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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 satQ390K, satG321R and satG325R variants led to a 2–3.2 -fold increase in Sb resistance in mrpA−/- parasites. Only satG321R and satG325R induced increased Sb resistance in wild-type parasites. These results reinforce and expand knowledge on the complex nature of Sb resistance in Leishmania parasites., Graphical abstract Image 1, Highlights • mrpA is not an essential gene in Leishmania infantum parasites. • Disruption of mrpA leads to an antimony-hypersensitive phenotype in promastigotes and amastigotes. • L. infantum deploys secondary survival mechanisms to increase antimony tolerance in the absence of MRPA. • Overexpression of ABCC2 confers resistance to antimony in mrpA null mutants. • Different mutations in the sat gene confer resistance to antimony.

Increasing drug resistance towards first line antimony-derived compounds has forced the introduction of novel therapies in leishmaniasis endemic areas including amphotericin B and miltefosine. However, their use is threatened by the emergence and spread of drug-resistant strains. In order to discover stage-dependent resistance genes, we have adapted the Cos-Seq approach through the introduction of macrophage infections in the pipeline. A L. infantum intracellular amastigote population complemented with a L. infantum cosmid library was submitted to increasing concentrations of miltefosine, amphotericin B and pentavalent antimonials in experimental infections of THP-1 cells. For each step of selection, amastigotes were extracted and cosmids were isolated and submitted to next-generation sequencing, followed by subsequent gene-enrichment analyses. Cos-Seq screen in amastigotes revealed four highly enriched loci for antimony, five for miltefosine and one for amphotericin B. Of these, a total of seven cosmids were recovered and tested for resistance in both promastigotes and amastigotes. Candidate genes within the pinpointed genomic regions were validated using single gene overexpression in wild-type parasites and/or gene disruption by means of a CRISPR-Cas9-based approach. This led to the identification and validation of a stage-independent antimony-resistance gene (LinJ.06.1010) coding for a putative leucine rich repeat protein and a novel amastigote-specific miltefosine-resistance gene (LinJ.32.0050) coding for a member of the SEC13 family of WD-repeat proteins. This study further reinforces the power of Cos-Seq approach to discover novel drug-resistance genes, some of which are life-stages specific., Graphical abstract Image 1, Highlights • The Cos-Seq led to the discovery of several new genomic regions selected with drugs. • This work led to the validation of novel drug-resistance genes in Leishmania. • Gene LinJ.06.1010 is involved in antimony resistance in both life stages of the parasite. • Gene LinJ.32.0050 is involved in miltefosine resistance in amastigotes. • The amastigote screen is labour intensive but complements screens in promastigotes.

Current antileishmanial treatment is hampered by limitations, such as drug toxicity and the risk of treatment failure, which may be related to parasitic drug resistance. Given the urgent need for novel drugs, the Drugs for Neglected Diseases initiative (DNDi) has undertaken a drug discovery program, which has resulted in the identification of aminopyrazoles, a highly promising antileishmanial chemical series. Multiple experiments have been performed to anticipate the propensity for resistance development., Current antileishmanial treatment is hampered by limitations, such as drug toxicity and the risk of treatment failure, which may be related to parasitic drug resistance. Given the urgent need for novel drugs, the Drugs for Neglected Diseases initiative (DNDi) has undertaken a drug discovery program, which has resulted in the identification of aminopyrazoles, a highly promising antileishmanial chemical series. Multiple experiments have been performed to anticipate the propensity for resistance development. Resistance selection was performed by successive exposure of Leishmania infantum promastigotes (in vitro) and intracellular amastigotes (both in vitro and in golden Syrian hamsters). The stability of the resistant phenotypes was assessed after passage in mice and Lutzomyia longipalpis sandflies. Whole-genome sequencing (WGS) was performed to identify mutated genes, copy number variations (CNVs), and somy changes. The potential role of efflux pumps (the MDR and MRP efflux pumps) in the development of resistance was assessed by coincubation of aminopyrazoles with specific efflux pump inhibitors (verapamil, cyclosporine, and probenecid). Repeated drug exposure of amastigotes did not result in the emergence of drug resistance either in vitro or in vivo. Selection at the promastigote stage, however, was able to select for parasites with reduced susceptibility (resistance index, 5.8 to 24.5). This phenotype proved to be unstable after in vivo passage in mice and sandflies, suggesting that nonfixed alterations are responsible for the elevated resistance. In line with this, single nucleotide polymorphisms and indels identified by whole-genome sequencing could not be directly linked to the decreased drug susceptibility. Copy number variations were absent, whereas somy changes were detected, which may have accounted for the transient acquisition of resistance. Finally, aminopyrazole activity was not influenced by the MDR and MRP efflux pump inhibitors tested. The selection performed does not suggest the rapid development of resistance against aminopyrazoles in the field. Karyotype changes may confer elevated levels of resistance, but these do not seem to be stable in the vertebrate and invertebrate hosts. MDR/MRP efflux pumps are not likely to significantly impact the activity of the aminopyrazole leads.

