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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.

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.

Protozoa and helminths have by far the greatest impact in terms of morbidity worldwide. The status of protozoa and helminths control, both in human and veterinary medicine, is challenged as the current medications against these parasites are losing their efficacy due to increasing and even further spreading drug resistance. Despite this alarming statement and the high burden imposed by parasites, research progress in parasitic diseases lags behind many other infectious diseases. Recent innovative technologies may significantly impact parasite diagnostics and their control in the near future, catalyzed by a better knowledge in drug resistance mechanisms. The present chapter review drug resistance assays in major protozoan and helminthic diseases, point-of-care tests and multiplexing assays for drug resistance testing, and opportunities for innovations in the field.

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.

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 .

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., Author Summary The parasite Leishmania relies on gene rearrangements to survive stressful conditions. However, maintaining a minimum level of genomic integrity is crucial for cell survival. Studies in other organisms have provided evidence that the DNA repair proteins MRE11 and RAD50 are involved in chromosomes organization, protection of chromosomes ends and therefore in the maintenance of genomic integrity. In this manuscript, we present the conditional inactivation of the Leishmania infantum RAD50 gene that was only possible in MRE11 deficient cells and suggest the genetic background is crucial for RAD50 inactivation. We demonstrate the occurrence of chromosomal translocations in the MRE11 and RAD50 deficient cells and described a MRE11-independent microhomology-mediated end-joining mechanism at the level of translocation breakpoints. We also suggest a possible involvement of RAD50 in subtelomeric regions stability. Our results highlight that both MRE11 and RAD50 are important for the maintenance of genomic integrity in Leishmania.

The ATP-binding cassette (ABC) protein superfamily is one of the largest evolutionarily conserved families and is found in all kingdoms of life. The recent completion of the Leishmania genome sequence allowed us to analyze and classify its encoded ABC proteins. The complete sequence predicts a data set of 42 open reading frames (ORFs) coding for proteins belonging to the ABC superfamily, with representative members of every major subfamily (from ABCA to ABCH) commonly found in eukaryotes. Comparative analysis showed that the same ABC data set is found between Leishmania major and Leishmania infantum and that some orthologues are found in the genome of the related parasites Trypanosoma brucei and Trypanosoma cruzi . Customized DNA microarrays were made to assess ABC gene expression profiling throughout the two main Leishmania life stages. Two ABC genes ( ABCA3 and ABCG3 ) are preferentially expressed in the amastigote stage, whereas one ABC gene ( ABCF3 ) is more abundantly expressed in promastigotes. Microarray-based expression profiling experiments also revealed that three ABC genes ( ABCA3 , ABCC3 , and ABCH1 ) are overexpressed in two independent antimony-resistant strains compared to the parental sensitive strain. All microarray results were confirmed by real-time reverse transcription-PCR assays. The present study provides a thorough phylogenic classification of the Leishmania ABC proteins and sets the basis for further functional studies on this important class of proteins.

Resistance to penicillin is widespread in the Gram-positive bacterium Streptococcus pneumoniae, and while several mutations are known to be implicated in resistance other mechanisms are likely to occur. We used a proteomic screen of two independent mutants in which resistance was selected in vitro. We found a number of differentially expressed proteins including PstS, a subunit of the phosphate ABC transporter of S. pneumoniae. This protein was increased in both mutants, a phenotype correlated to increased RNA expression of the entire phosphate ABC transporter operon. Inactivation of the pstS gene led to increased susceptibility to penicillin in the wild-type strain. To further link the expression of the ABC phosphate transporter with penicillin resistance, we looked at pstS mRNA levels in 12 independent clinical isolates sensitive and resistant to penicillin and found an excellent correlation between resistance and increased expression of pstS. Inactivation of pstS in one of the clinical isolates significantly reduced penicillin resistance. Global approaches are ideally suited for the discovery of novel factors in the biology of resistance.

