Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability.     

Antimonial treatment of visceral leishmaniasis: are current in vitro susceptibility assays adequate for prognosis of in vivo therapy outcome?

In most of the Indian subcontinent, the first line treatment for visceral leishmaniasis (VL) is sodium stibogluconate (SSG), an antimonial drug, but the efficacy of the drug varies according to region. We aimed to characterize the in vitro antimony susceptibility of clinical isolates of Nepalese VL patients, and to correlate this in vitro parasite phenotype to clinical therapy outcome. Thirty-three clinical isolates of L. donovani were taken from patients with known disease history. These isolates were typed and the susceptibility of intracellular amastigotes to pentavalent (SbV) and trivalent (SbIII) antimonials was determined. We observed (i) 22 SbV-resistant isolates out of 33 tested and (ii) 3 SbIII-resistant isolates out of 12 tested. Amongst the latter, there were three combinations of in vitro phenotypes: (i) parasites sensitive (n=4) or (ii) resistant to both drugs (n=3) and (iii) resistant to SbV only (n=5). There was no geographical clustering in terms of in vitro susceptibility. The relation between the in vitro susceptibility to antimonials and the corresponding in vivo treatment outcome was ambiguous. Our results highlight the need to adjust the currently used Leishmania drug susceptibility assays if they are to be used for prognosis of in vivo SSG treatment outcome.     

Antimonial resistance in Leishmania donovani is associated with increased in vivo parasite burden

Leishmania donovani is an intracellular protozoan parasite that causes visceral leishmaniasis (VL). Antimonials (SSG) have long been the first-line treatment against VL, but have now been replaced by miltefosine (MIL) in the Indian subcontinent due to the emergence of SSG-resistance. Our previous study hypothesised that SSG-resistant L. donovani might have increased in vivo survival skills which could affect the efficacy of other treatments such as MIL. The present study attempts to validate these hypotheses. Fourteen strains derived from Nepalese clinical isolates with documented SSG-susceptibility were infected in BALB/c mice to study their survival capacity in drug free conditions (non-treated mice) and in MIL-treated mice. SSG-resistant parasites caused a significant higher in vivo parasite load compared to SSG-sensitive parasites. However, this did not seem to affect the strains' response to MIL-treatment since parasites from both phenotypes responded equally well to in vivo MIL exposure. We conclude that there is a positive association between SSG-resistance and in vivo survival skills in our sample of L. donovani strains which could suggest a higher virulence of SSG-R strains compared to SSG-S strains. These greater in vivo survival skills of SSG-R parasites do not seem to directly affect their susceptibility to MIL. However, it cannot be excluded that repeated MIL exposure will elicit different adaptations in these SSG-R parasites with superior survival skills compared to the SSG-S parasites. Our results therefore highlight the need to closely monitor drug efficacy in the field, especially in the context of the Kala-azar elimination programme ongoing in the Indian subcontinent.     

Inhibition of Leishmania donovani promastigote DNA topoisomerase I and human monocyte DNA topoisomerases I and II by antimonial drugs and classical antitopoisomerase agents

We have compared the inhibitor sensitivities of DNA topoisomerase I (TOPI) from Leishmania donovani promastigotes and TOPs I and II of human monocytes using pentavalent and trivalent antimonials (SbV, SbIII) and classical TOP inhibitors. Bis-benzimidazoles (Hoechst-33258 and -33342) were potent inhibitors of both parasite and human TOPI, but Hoechst-33342 was markedly less cytotoxic to promastigotes than to monocytes in vitro. Leishmania donovani was also considerably less sensitive than monocytes to camptothecin, both at enzyme and cellular levels. Sodium stibogluconate (SSG) was the only antimonial to inhibit TOPI, exhibiting a significant (P < 0.05) 3-fold greater potency against the L. donovani enzyme but showed low cytotoxicities against intact promastigotes. The SbV meglumine antimoniate failed to inhibit TOPI and showed negligible cytotoxicities, whereas SbIII drugs were lethal to parasites and monocytes yet poor inhibitors of TOPI. Monocyte TOPII was inhibited by bis-benzimidazoles and insensitive to antimonials and camptothecin. The disparity between the high leishmanicidal activity and low anti-TOPI potency of SbIII indicates that in vivo targeting of L. donovani TOPI by the reductive pathway of antimonial activation is improbable. Nevertheless, the potent direct inhibition of TOPI by SSG and the differential interactions of camptothecin with L. donovani and human TOPI support the possibility of developing parasite-specific derivatives.     

