKGROUND: The human protozoan parasites Leishmania are prototrophic for pyrimidines with the ability of both de novo biosynthesis and uptake of pyrimidines. METHODOLOGY/PRINCIPAL FINDINGS: Five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU) in the hope to better understand the metabolism of pyrimidine in Leishmania. Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed in selected mutants the amplification of DHFR-TS and a deletion of part of chromosome 10. Point mutations in uracil phosphorybosyl transferase (UPRT), thymidine kinase (TK) and uridine phosphorylase (UP) were also observed in three individual resistant mutants. Transfection experiments confirmed that these point mutations were responsible for 5-FU resistance. Transport studies revealed that one resistant mutant was defective for uracil and 5-FU import. CONCLUSION/SIGNIFICANCE: This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in Leishmania.

Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species.

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage.

ABC transporters involved in drug resistance in human parasites.

The ABC (ATP-binding cassette) protein superfamily is a ubiquitous and functionally versatile family of proteins that is conserved from archaea to humans. In eukaryotes, most of these proteins are implicated in the transport of a variety of molecules across cellular membranes, whereas the remaining ones are involved in biological processes unrelated to transport. The biological functions of several ABC proteins have been described in clinically important parasites and nematode worms and include vesicular trafficking, phospholipid movement, translation and drug resistance. This chapter reviews our current understanding of the role of ABC proteins in drug resistance and treatment failure in apicomplexan, trypanosomatid and amitochondriate parasites of medical relevance as well as in helminths.

Whole genome analysis of linezolid resistance in Streptococcus pneumoniae reveals resistance and compensatory mutations.

BACKGROUND: Several mutations were present in the genome of Streptococcus pneumoniae linezolid-resistant strains but the role of several of these mutations had not been experimentally tested. To analyze the role of these mutations, we reconstituted resistance by serial whole genome transformation of a novel resistant isolate into two strains with sensitive background. We sequenced the parent mutant and two independent transformants exhibiting similar minimum inhibitory concentration to linezolid. RESULTS: Comparative genomic analyses revealed that transformants acquired G2576T transversions in every gene copy of 23S rRNA and that the number of altered copies correlated with the level of linezolid resistance and cross-resistance to florfenicol and chloramphenicol. One of the transformants also acquired a mutation present in the parent mutant leading to the overexpression of an ABC transporter (spr1021). The acquisition of these mutations conferred a fitness cost however, which was further enhanced by the acquisition of a mutation in a RNA methyltransferase implicated in resistance. Interestingly, the fitness of the transformants could be restored in part by the acquisition of altered copies of the L3 and L16 ribosomal proteins and by mutations leading to the overexpression of the spr1887 ABC transporter that were present in the original linezolid-resistant mutant. CONCLUSIONS: Our results demonstrate the usefulness of whole genome approaches at detecting major determinants of resistance as well as compensatory mutations that alleviate the fitness cost associated with resistance.

Proteomic and transcriptomic analysis of linezolid resistance in Streptococcus pneumoniae.

Linezolid is an oxazolidinone antibiotic that inhibits the initiation of translation. Although resistance to linezolid is an uncommon event, it has been reported in clinical isolates. The genome sequence of Streptococcus pneumoniae linezolid-resistant mutants recently revealed mutations associated with resistance. A proteomic and transcriptomic screen now reveals a possible increase in the metabolism and transport of carbohydrates in these linezolid-resistant S. pneumoniae mutants. Several glycolytic proteins were shown to be overexpressed in the resistant strains, along with other enzymes and transporters involved in the metabolism of sugars. An increase in energy needs appears to be required to sustain extended levels of resistance to linezolid as the disruption of two ABC transporters putatively involved in the import of carbohydrates leads to a 2-fold sensitization to linezolid. Furthermore, the disruption of the catabolite control protein A, a regulator of the metabolism of sugars whose expression is highly increased in one linezolid-resistant mutant, resulted in a 2-fold increase in linezolid susceptibility. This global scale analysis of gene and protein expression profiling highlights metabolism alterations associated with linezolid resistance in S. pneumoniae.

Gene expression profiling and molecular characterization of antimony resistance in Leishmania amazonensis.