Antimonial compounds are the mainstay for the treatment of infections with the protozoan parasite Leishmania . We present our studies on Leishmania infantum amastigote parasites selected for resistance to potassium antimonyl tartrate [Sb(III)]. Inside macrophages, the Sb(III)-selected cells are cross-resistant to sodium stibogluconate (Pentostam), the main drug used against Leishmania . Putative alterations in the level of expression of more than 40 genes were compared between susceptible and resistant axenic amastigotes using customized DNA microarrays. The expression of three genes coding for the ABC transporter MRPA (PGPA), S -adenosylhomocysteine hydrolase, and folylpolyglutamate synthase was found to be consistently increased. The levels of cysteine were found to be increased in the mutant. Transfection of the MRPA gene was shown to confer sodium stibogluconate resistance in intracellular parasites. This MRPA-mediated resistance could be reverted by using the glutathione biosynthesis-specific inhibitor buthionine sulfoximine. These results highlight for the first time the role of MRPA in antimony resistance in the amastigote stage of the parasite and suggest a strategy for reversing resistance.

Graphical abstract, Highlights • First large-scale quantitative proteomic study of amphotericin B resistance in Leishmania. • Identification of 97 differentially expressed proteins. • Upregulation of glycolysis and TCA cycle pathways. • Upregulation in reactive oxygen species scavenging and heat-shock proteins., 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.

The Leishmania ATP-binding cassette (ABC) transporter PGPA is involved in metal resistance (arsenicals and antimony), although the exact mechanism by which PGPA confers resistance to antimony, the first line drug against Leishmania, is unknown. The results of co-transfection experiments, transport assays, and the use of inhibitors suggest that PGPA recognizes metals conjugated to glutathione or trypanothione, a glutathione-spermidine conjugate present in Leishmania. The HA epitope tag of the influenza hemagglutinin as well as the green fluorescent protein were fused at the COOH terminus of PGPA. Immunofluorescence, confocal, and electron microscopy studies of the fully functional tagged molecules clearly indicated that PGPA is localized in membranes that are close to the flagellar pocket, the site of endocytosis and exocytosis in this parasite. Subcellular fractionation of Leishmania tarentolae PGPAHA transfectants was performed to further characterize this ABC transporter. The basal PGPA ATPase activity was determined to be 115 nmol/mg/min. Transport experiments using radioactive arsenite-glutathione conjugates clearly showed that PGPA recognizes and actively transports thiol-metal conjugates. Overall, the results are consistent with PGPA being an intracellular ABC transporter that confers arsenite and antimonite resistance by sequestration of the metal-thiol conjugates.

Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX.

The mechanism of resistance to the metal arsenite has been studied and compared in L. mexicana, L. tropica, and L. tarentolae selected in a step by step manner for arsenite resistance. Amplification of the ABC transporter gene pgpA was found to be a frequent resistance mechanism in all species. Transfection of pgpA genes into different species indicated that both the origin of the pgpA gene and the recipient strain into which the gene is transfected seem important for resistance. An increase in the levels of trypanothione was also correlated with metal resistance in different Leishmania species. The mechanism used to increase the levels of trypanothione seems to differ, however, between the different species. This study points to a key role of transporters and thiol levels in metal resistance in Leishmania.

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., Author Summary The human protozoan parasites Leishmania present the ability of both de novo biosynthesis and uptake of pyrimidines. The pyrimidine pathway is not well understood in these parasites. In the hope to better understand the pyrimidine pathway in Leishmania, five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU). Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed the amplification of the main target enzyme DHFR-TS, and point mutations in three important metabolic enzymes. Transfection experiments confirmed that these point mutations were responsible for 5-FU resistance. Transport studies also revealed that one resistant mutant was defective for uracil and 5-FU import. Overall, this study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in these protozoa.

Multidrug resistance in tumor cells is often caused by the increased efflux of a wide variety of drugs, mediated by P glycoprotein, a member of the superfamily of ATP-binding cassette (ABC) transporters. The genes encoding members of this superfamily have also been isolated from drug-resistant microorganisms, and the role of microbial ABC transporters in drug resistance is being investigated.

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.

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.