Our understanding of folate metabolism in Leishmania has greatly benefited from studies of resistance to the inhibitor methotrexate (MTX). Folates are reduced in Leishmania by the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS) and by pteridine reductase (PTR1). To further our understanding of folate metabolism in Leishmania, a Cos-seq genome-wide gain of function screen was performed against MTX and against the two thymidylate synthase (TS) inhibitors 5-fluorouracil and pemetrexed. The screen revealed DHFR-TS and PTR1 but also the nucleoside transporter NT1 and one hypothetical gene derived from chromosome 31. For MTX, the concentration of folate in the culture medium affected the enrichment pattern for genes retrieved by Cos-seq. We generated a L. infantum DHFR-TS null mutant that was thymidine auxotroph, a phenotype that could be rescued by the addition of thymidine or by transfection of the flavin dependent bacterial TS gene ThyX. In these DHFR-TS null mutants it was impossible to obtain a chromosomal null mutant of PTR1 except if DHFR-TS or PTR1 were provided episomally. The transfection of ThyX however did not allow the elimination of PTR1 in a DHFR-TS null mutant. Leishmania can survive without copies of either DHFR-TS or PTR1 but not without both. Provided that our results observed with the insect stage parasites are also replicated with intracellular parasites, it would suggest that antifolate therapy in Leishmania would only work if both DHFR-TS and PTR1 would be targeted simultaneously., Author summary The protozoan parasite Leishmania is auxotroph for folate and unconjugated pterins and salvages both from the mammalian host. Two enzymes of the folate metabolism pathway, namely the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) and the pteridine reductase 1 (PTR1), are being evaluated for drug discovery and repurposing of existing anti-metabolites. Despite their apparent potential, development of DHFR-TS and PTR1 targeted chemotherapy against Leishmania is still awaiting. Here we revisited folate metabolism at the genomic level and report on the identification of known resistance genes alongside some new ones. Through gene disruption studies we found that L. infantum DHFR-TS null mutants are thymidine auxotroph and that these can be rescued by the bacterial flavin dependent thymidylate synthase ThyX. We also found that PTR1 is essential in the absence of a functional DHFR-TS even in the presence of ThyX or thymidine supplementation, indicating the essential role of reduced pterins or folate beyond thymidine synthesis. This study indicates that simultaneous targeting of DHFR-TS and PTR1 will be required for the development of anti-folate chemotherapy against Leishmania.

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.

Clostridium difficile is the most frequently identified enteric pathogen in patients with nosocomial antibiotic-associated diarrhoea and pseudomembranous colitis. Several clinically isolated C. difficile strains are resistant to antibiotics other than metronidazole and vancomycin. Recently, bacteriocins of lactic acid bacteria have been proposed as an alternative or complementary treatment. The aim of this study was to investigate the inhibitory effect of nisin, a bacteriocin produced by several strains of Lactococcus lactis, against clinical isolates of C. difficile. Nisin Z obtained from culture of L. lactis subsp. lactis biovar. diacetylactis was tested along with commercial nisin A. The effect of nisin A on C. difficile spores was also examined. Nisin A and Z both inhibited the growth of all C. difficile isolates, and MICs were estimated at 6.2 μg ml(-1) for nisin Z and 0.8 μg ml(-1) for nisin A. In addition, C. difficile spores were also susceptible to nisin A (25.6 μg ml(-1)), which reduced spore viability by 40-50%. These results suggested that nisin and hence nisin-producing Lactococcus strains could be used to treat C. difficile-associated diarrhoea.

To achieve drug resistance Leishmania parasite alters gene copy number by using its repeated sequences widely distributed through the genome. Even though homologous recombination (HR) is ascribed to maintain genome stability, this eukaryote exploits this potent mechanism driven by the Rad51 recombinase to form beneficial extrachromosomal circular amplicons. Here, we provide insights on the formation of these circular amplicons by analyzing the functions of the Rad51 paralogs. We purified three Leishmania infantum Rad51 paralogs homologs (LiRad51-3, LiRad51-4 and LiRad51-6) all of which directly interact with LiRad51. LiRad51-3, LiRad51-4 and LiRad51-6 show differences in DNA binding and annealing capacities. Moreover, it is also noteworthy that LiRad51-3 and LiRad51-4 are able to stimulate Rad51-mediated D-loop formation. In addition, we succeed to inactivate the LiRad51-4 gene and report a decrease of circular amplicons in this mutant. The LiRad51-3 gene was found to be essential for cell viability. Thus, we propose that the LiRad51 paralogs play crucial functions in extrachromosomal circular DNA amplification to circumvent drug actions and preserve survival.

SUMMARYAn ideal culture medium forLeishmaniapromastigotes should retain the basic characteristics of promastigotes found in sandflies (morphology and infectivity). Furthermore, the media should not create a bias in experimental settings, thus enabling the proper extrapolation of results. To assess this we studied several established media for promastigote growth. We analysed morphology, viability, cell cycle progression, metacyclic profile, capacity to differentiate into axenic amastigotes and infectivity. Furthermore, using a rational approach from the evaluated media we developed a simple serum-free medium (cRPMI). We report that parasites growing in different media present different biological characteristics and distinctin vitroandin vivoinfectivities. The developed medium, cRPMI, proved to be a less expensive substitute for traditional serum-supplemented media for thein vitromaintenance of promastigotes. In fact, cRPMI is ideal for the maintenance of parasites in the laboratory, diminishing the expected loss of virulence over time typical of the parasite cultivation. Ultimately this report is a clear warning that the normalization of culture media should be a real concern in the field as media-specific phenomena are sufficient to induce biological bias with consequences in infectivity and general parasite biology.

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.

Prior analyses of the proteome of the protozoan parasite Leishmania have underrepresented basic proteins. Here, we applied protein fractionation by isoelectric point (pI) using free-flow electrophoresis (FFE) to study stage-specific expression of basic proteins in this pathogen. Overall, we resolved 2469 protein spots in both the flagellated promastigote and the nonmotile amastigote forms in the basic range by two-dimensional gel electrophoresis (2-DE). Highly basic proteins were enriched by FFE fractionation, allowing many to be identified and characterized for the first time by proteomics analysis. Among proteins upregulated in the promastigote stage, we found glycolytic enzymes and flagellar proteins. Proteins upregulated in the amastigote stage included enzymes involved in gluconeogenesis and fatty acid beta-oxidation. In both life stages, many proteins were found in multiple spots or as proteolytic fragments, suggesting that extensive post-translational modification and processing occur. Interestingly, evidence was obtained suggesting that some of these processes may be stage-specific.