Drug uptake and modulation of drug resistance in Leishmania by an aquaglyceroporin

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.     

Leishmania spp.: development of pentostam-resistant clones in vitro by discontinuous drug exposure

Antimony unresponsiveness in mucocutaneous and visceral leishmaniasis is a serious clinical problem. Information on the mechanisms and characteristics of drug resistance in parasites that suggest chemotherapeutic strategies to overcome resistance is of practical importance. We developed nine lines of Leishmania resistant to drugs, the major emphasis being on pentavalent antimony (Sb) complexed to carbohydrate in the form of sodium stibogluconate (Pentostam), one of the only two antileishmanial agents with a clearly favorable therapeutic index. Resistance to Pentostam (33- to 212-fold increase) was obtained in promastigotes of Leishmania in vitro by exposure to gradually increasing concentrations of drug over several passages. Resistance to Sb was found to be either stable or unstable. Stable resistance to Sb required (greater than 3) exposures of the initial sensitive clones to Pentostam and tended to stabilize with increased time under pressure. In general, resistance obtained in a clone after only a few (less than or equal to 3) step treatments was low and unstable. Differences in the susceptibility to Pentostam were found between strains isolated from patients with American cutaneous leishmaniasis. In addition, natural isolates of Leishmania from patients represented a heterogeneous population of parasites as demonstrated by a biphasic concentration response to Sb (typical of mixed population dynamics) and by marked differences in susceptibility to Pentostam among clones prepared from single isolates. These results suggest that the emergence of parasite resistance to antimonial treatment is a potential risk of inadequate dose therapy.     

Sensitivity of Leishmania braziliensis promastigotes to meglumine antimoniate (glucantime) is higher than that of other Leishmania species and correlates with response to therapy in American tegumentary leishmaniasis

The first line drugs for the treatment of leishmaniasis are antimonial derivatives. Poor clinical response may be credited to factors linked to the host, the drug, or the parasite. We determined the sensitivity of Leishmania sp. promastigotes and amastigotes by counting parasites exposed to increasing concentrations of meglumine antimoniate (Glucantime). Leishmania braziliensis promastigotes were significantly more sensitive than those belonging to other species. The sensitivity of L. braziliensis isolates from patients with unfavorable clinical outcome, such as therapeutic failure or relapse, was significantly lower than those from patients who had clinical cure. Poor clinical response to therapy (therapeutic failure or relapse) was also associated with inadequate antimonial therapy. We also found a significant and positive correlation between promastigotes and intracellular amastigotes with regard to their in vitro susceptibilities to meglumine antimoniate. Our data provide evidence for an association between the sensitivity of promastigotes to antimonials in vitro and clinical response to therapy in American tegumentary leishmaniasis. The high sensitivity of the local L. braziliensis to meglumine antimoniate in vitro provides an explanation for the good clinical response of cutaneous leishmaniasis in the municipality of Rio de Janeiro, Brazil, even when low-dose regimens are employed.     

Proteome mapping of the protozoan parasite Leishmania and application to the study of drug targets and resistance mechanisms

Leishmania is a protozoan parasite responsible for significant morbidity and mortality worldwide. Few parasites have been subjected to proteomic analysis to date, but a genome sequencing project for Leishmania major is currently underway, making these studies possible. Here we present a high resolution proteome for L. major comprising almost 3700 spots, making it the most complete two-dimensional gel representation of a parasite proteome generated to date. We have identified a number of landmark proteins by mass spectrometry and show that several of these are valid for the related species Leishmania donovani infantum. We have also observed several forms and fragments of alpha- and beta-tubulins and show that the number and amount of these fragments increase with the age of the parasite culture. Trypanothione reductase (TRYR), which replaces glutathione reductase in trypanosomatid parasites, is an essential protein specific to these parasites and as such is under considerable scrutiny as a drug target. Two-dimensional gel analysis of a L. major strain overexpressing TRYR revealed increased amounts of five spots, all at the predicted molecular weight for TRYR and differing by 0.08 pH units in pI. Mass spectrometry identified four of these as TRYR, leading to the novel suggestion that it could be post-translationally modified. Finally quantitative comparative analysis of a methotrexate-resistant mutant of L. major generated in vitro found that a known primary resistance mediator, the pteridine reductase PTR1, was overexpressed. This constitutes the first proteomic analysis of drug resistance in a parasite and also the clearest identification of a primary drug resistance mechanism using this approach. Together these results provide a framework for further proteomic studies of Leishmania species and demonstrate that these tools are valuable for the essential study of potential drug targets and drug resistance mechanisms.     