BACKGROUND: Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using DNA microarrays is a suitable approach to study simultaneous events leading to a drug-resistance phenotype. Genomic analysis has been performed primarily with Old World Leishmania species and here we investigate molecular alterations in antimony resistance in the New World species L. amazonensis. METHODS/PRINCIPAL FINDINGS: We selected populations of L. amazonensis promastigotes for resistance to antimony by step-wise drug pressure. Gene expression of highly resistant mutants was studied using DNA microarrays. RNA expression profiling of antimony-resistant L. amazonensis revealed the overexpression of genes involved in drug resistance including the ABC transporter MRPA and several genes related to thiol metabolism. The MRPA overexpression was validated by quantitative real-time RT-PCR and further analysis revealed that this increased expression was correlated to gene amplification as part of extrachromosomal linear amplicons in some mutants and as part of supernumerary chromosomes in other mutants. The expression of several other genes encoding hypothetical proteins but also nucleobase and glucose transporter encoding genes were found to be modulated. CONCLUSIONS/SIGNIFICANCE: Mechanisms classically found in Old World antimony resistant Leishmania were also highlighted in New World antimony-resistant L. amazonensis. These studies were useful to the identification of resistance molecular markers.

Genome sequencing of linezolid-resistant Streptococcus pneumoniae mutants reveals novel mechanisms of resistance.

Linezolid is a member of a novel class of antibiotics, with resistance already being reported. We used whole-genome sequencing on three independent Streptococcus pneumoniae strains made resistant to linezolid in vitro in a step-by-step fashion. Analysis of the genome assemblies revealed mutations in the 23S rRNA gene in all mutants including, notably, G2576T, a previously recognized resistance mutation. Mutations in an additional 31 genes were also found in at least one of the three sequenced genomes. We concentrated on three new mutations that were found in at least two independent mutants. All three mutations were experimentally confirmed to be involved in antibiotic resistance. Mutations upstream of the ABC transporter genes spr1021 and spr1887 were correlated with increased expression of these genes and neighboring genes of the same operon. Gene inactivation supported a role for these ABC transporters in resistance to linezolid and other antibiotics. The hypothetical protein spr0333 contains an RNA methyltransferase domain, and mutations within that domain were found in all S. pneumoniae linezolid-resistant strains. Primer extension experiments indicated that spr0333 methylates G2445 of the 23S rRNA and mutations in spr0333 abolished this methylation. Reintroduction of a nonmutated version of spr0333 in resistant bacteria reestablished G2445 methylation and led to cells being more sensitive to linezolid and other antibiotics. Interestingly, the spr0333 ortholog was also mutated in a linezolid-resistant clinical Staphylococcus aureus isolate. Whole-genome sequencing and comparative analyses of S. pneumoniae resistant isolates was useful for discovering novel resistance mutations.

Intracellular localization of the ABCC proteins of Leishmania and their role in resistance to antimonials.

The ABCC subfamily of proteins is composed of nine members in Leishmania. We report that all of these proteins have an intracellular localization and that the overexpression of at least four members, ABCC3, ABCC4, ABCC5, and ABCC7, can confer resistance to antimonials, the first-line drug against Leishmania.

Gene expression modulation is associated with gene amplification, supernumerary chromosomes and chromosome loss in antimony-resistant Leishmania infantum.

Antimonials remain the first line drug against the protozoan parasite Leishmania but their efficacy is threatened by resistance. We carried out a RNA expression profiling analysis comparing an antimony-sensitive and -resistant (Sb2000.1) strain of Leishmania infantum using whole-genome 70-mer oligonucleotide microarrays. Several genes were differentially expressed between the two strains, several of which were found to be physically linked in the genome. MRPA, an ATP-binding cassette (ABC) gene known to be involved in antimony resistance, was overexpressed in the antimony-resistant mutant along with three other tandemly linked genes on chromosome 23. This four gene locus was flanked by 1.4 kb repeated sequences from which an extrachromosomal circular amplicon was generated in the resistant cells. Interestingly, gene expression modulation of entire chromosomes occurred in the antimony-resistant mutant. Southern blots analyses and comparative genomic hybridizations revealed that this was either due to the presence of supernumerary chromosomes or to the loss of one chromosome. Leishmania parasites with haploid chromosomes were viable. Changes in copy number for some of these chromosomes were confirmed in another antimony-resistant strain. Selection of a partial revertant line correlated antimomy resistance levels and the copy number of aneuploid chromosomes, suggesting a putative link between aneuploidy and drug resistance in Leishmania.

Functional analysis and complex gene rearrangements of the folate/biopterin transporter (FBT) gene family in the protozoan parasite Leishmania.