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 .

During its life cycle, the protozoan parasite Leishmania experiences oxidative stress when interacting with macrophages. Reduced pterins are known scavengers of reactive oxygen and nitrogen intermediates. Leishmania has a pteridine reductase, PTR1, whose main function is to provide reduced pterins. We investigated the role of PTR1 in resistance to oxidative and nitrosative stress in Leishmania tarentolae, Leishmania infantum, and Leishmania major PTR1(-/-) mutants. The PTR1(-/-) cells of the three species were more sensitive to H2O2- and NO-induced stress. Using a fluorescent probe allowing ROI quantification, we demonstrated an increase in intracellular oxidant molecules in the PTR1(-/-) mutants. The disruption of PTR1 increased metacyclogenesis in L. infantum and L. major. We purified metacyclic parasites from PTR1(-/-) mutants and control cells and tested their intracellular survival in the J774 mouse cell line and in human monocyte-derived macrophages. Our results showed that PTR1(-/-) null mutants survived less in both macrophage models compared to control cells and this decrease was more pronounced in macrophages activated for oxidant production. This study demonstrates that one physiological role of reduced pterins in Leishmania is to deal with oxidative and nitrosative species, and a decreased ability to provide reduced pterins leads to decreased intracellular survival.

BACKGROUND Visceral leishmaniasis (VL) is an opportunistic infection that can occur among patients infected with human immunodeficiency virus type 1 (HIV-1) in areas where both infections are endemic. Highly active antiretroviral therapy has decreased the incidence of VL in southern Europe among HIV-1-infected patients, but VL is still observed among patients with low CD4 cell counts, and most coinfected patients receiving highly active antiretroviral therapy experienced relapse, despite initial treatment with liposomal amphotericin B. METHODS Through long-term monitoring of VL in 10 patients with HIV-1 infection and/or AIDS, we compared parasite strains derived from primary and secondary episodes of VL. All the patients have received many courses of amphotericin B treatment and/or prophylaxis. RESULTS Through molecular techniques, we have shown that secondary episodes of VL can be attributable to relapse (7 of 10 episodes) or reinfection (3 of 10). We developed an assay to measure amphotericin B susceptibility and found no evidence of decreased susceptibility among strains isolated from patients, some of whom were infected with the same isolate for up to 10 years. CONCLUSIONS This apparent absence of resistance, as determined by in vitro susceptibility testing, has important consequences and suggests that amphotericin B will remain a useful drug of choice against VL, even after repetitive treatments or prophylactic use.

Visceral leishmaniasis is now recognized as an opportunistic disease in individuals infected with human immunodeficiency virus type 1 (HIV-1). Although the usefulness of HIV-1 protease inhibitors (PIs) in antiretroviral regimens is well documented, little is known about their potential impact in the setting of Leishmania/HIV-1 coinfections. We now report that, although selected PIs do not inhibit the growth of Leishmania infantum promastigotes alone in culture, these drugs significantly inhibit the intracellular survival of parasites in phorbol myristate acetate‐differentiated THP-1 macrophages and human primary monocyte-derived macrophages (MDMs). Furthermore, a field isolate of Leishmania donovani resistant to sodium stibogluconate (SbV), one of the drugs most commonly used to treat leishmaniasis, is equally susceptible to the tested PIs compared with a sensitivestrain,thussuggestingthatresistancetoSbVdoesnotresultincross-resistancetoPIs.Importantly,the efficacyofPIstoreducetheintracellulargrowthofLeishmaniaparasitesisalsoobservedinMDMscoinfectedwith HIV-1. In the wake of the extensive and rapid expansion of the worldwide AIDS pandemic, several infectious diseases have developed into AIDS-associated opportunistic infections. This phenomenon complicates the therapeutic measures aimed at controlling HIV-1, the causative agent of AIDS, and the various opportunistic pathogens

Cutaneous leishmaniasis (CL) is a major health problem in endemic areas of Iran. The pentavalent antimony (SbV) based drug Glucantime is the first line of treatment for CL in Iran, but recently SbV-resistant Leishmania tropica isolates derived from unresponsive patients were reported. We show in this study that these resistant parasites are cross-resistant to the other SbV-containing drug Pentostam and at least for one isolate also to amphotericin B. However, these resistant isolates were shown to be sensitive to miltefosine and paromomycin. The latter two drugs could thus be useful alternatives for the treatment of leishmaniasis in Iran even for SbV-resistant isolates.

The therapeutic mainstay against the protozoan parasite Leishmania is still based on the antiquated pentavalent antimonials (Sb(V)), but resistance is increasing in several parts of the world. Resistance is now partly understood in laboratory isolates, but our understanding of resistance in field isolates is lagging behind. We describe here a comparative analysis of a genetically related pair of Sb(V)-sensitive and -resistant Leishmania donovani strains isolated from kala-azar patients. The resistant isolate exhibited cross-resistance to other unrelated Leishmania drugs including miltefosine and amphotericin B. A comparative proteomics screen has highlighted a number of proteins differentially expressed suggesting that programmed cell death (PCD) is modified in the resistant parasite. Indeed drug-induced PCD progression was altered in the Sb(V)-resistant strain as determined using early and late markers of apoptosis. Two proteins, the heat shock protein HSP83 and the small kinetoplastid calpain-related protein (SKCRP14.1) were shown to be intimately implicated in the drug-induced PCD phenotype. HSP83 increased drug resistance and reduced drug-mediated PCD activation by interfering with the mitochondrial membrane potential, whereas SKCRP14.1 promoted antimonial-induced PCD but protected against miltefosine-induced PCD. This study highlights the important role of PCD in drug susceptibility/resistance in the protozoan parasite Leishmania.