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., Author Summary Leishmania are unicellular microorganisms that can be transmitted to humans by the bite of sandflies. They cause a spectrum of diseases called leishmaniasis, which are classified as neglected tropical diseases by the World Health Organization. The treatment of leishmaniasis is based on the administration of antimony-containing drugs. These drugs have been used since 1947 and still constitute the mainstay for leishmaniasis treatment in several countries. One of the problems with these compounds is the emergence of resistance. Our work seeks to understand how these parasites become resistant to the drug. We studied antimony-resistant Leishmania amazonensis mutants. We analyzed gene expression at the whole genome level in antimony-resistant parasites and identified mechanisms used by Leishmania for resistance. This work could help us in developing new strategies for treatment in endemic countries where people are unresponsive to antimony-based chemotherapy. The identification of common mechanisms among different species of resistant parasites may also contribute to the development of diagnostic kits to identify and monitor the spread of resistance.

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.

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.

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.

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.

Resistance to penicillin is widespread in the Gram-positive bacterium Streptococcus pneumoniae, and while several mutations are known to be implicated in resistance other mechanisms are likely to occur. We used a proteomic screen of two independent mutants in which resistance was selected in vitro. We found a number of differentially expressed proteins including PstS, a subunit of the phosphate ABC transporter of S. pneumoniae. This protein was increased in both mutants, a phenotype correlated to increased RNA expression of the entire phosphate ABC transporter operon. Inactivation of the pstS gene led to increased susceptibility to penicillin in the wild-type strain. To further link the expression of the ABC phosphate transporter with penicillin resistance, we looked at pstS mRNA levels in 12 independent clinical isolates sensitive and resistant to penicillin and found an excellent correlation between resistance and increased expression of pstS. Inactivation of pstS in one of the clinical isolates significantly reduced penicillin resistance. Global approaches are ideally suited for the discovery of novel factors in the biology of resistance.

Leishmaniasis is a protozoan parasitic disease that affects 12 million people worldwide. The first line choice for the treatment of this disease is antimonial drugs. In the endemic regions, resistance to this class of drugs is a major impediment to treatment. Microbes often become resistant to drugs by mutation or down-regulation of uptake systems, but the uptake system for the antimonial drugs in Leishmania is unknown. In other organisms, aquaglyceroporins have been shown to facilitate uptake of trivalent metalloids. In this study, we report the identification and characterization of aquaglyceroporins from Leishmania major (LmAQP1) and Leishmania tarentolae (LtAQP1), respectively. These Leishmania proteins have the conserved signature motifs of aquaglyceroporins. Transfection of LmAQP1 into three species of Leishmania, L. tarentolae, Leishmania infantum, and L. major, produced hypersensitivity to both As(III) and Sb(III) in all three strains. Increased production of LmAQP1 was detected by immunoblotting. Drug-resistant parasites with various mutations leading to resistance mechanisms became hypersensitive to both metalloids after expression of LmAQP1. Increased rates of uptake of As(III) or Sb(III) correlated with metalloid sensitivity of the wild type and drug-resistant transfectants. Transfection of LmAQP1 in a Pentostam-resistant field isolate also sensitized the parasite in the macrophage-associated amastigote form. One allele of LmAQP1 was disrupted in L. major, and the resulting cells became 10-fold more resistant to Sb(III). This is the first report of the uptake of a metalloid drug by an aquaglyceroporin in Leishmania, suggesting a strategy to reverse resistance in the field.

Publisher Summary This chapter describes the amplicons that can be detected and isolated. It also explains how the function of heavy metal resistance genes in Leishmania can be studied by gene transfection and gene disruption. Resistance to oxyanions in Leishmania is a complex phenomenon. The ease of detection of gene amplification has permitted the isolation of genes involved in resistance including one encoding for the ABC transporter PgpA and for a gene collaborating with it. Several other amplified genes in L. tarentolae , resistant to oxyanions have been isolated using the methodology described in the chapter. Studies of drug resistance mechanisms in parasites are correlative with the ability to introduce genes by electroporation. By disrupting the genes by homologous recombination, the role of genes in resistance can be directly studied. With the increased understanding of resistance mechanisms in laboratory strains, the resistance mechanisms prevailing in field strains isolated from patients who are unresponsive to pentavalent antimony treatment are tested. This is beneficial for the millions of people suffering from Leishmania infections.