The protozoan parasite Leishmania is a folic acid auxotroph. Previous work has led to the characterization of the main folate transporter FT1. FT1 is part of the folate/biopterin transporter (FBT) family and Leishmania with its 14 members is, of all sequenced organisms, the one with the most FBTs. We developed a real-time TaqMan RT-PCR assay to follow the expression of these FBT genes during growth phases, life cycles and in methotrexate-resistant mutants of Leishmania infantum. FT1 is expressed preferentially in the logarithmic phase which is consistent with the higher accumulation of folate in that stage. FT1 RNA levels even seemed to be related to folate concentration in the medium. Surprisingly, several of the FBT genes were expressed preferentially in the stationary phase of growth, a stage with minimal folate accumulation. It suggests that these FBT members may transport other related substrates. Resistance to methotrexate is associated with FT1 inactivation and upregulation of other FBT genes. Inactivation of FT1 is due either to a gene deletion mediated by homologous recombination between conserved FBT sequences or by segmental gene conversion. This study highlighted the multiplicity of FBT genes in Leishmania, their complex RNA expression, and novel gene rearrangements associated with FT1 inactivation and antifolate resistance.

Modulation of gene expression in drug resistant Leishmania is associated with gene amplification, gene deletion and chromosome aneuploidy.

BACKGROUND: Drug resistance can be complex, and several mutations responsible for it can co-exist in a resistant cell. Transcriptional profiling is ideally suited for studying complex resistance genotypes and has the potential to lead to novel discoveries. We generated full genome 70-mer oligonucleotide microarrays for all protein coding genes of the human protozoan parasites Leishmania major and Leishmania infantum. These arrays were used to monitor gene expression in methotrexate resistant parasites. RESULTS: Leishmania is a eukaryotic organism with minimal control at the level of transcription initiation and few genes were differentially expressed without concomitant changes in DNA copy number. One exception was found in Leishmania major, where the expression of whole chromosomes was down-regulated. The microarrays highlighted several mechanisms by which the copy number of genes involved in resistance was altered; these include gene deletion, formation of extrachromosomal circular or linear amplicons, and the presence of supernumerary chromosomes. In the case of gene deletion or gene amplification, the rearrangements have occurred at the sites of repeated (direct or inverted) sequences. These repeats appear highly conserved in both species to facilitate the amplification of key genes during environmental changes. When direct or inverted repeats are absent in the vicinity of a gene conferring a selective advantage, Leishmania will resort to supernumerary chromosomes to increase the levels of a gene product. CONCLUSION: Aneuploidy has been suggested as an important cause of drug resistance in several organisms and additional studies should reveal the potential importance of this phenomenon in drug resistance in Leishmania.

A protein of the leucine-rich repeats (LRRs) superfamily is implicated in antimony resistance in Leishmania infantum amastigotes.

Pentavalent antimonial containing drugs (SbV) are the mainstay for the control of the protozoan parasite Leishmania but resistance to this class of drug is now prevalent in several endemic areas. We describe here the use of functional cloning where an expression cosmid bank derived from Leishmania infantum was transfected in L. infantum axenic amastigotes and selected for potassium antimonyl tartrate (SbIII) resistance. This strategy allowed the isolation of a cosmid encoding for a novel resistance protein, LinJ34.0570, which belongs to the superfamily of leucine-rich repeat (LRR) proteins. Parasites overexpressing this LRR protein, which is part of the LRR_CC subfamily, were resistant to SbIII as axenic amastigotes and to SbV as intracellular parasites. This work pinpoints a novel protein that can contribute to antimonial resistance in Leishmania.

Modulation of gene expression in human macrophages treated with the anti-leishmania pentavalent antimonial drug sodium stibogluconate.

Within the mammalian host, Leishmania donovani is an obligatory intracellular protozoan parasite that resides and multiplies exclusively in the phagolysosomes of macrophages. Leishmania control relies primarily on chemotherapy, with the mainstay being pentavalent antimony (SbV) complexed to carbohydrates in the form of sodium stibogluconate (Pentostam) or meglumine antimoniate (Glucantime). The mode of action of SbV is still not known precisely. To explore the effect of SbV on macrophage gene expression, a microarray analysis was performed using Affymetrix focus arrays to compare gene expression profiles in noninfected and L. donovani-infected THP-1 monocytic cells treated or not treated with sodium stibogluconate. Under our experimental conditions, SbV changed the expression of a few host genes, and this was independent of whether cells were infected or not infected with Leishmania. Leishmania infection had a greater effect on the modulation of host gene expression. Statistical analyses have indicated that the expression of eight genes was modified by at least twofold upon SbV treatment, with six genes upregulated and two genes downregulated. One gene whose expression was affected by SbV was the heme oxygenase gene HMOX-1, and this change was observed both in the monocytic cell line THP-1 and in primary human monocyte-derived macrophages. Another pathway that was affected was the glutathione biosynthesis pathway, where the expression of the glutamate-cysteine ligase modifier subunit was increased upon SbV treatment. Our analysis has suggested that, under our experimental conditions, the expression of a few genes is altered upon SbV treatment, and some of these encoded proteins may be implicated in the yet-to-be-defined mode of action of SbV.