Live-vector human immunodeficiency virus (HIV) vaccines are an integral part of a number of HIV vaccine regimens currently under evaluation that have yielded promising results in pre-clinical testing. In this report, a non-pathogenic protozoan parasitic vector,Leishmania tarentolae, which shares common target cells with HIV-1, was used to express full-length HIV-1 Gag protein. Immunization of BALB/c mice with recombinantL. tarentolaeled to the expansion of HIV-1 Gag-specific T cells and stimulated CD8+T cells to produce gamma interferon in response to specific viral Gag epitopes. A booster immunization with recombinantL. tarentolaeelicited effector memory HIV-1 Gag-specific CD4+T lymphocytes and increased antibody titres against HIV-1 Gag. Most importantly, immunization of human tonsillar tissue culturedex vivowith Gag-expressingL. tarentolaevaccine vector elicited a 75 % decrease in virus replication following exposure of the immunized tonsils to HIV-1 infection. These results demonstrated that recombinantL. tarentolaeis capable of eliciting effective immune responses in mice and human systems, respectively, and suggest that this novel non-pathogenic recombinant vaccine vector shows excellent promise as a vaccination strategy against HIV-1.

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 resistance of clinical isolates of Leishmania donovani to sodium antimony gluconate (SAG), the mainstay of treatment in Indian visceral leishmaniasis, has become a critical issue in India. The present work investigates the mechanism of resistance to SAG in parasites isolated from patients who are unresponsive to SAG.Susceptibility to SAG as determined in vitro with intracellular amastigotes correlated well with the clinical response. The ABC transporter gene MRPA was amplified in resistant field isolates as part of an extrachromosomal circle. Co-amplification of the pterin reductase gene (PTR1) and MRPA suggests amplification of the H locus in SAG-resistant isolates. Amplification of MRPA was correlated to increased RNA as determined by real-time PCR. MRPA is an ABC-thiol transporter, and cysteine and glutathione were increased in the resistant isolates. Ornithine decarboxylase (a rate limiting enzyme in polyamine biosynthesis), and gamma-glutamylcysteine synthetase (a rate limiting enzyme in glutathione biosynthesis), the two building blocks of the main cellular thiol trypanothione, were overexpressed in some of the resistant isolates.A variety of resistance mechanisms to SAG, most of them consistent with a model based on the study of resistance in vitro, were present in clinical isolates from the same geographical region.

Serine hydroxymethyltransferase (SHMT) catalyses the reversible conversion of serine and tetrahydrofolate to glycine and methylene-tetrahydrofolate. The recent completion of the genome sequence of Leishmania major revealed the presence of two genes coding for two isoforms of this protein. In silico analysis showed that one isoform had an extension at its N-terminus and was predicted to localize to the mitochondrion. The situation is different in other kinetoplastid parasites with only one SHMT encoding gene in Trypanosoma cruzi and no SHMT encoding gene in Trypanosoma brucei. The two L. major SHMT genes were cloned in frame with the green fluorescent protein and the resulting fusion proteins showed differential localization: the short form (SHMT-S) was found in the cytosol while the long one (SHMT-L) was found in an organelle that has hallmarks of the parasite mitochondrion. Indeed, SHMT-L had a similar cellular fractionation pattern as the mitochondrial HSP60 as determined by digitonin fractionation. Both SHMT-S and SHMT-L genes were expressed preferentially in the amastigote stage of the parasite and the RNA levels of SHMT-L could be modulated by glycine, serine, and folate. Overexpression of SHMT-S increased resistance to the antifolate methotrexate and to a lower level to the inhibitor thiosemicarbazide in a rich folate containing medium. These findings suggest that folate metabolism is compartmentalised in Leishmania and that SHMT RNA levels are responsive to environmental conditions.

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.

Protozoan parasites of the genus Leishmania are found as promastigotes in the sandfly vector and as amastigotes in mammalian macrophages. Mechanisms controlling stage-regulated gene expression in these organisms are poorly understood. Here, we applied a comprehensive approach consisting of protein prefractionation, global proteomics and targeted DNA microarray analysis to the study of stage differentiation in Leishmania. By excluding some abundant structural proteins and reducing complexity, we detected and identified numerous novel differentially expressed protein isoforms in L. infantum. Using 2-D gels, over 2200 protein isoforms were visualized in each developmental stage. Of these, 6.1% were strongly increased or appeared unique in the promastigote stage, while the relative amounts of 12.4% were increased in amastigotes. Amastigote-specific protein isoform and mRNA expression trends correlated modestly (53%), while no correlation was found for promastigote-specific spots. Even where direction of regulation was similar, fold-changes were more modest at the RNA than protein level. Many proteins were present in multiple spots, suggesting that PTM is extensive in this organism. In several cases, different isoforms appeared to be specific to different life stages. Our results suggest that post-transcriptional controls at translational and post-translational levels could play major roles in differentiation in Leishmania parasites.

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.