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.

P-glycoprotein gene amplification has been described in several drug-resistant parasitic protozoa. The first P-glycoprotein related gene described in Leishmania was ltpgpA , a gene frequently amplified in arsenite resistant Leishmania . Hybridization experiments indicated that ltpgpA was part of a gene family. In addition to ltpgpA , four novel genes were cloned that are present in two loci: ltpgpB and ltpgpC tandemly linked to ltpgpA on a 800-kb chromosome; and ltpgpD and ltpgpE closely linked on a chromosome ranging from 950 kb to 1400 kb, depending on the Leishmania species. Another P-glycoprotein gene, homologous to the more recently described ldmdrl , was linked to ltpgpD and ltpgpE . Nucleotide sequencing of ltpgpB and ltpgpE revealed that the Leishmania P-glycoprotein-related genes have diverged considerably from the main branch of P-glycoproteins and are more homologous to the recently described multidrug resistance-associated protein found in multidrug-resistant human lung cancer cell lines. Cross-resistance studies and gene transfection experiments indicated that under the conditions tested only ltpgpA and ldmdrl are involved in resistance to arsenite and antimonials or hydrophobic drugs such as vinblastine respectively.

Penicillin resistance in Streptococcus pneumoniae is mediated by a mosaic of genes encoding altered penicillin-binding proteins (PBP). Nonetheless, S. pneumoniae has also developed non-PBP mechanisms implicated in penicillin resistance. In this study, whole genome sequencing of resistant organisms was used to discover mutations implicated in resistance to penicillin.

Gene expression and DNA copy number analyses using full genome oligonucleotide microarrays of Leishmania reveal molecular mechanisms of methotrexate resistance., 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.

The parasite Leishmania exhibits a dimorphic life cycle with promastigotes found in the insect vector and amastigotes residing within mammalian macrophages. In the insect, promastigotes undergo metacyclogenesis with two main stages, the procyclic and metacyclic stages. Reduced pterins, provided by the pteridine reductase PTR1, are important factors controlling metacyclogenesis. We applied here the liquid-based free-flow electrophoresis technique for the analysis of L. infantum wild-type and PTR1 null-mutant procyclic and metacyclic proteomes. Significant modulations in energy metabolism, antioxidant and stress-related defences, genetic information processing, protein degradation and motility were observed between procyclic and metacyclic forms, in addition to numerous hypothetical proteins.

In the protozoan parasite Leishmania, drug resistance can be a complex phenomenon. Several metabolic pathways and membrane transporters are implicated in the resistance phenotype. To monitor the expression of these genes, we generated custom DNA microarrays with PCR fragments corresponding to 44 genes involved with drug resistance. Transcript profiling of arsenite and antimony resistant mutants with these arrays pinpointed a number of genes overexpressed in mutants, including the ABC transporter PGPA, the glutathione biosynthesis genes gamma-glutamylcysteine synthetase (GSH1) and the glutathione synthetase (GSH2). Competitive hybridisations with total RNA derived from sensitive and methotrexate resistant cells revealed the overexpression of genes coding for dihydrofolate reductase (DHFR-TS), pteridine reductase (PTR1) and S-adenosylmethionine synthase (MAT2) and a down regulation of one gene of the folate transporter (FT) family. By labelling the DNA of sensitive and resistant parasites we could also detect several gene amplification events using DNA microarrays including the amplification of the S-adenosyl homocysteine hydrolase gene (SAHH). Alteration in gene expression detected by microarrays was validated by northern blot analysis, while Southern blots indicated that most genes overexpressed were also amplified, although other mechanisms were also present. The microarrays were useful in the study of resistant parasites to pinpoint several genes linked to drug resistance.