The protozoan parasite Leishmania relies on the uptake of folate and pterin from the environment to meet its nutritional requirements. We show here that a novel gene (folate transporter 1 (FT1)) deleted in a Leishmania infantum methotrexate-resistant mutant corresponds to the main folate transporter (Km, 410 nm). FT1 was established as the main folate transporter by both gene transfection and by targeted gene deletion. Modulation of the expression of FT1 by these manipulations altered the susceptibility of Leishmania cells to methotrexate. Folate transport was stage-regulated with higher activity in the logarithmic phase and less in the stationary phase. FT1 fused to green fluorescent protein led to the observation that FT1 was located in the plasma membrane in the logarithmic phase but was retargeted to an intracellular organelle followed by a degradation of the protein in stationary phase. Leishmania has several folate transporters with different characteristics, and the growth stage-related activity of at least one transporter is regulated post-translationally.

Arsenicals and antimonials are first line drugs for the treatment of trypanosomal and leishmanial diseases. To create the active form of the drug, Sb(V) must be reduced to Sb(III). Because arsenic and antimony are related metalloids, and arsenical resistant Leishmania strains are frequently cross-resistant to antimonials, we considered the possibility that Sb(V) is reduced by a leishmanial As(V) reductase. The sequence for the arsenate reductase of Saccharomyces cerevisiae, ScAcr2p, was used to clone the gene for a homologue, LmACR2, from Leishmania major. LmACR2 was able to complement the arsenate-sensitive phenotype of an arsC deletion strain of Escherichia coli or an ScACR2 deletion strain of Saccharomyces cerevisiae. Transfection of Leishmania infantum with LmACR2 augmented Pentostam sensitivity in intracellular amastigotes. LmACR2 was purified and shown to reduce both As(V) and Sb(V). This is the first report of an enzyme that confers Pentostam sensitivity in intracellular amastigotes of Leishmania. We propose that LmACR2 is responsible for reduction of the pentavalent antimony in Pentostam to the active trivalent form of the drug in Leishmania.

The protozoan parasite Leishmania is a folate and pterin auxotroph. The main biopterin transporter (BT1) and pterin reductase (PTR1) have already been characterized in Leishmania. In this study, we have succeeded in generating a BT1 and PTR1 null mutant in the same Leishmania tarentolae strain. These cells are viable with growth properties indistinguishable from wildtype cells. However, in response to the inactivation of BT1 and PTR1, at least one of the folate transporter genes was deleted, and the level of the folylpolyglutamate synthetase activity was increased, leading to increased polyglutamylation of both folate and methotrexate (MTX). Secondary events following gene inactivation should be considered when analyzing a phenotype in Leishmania. The BT1/PTR1 null mutant is hypersensitive to MTX, but in a step-by-step fashion, we could induce resistance to MTX in these cells. Several resistance mechanisms were found to co-exist including a reduced folate and MTX accumulation, demonstrating that cells with no measurable biopterin uptake but also greatly reduced folate uptake are viable, despite their auxotrophy for each of these substrates. The resistant cells have also amplified the gene coding for the MTX target dihydrofolate reductase. Finally, we found a marked reduction in MTX polyglutamylation in resistant cells. These studies further highlight the formidable ability of Leishmania cells to bypass the blockage of key metabolic pathways.

Leishmaniais an intracellular pathogen that replicates inside macrophages. Activated macrophages produce a specific subset of cytokines that play an important role in the control ofLeishmaniainfections. As part of our interest in developing suicide parasites that produce abortive infections for the purposes of vaccination, we engineered recombinantLeishmania majorstrains producing biologically active granulocyte-macrophage colony-stimulating factor (GM-CSF). We showed that GM-CSF is being produced in the phagosomes of infected macrophages and that it can be detected in the culture supernatants of both infected macrophages and extracellular parasites. Our data support the notion that GM-CSF secreted by both developmental forms of recombinantL. majorcan activate macrophages to produce high levels of proinflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and IL-18 and various chemokines including RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MIP-2/CXCL2, and MCP-1/CCL2, which enhance parasite killing. Indeed, GM-CSF-expressing parasites survive poorly in macrophages in vitro and produce delayed lesion development in susceptible BALB/c mice in vivo. Selective killing of intracellularLeishmaniaexpressing cytokine genes capable of activating cellular responses may constitute a promising strategy to control and/or prevent parasitic infections.

Folates are polyglutamylated in most organisms, and in cancer cells the polyglutamylation of folates and of the antifolate methotrexate (MTX) is an important determinant of MTX susceptibility. The folylpolyglutamate synthetase (FPGS) responsible for polyglutamylation of folates was recently characterized in the parasite Leishmania. We show here that MTX is polyglutamylated in Leishmania tarentolae and that triglutamates are the predominant form. The glutamate chain length of MTX increases significantly in Leishmania cells transfected with the FPGS gene and decreases in cells with one FPGS allele disrupted. Modulation in the expression of the FPGS gene also has a profound effect on MTX susceptibility and this effect was found to be dependent on the folate concentration of the medium. In the folate-rich medium SDM-79, overexpression of FPGS will confer MTX resistance while in M-199 medium, which has much less folates, FPGS transfectants are more sensitive to MTX. Cells with one allele of FPGS disrupted are more resistant to MTX in low folate medium. The modulation of FPGS expression affects both the short-term and long-term accumulation of folate and MTX, showing a marked decrease in accumulation in the FPGS haploid mutant. This differential accumulation was mediated by decreased influx of the drug into the cell. Finally, the analysis of MTX-resistant Leishmania mutants indicated that the presence of shorter glutamate chains on MTX is correlated with MTX resistance.

The protozoan parasites Leishmania utilize a pteridine-reducing enzyme, pteridine reductase (PTR1), to bypass antifolate inhibition. The crystal structure of PTR1 from L. tarentolae has been solved as a binary complex with NADPH at 2.8 A resolution. The structure was solved by molecular-replacement techniques using the recently reported L. major PTR1 structure as a search model. Comparisons of the present structure with the L. major PTR1 allowed us to identify regions of flexibility in the molecule. PTR1 is a member of the growing family of short-chain dehydrogenases (SDR) which share the characteristic Tyr(Xaa)(3)Lys motif in the vicinity of the active site. The functional enzyme is a tetramer and the crystallographic asymmetric unit contains a tetramer with 222 point-group symmetry.

Drug resistance is complicating the treatment of parasitic diseases. We review here the basic mechanisms of parasite resistance in malaria, sleeping sickness, leishmaniasis and common helminthiases. Parasites resort to multiple biochemical means to achieve resistance and we have begun to isolate and characterize the genes/proteins implicated in resistance. Understanding drug resistance is essential for the control of parasitic diseases.

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.

Protocols for DNA electroporation in Leishmania promastigote cells are well established. More recently, in vitro culture of axenic Leishmania amastigotes became possible. We have established conditions for DNA transformation of axenically grown Leishmania infantum amastigotes. Parameters for DNA electroporation of Leishmania axenic amastigotes were systematically studied using luciferase-mediated transient transfection. Cell lines expressing stable luciferase activity were then selected, and their ability to be used in an in vitro drug screening procedure was determined. A model was established, using axenic amastigotes expressing luciferase activity, for rapidly determining the activity of drugs directly against both axenic and intracellular amastigotes. For intracellular amastigotes, the 50% effective concentrations of pentamidine, sodium stibogluconate (Pentostam), meglumine (Glucantime), and potassium antimonyl tartrate determined with the luciferase assay were 0.2 μM (0.12 μg/ml), 55 μg/ml, 95 μg/ml, and 0.12 μg/ml, respectively; these values are in agreement with values determined by more labor-intensive staining methods. We also showed the usefulness of luciferase-expressing parasites for analyzing drug resistance. The availability of luciferase-expressing amastigotes for use in high-throughput screening should facilitate the search for new antileishmanial drugs.

Roy, G., Kundig, C., Olivier, M., Papadopoulou, B., and Ouellette, M. 2001. Adaptation of Leishmania cells to in vitro culture results in a more efficient reduction and transport of biopterin. Experimental Parasitology97, 161–168. Leishmania major and Leishmania donovani cells freshly isolated from infected animals divided slowly as axenic promastigotes but the addition of biopterin in the culture medium greatly enhanced their growth. However, when cells were subjected to serial passages and adapted to culture, this growth-promoting effect of biopterin was no longer observed. Genetic analysis of these culture-adapted Leishmania cells demonstrated that the genes coding for the pterin reductase PTR1 or for the biopterin transporter BT1 were over-expressed. This suggests that Leishmania cells adapted to culture were more efficient in utilizing biopterin, an essential growth factor in Leishmania.

The protozoan parasite Leishmania resists the antifolate methotrexate (MTX) by amplifying the R locus dihydrofolate reductase-thymidylate synthase ( dhfr-ts ) gene, the H locus ptr1 pterin reductase gene, and finally by mutation in a common folate/MTX transporter. Amplification of dhfr-ts has never been observed in Leishmania tarentolae MTX resistant mutants while ptr1 amplification is common. We have selected a L.tarentolae ptr1 null mutant for MTX resistance and observed dhfr-ts amplification in this mutant demonstrating that once a preferred resistance mechanism is unavailable, a second one will take over. By introducing the ptr1 gene at the R locus and the dhfr-ts gene at the H locus by gene targeting, we investigated the role of the resistance gene and the locus on the rate of gene amplification. Transfection studies indicated that ptr1 gave higher levels of MTX resistance than dhfr-ts. Consistent with this, when ptr1 was present as part of either the H locus or the R locus it was invariably amplified, while dhfr-ts was only amplified when ptr1 was inactivated. When dhfr-ts was present in a ptr1 null background on both the H locus and the R locus, amplification from the H locus was more frequent suggesting that both the gene and the locus are determining the frequency of gene amplification in Leishmania.

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.

Pentamidine is a second-line agent in the treatment of leishmaniasis whose mode of action and resistance mechanism are not well understood. In this work, we show that the intracellular ABC protein PRP1 (pentamidine resistance protein 1) (ABCC7) can confer resistance to pentamidine in Leishmania sp. parasites in the intracellular stage.

Gene amplification is frequently observed in Leishmania cells selected for drug resistance. By gene targeting we have tagged both alleles of the H locus of Leishmania tarentolae with the neomycin and hygromycin phosphotransferase genes ( neo and hyg ). Selection of these recombinant parasites for low level methotrexate resistance led to amplification of the H locus as part of linear amplicons. The availability of tags has permitted us to determine that both alleles can be amplified in the same cell and that chromosomal deletions are frequent. When methotrexate concentration was increased in subsequent selection steps, circles were observed in several mutants. We have introduced a hyg marker into linear amplicons to test whether the circles originated from linear amplicons. After selection with a high methotrexate concentration, circles with the hyg marker were observed, showing that circles can indeed be formed from linear amplicons. The tagging of H locus alleles permits appreciation of the extent of genetic rearrangements leading to amplicon formation in Leishmania cells selected for drug resistance.

Leishmania tarentolae promastigotes were selected step by step for resistance to sodium stibogluconate (Pentostam). Mutants resistant to antimony-containing drugs and cross-resistant to arsenite were therefore obtained. Amplification of one common locus was observed in several independent sodium stibogluconate-resistant mutants, and the locus amplified was novel. The copy number of the amplified locus was related to the level of resistance to pentavalent antimony. The gene responsible for antimony resistance was isolated by transfection and was shown to correspond to an open reading frame coding for 770 amino acids. The putative gene product did not exhibit significant homology with sequences present in data banks, and the putative role of this protein in antimony resistance is discussed.

The Leishmania short-chain dehydrogenase/reductase gene ptr1 has been isolated while characterizing antifolate-resistant Leishmania mutants. PTR1 is active as a tetramer and can reduce pterins and folates. PTR1 has several of the hallmarks of the short chain dehydrogenase/reductase family, including a glycine rich co-factor binding site at its N-terminus, and the consensus catalytic site TyrXaa3Lys. To start probing the structure/function of PTR1, we have generated by site-directed mutagenesis five mutants, either in the co-factor-binding site, Y38D or in the catalytic site Y195F, Y195W, K199R or in a PTR1-specific region, Y175F. The mutated versions of PTR1 were studied in vivo in Leishmania and at the biochemical level using purified proteins. The Y175F mutant showed properties similar to wild-type PTR1 in every aspect tested, but all the other mutants were inactive even if they were purified as tetramers. To test the ability of the mutated PTR1 versions to bind its co-factor and substrates, trypsin digestion experiments were carried out under conditions upon which binding will prevent wild-type PTR1 of being digested by trypsin. The wild-type PTR1 as well as Y175F and Y195F mutants were protected against trypsin digestion whereas Y38D and K199R mutants were not. Mutations in regions involved in co-factor binding (Y38D) or in catalytic site (K199R) altered the binding of the ligands, explaining why those protein versions are inactive.

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.

Resistance to the oxyanion arsenite in the parasite Leishmania is multifactorial. We have described previously the frequent amplification of the ABC transporter gene pgpA , the presence of a non‐PgpA thiol–metal efflux pump and increased levels of glutathione and trypanothione in resistant cells. Other loci are also amplified, although their role in resistance is unknown. By gene transfection, we have characterized one of these novel genes. It corresponds to gsh1 , which encodes γ‐glutamylcysteine synthetase, an enzyme involved in the rate‐limiting step of glutathione biosynthesis. Transfection of gsh1 in wild‐type cells increased the levels of glutathione and trypanothione to levels found in resistant mutants. These transfectants were not resistant to metals. However, when gsh1 was transfected in partial revertants, it conferred resistance. As pgpA is frequently co‐amplified with gsh1 , we co‐transfected the two genes into both wild‐type and partial revertants. Arsenite resistance levels in wild‐type cells could be accounted for by the contribution of PgpA alone. In the partial revertant, the gsh1 and pgpA gene product acted synergistically. These results support our previous suggestion that PgpA recognizes metals conjugated to thiols. Furthermore, amplification of gsh1 overcomes the rate‐limiting step in the synthesis of trypanothione, contributing to resistance. In addition, the results suggest that at least one more factor acts synergistically with the gsh1 gene product.

Overproduction of the short-chain dehydrogenase/reductase PTR1 confers resistance to the dihydrofolate reductase inhibitor methotrexate in the protozoan parasite Leishmania. Genetic analysis has previously implicated PTR1 in pterin and folate metabolism. PTR1 was purified from a fusion protein expressed in Escherichia coli. Purified PTR1 exhibits NADPH-dependent biopterin, dihydrobiopterin, folate, and dihydrofolate reductase activities. The highest activity was found with the most oxidized pterins. The active protein was found to be a tetramer as demonstrated by gel-filtration chromatography. Kinetic constants (K(m)), as determined by double-reciprocal plots, were calculated for NADPH and for several of PTR1's substrates. The PTR1 of Leishmania tarentolae had a K(m) of 16.9 microM for the cofactor NADPH and K(m) values ranging from 3.5 to 85 microM for the various substrates. The dissociation constant (KD), as determined by fluorescence titration, for NADPH was estimated to be 130 microM. The biochemical characterization of this important and novel enzyme involved in folate and pterin metabolism of Leishmania should be useful for structure-function analysis and for developing specific inhibitors against this putative important chemotherapeutic target.

Parasitic protozoa belonging to the order Kinetoplastida contain trypanothione as their major thiol. Trypanothione reductase (TR), the enzyme responsible for maintaining trypanothione in its reduced form, is thought to be central to the redox defence systems of trypanosomatids. To investigate further the physiological role of TR in Leishmania, we attempted to create TR-knockout mutants by gene disruption in L. donovani and L. major strains using the selectable markers neomycin and hygromycin phosphotransferases. TR is likely to be an important gene for parasite survival since all our attempts to obtain a TR null mutant in L. donovani failed. Instead, we obtained mutants with a partial trisomy for the TR locus where, despite the successful disruption of two TR alleles by gene targeting, a third TR copy was generated as a result of genomic rearrangements involving the translocation of a TR-containing region to a larger chromosome. Mutants of L. donovani and L. major possessing only one wild-type TR allele express less TR mRNA and have lower TR activity compared with wild-type cells carrying two copies of the TR gene. Significantly, these mutants show attenuated infectivity with a markedly decreased capacity to survive intracellularly within macrophages, provided that the latter are producing reactive oxygen intermediates.

The thymidine kinase gene of Herpes simplex type-1 virus was transfected into several Leishmania species to create drug-sensitive mutants. Expression of the thymidine kinase gene is not by itself harmful to Leishmania cells but it is capable of phosphorylating ganciclovir, a nucleoside analog, into a highly toxic product. In addition to the generation of Leishmania promastigotes highly sensitive to ganciclovir, the thymidine kinase gene was expressed similarly by amastigotes engulfed either by murine or by human macrophages. Leishmania major amastigotes expressing thymidine kinase were eliminated by 85% when treated with ganciclovir. Selective killing of parasites expressing suicide genes at their infective stage could suggest novel strategies for controlling parasitic infections.

Transfection of pgpA into Leishmania confers resistance to arsenite and antimonials. By gene targeting mediated by homologous recombination the two alleles of the pgpA gene of a L. tarentolae wild-type cell were disrupted sequentially with the neomycin and hygromycin phosphotransferase genes. This pgpA null mutant showed an increased sensitivity to arsenite and antimonite. In addition, the L. tarentolae pgpA null mutant exhibited a decreased intracellular survival inside murine macrophages. The observed phenotypes were reverted to levels not statistically different than wild-type when an intact pgpA gene was introduced into the null mutant. Disruption of the pgpA chromosomal locus in an arsenite resistant mutant indicated that PgpA is not essential for resistance to oxyanions, although it might be required in the early steps of selection when resistance is being established.

The main line of defense now available against parasitic protozoa--which are responsible for major diseases of humans and domestic animals--is chemotherapy. This defense is being eroded by drug resistance and, with few new drugs in the pipeline, prevention and circumvention of resistance are medical and veterinary priorities. Although studies of resistance mechanisms in parasites have lagged behind similar studies in bacteria and cancer cells, the tools to tackle this problem are rapidly improving. Transformation with exogenous DNA is now possible with all major parasitic protozoa of humans. Hence, putative resistance genes can be tested in sensitive protozoa, allowing an unambiguous reconstruction of resistance mechanisms. Gene cloning, the polymerase chain reaction, and monoclonal antibodies against resistance-related proteins have made it possible to analyze potential resistance mechanisms in the few parasites that can be obtained from infected people. Hence, the prospect of applying new knowledge about resistance mechanisms to parasites in patients is good, even though today virtually all knowledge pertains to parasites selected for resistance in the laboratory. Resistance mechanisms highlighted in this review include: 1. Decrease of drug uptake because of the loss of a transporter required for uptake. This decrease contributes to resistance to arsenicals and diamidines in African trypanosomes. 2. The export of drugs from the parasite by P-glycoproteins and other traffic ATPases. This export could potentially be an important mechanism of resistance, as these proteins are richly represented in the few protozoa analyzed. There are indications that such transmembrane transporters can be involved in resistance to emetine in Entamoeba spp., to mefloquine in Plasmodium spp., and to antimonials in Leishmania spp. 3. The possible involvement of the P-glycoprotein encoded by the Plasmodium falciparum pfmdr1 gene in chloroquine resistance. We present the available data that lead to the conclusion that overproduction of the wild-type version of this protein results in chloroquine hypersensitivity rather than resistance. 4. The involvement of the PgpA P-glycoprotein of Leishmania spp. in low-level resistance to arsenite and antimonials. We raise the possibility that this protein transports glutathione conjugates of arsenite and antimonials rather than the compounds themselves. 5. Loss of drug activation as the main mechanism of metronidazole resistance in Trichomonas and Giardia spp. Recent evidence indicates that a decrease of the proximal cellular electron donor for metronidazole activation, ferredoxin, is the main cause of resistance in Trichomonas. 6. Resistance arising through alteration of drug targets. The amino acid substitutions in the dihydrofolate reductase-thymidylate synthase of Plasmodium spp. are good examples of this mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Background Miltefosine (MF) is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance. Methods/Principal Findings Analysis of the genome assemblies revealed several independent point mutations in a P-type ATPase involved in phospholipid translocation. Mutations in two other proteins—pyridoxal kinase and α-adaptin like protein—were also observed in independent mutants. The role of these proteins in the MF resistance was evaluated by gene transfection and gene disruption and both the P-type ATPase and pyridoxal kinase were implicated in MF susceptibility. The study also highlighted that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes. Conclusions/Significance Whole genome sequencing was used to pinpoint known and new resistance markers associated with MF resistance in the protozoan parasite Leishmania. The study also demonstrated the polyclonal nature of a resistant population with individual cells with varying susceptibilities and genotypes., Author Summary Leishmania spp. are parasitic protozoa responsible for a spectrum of diseases known as leishmaniasis. There are few drugs available for the treatment of these diseases, and miltefosine is the first oral drug used in treatment of visceral leishmaniasis, a form of the disease that can be lethal if not treated. In this study, we seek to understand the mechanism of action and identify targets of the drug by generating promastigote mutants highly resistant to miltefosine. Two independent mutants were submitted to short read whole genome sequencing. Genome analysis of these mutants has permitted us to identify point mutations in three genes (P-type ATPase, pyridoxal kinase and α-adaptin like protein) that were also present in other independent miltefosine resistant mutants. Some of the new genes identified here could be useful as potential markers for miltefosine resistance in Leishmania. Moreover, our approach has permitted us to highlight that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes. This may have practical applications while studying resistance.

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.

Extrachromosomal amplicons are frequently observed in drug-resistant Leishmania. A dominant selectable marker, the neomycin phosphotransferase gene, was introduced by gene targeting in a circular amplicon derived from the H locus of Leishmania in a mutant cell. This recombinant amplicon was isolated and transfected in a wild-type cell. The amplicon was kept in the wild-type cells, provided the selective pressure was maintained, suggesting that it was capable of autonomous replication. Novel Leishmania expression vectors suited for stable transfections were made to isolate, by a high transformation assay, the putative origin of replication in the amplicons. However, these plasmids, which did not contain a single Leishmania nucleotide, were found as extrachromosomal circular oligomers in Leishmania transfectants. Their relative stability, in addition to changes in their methylation pattern, indicated that these plasmids were most likely replicating. No specific sequences seem to be required for replication (and expression) in Leishmania, therefore precluding the isolation of origins of replication by genetic transformation.