Antimonial-induced increase in intracellular Ca2+ through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani amastigotes

The capability of the obligate intracellular parasites like Leishmania donovani to survive within the host cell parasitophorous vacuoles as nonmotile amastigotes determines disease pathogenesis, but the mechanism of elimination of the parasites from these vacuoles are not well understood. By using the anti-leishmanial drug potassium antimony tartrate, we demonstrate that, upon drug exposure, intracellular L. donovani amastigotes undergo apoptotic death characterized by nuclear DNA fragmentation and externalization of phosphatidylserine. Changes upstream of DNA fragmentation included generation of reactive oxygen species like superoxide, nitric oxide, and hydrogen peroxide that were primarily concentrated in the parasitophorous vacuoles. In the presence of antioxidants like N-acetylcysteine or Mn(III) tetrakis(4-benzoic acid)porphyrin chloride, an inhibitor of inducible nitric-oxide synthase, a diminution of reactive oxygen species generation and improvement of amastigote survival were observed, suggesting a close link between drug-induced oxidative stress and amastigote death. Changes downstream to reactive oxygen species increase involved elevation of intracellular Ca2+ concentrations in both the parasite and the host that was preventable by antioxidants. Flufenamic acid, a non-selective cation channel blocker, decreased the elevation of Ca2+ in both the cell types and reduced amastigote death, thus establishing a central role of Ca2+ in intracellular parasite clearance. This influx of Ca2+ was preceded by a fall in the amastigote mitochondrial membrane potential. Therefore, this study projects the importance of flufenamic acid-sensitive non-selective cation channels as important modulators of antimonial efficacy and lends credence to the suggestion that, within the host cell, apoptosis is the preferred mode of death for the parasites.     

Confocal microscopic investigation of tubulin distribution and effect of paclitaxel on posttranslationally modified tubulins in sodium arsenite resistant Leishmania donovani

The affinity of arsenic towards the cytoskeleton leading to disturbance of tubulin polymerization is well known. Tubulin undergoes extensive posttranslational modifications which effect stability and dynamics of microtubules but little is known about the effect of antimicrotubule drugs on their distribution and function in kinetoplastid parasites such as Leishmania. The current study was undertaken to investigate the effect of continuous sodium arsenite exposure on the tubulin distribution profile in wild type and sodium arsenite resistant Leishmania donovani together with effect of paclitaxel, a tubulin-polymerizing agent, on that distribution using confocal microscopy. Immunofluorescence studies using specific monoclonal antibodies against alpha-tubulin and posttranslationally modified tubulins (acetylated and tyrosinated) have revealed distinct differences in the organization of microtubule arrays in wild type and sodium arsenite resistant L. donovani that is further affected by paclitaxel treatment. Microtubules are arranged in spiral arrays in wild type as compared to the longitudinal arrays in arsenite resistant L. donovani. The difference in microtubular structure organization may explain the parasite response to continuous drug pressure and illustrate the fundamental impact of arsenite on microtubules in arsenite resistant L. donovani.     

Substrate preferences and glucose uptake in glibenclamide-resistant Leishmania parasites

Several drug-resistant mammalian cell types exhibit increased glycolytic rates, preferential synthesis of ATP through oxidative phosphorylation, and altered glucose transport. Herein we analyzed the influence of parasite growth phase on energy substrate uptake and use in a Leishmania strain [NR(Gr)] selected for resistance against glibenclamide. Glibenclamide is an ABC-transporter blocker which modulates the function of glucose transporters in some mammalian cells. Our results demonstrate for the first time that compared to glibenclamide-sensitive Leishmania, exponential phase glibenclamide-resistant parasites exhibit decreased use of glucose as energy substrate, decreased glucose uptake and decreased glucose transporter expression. However, compared to glibenclamide-sensitive cells, stationary phase resistant parasites display an increased use of amino acids as energy substrate and an increased activity of the enzymes hexokinase, phosphoglucose isomerase, and especially NAD(+)-linked glutamate dehydrogenase. These results suggest that drug resistance in Leishmania involves a metabolic adaptation that promotes a stage dependent modulation of energy substrate uptake and use as a physiological response to the challenge imposed by drug pressure.     

Diverse viscerotropic isolates of Leishmania all express a highly conserved secretory nuclease during human infections

Previously, we characterized a gene encoding the unique nuclease (LdNuc(s)) from a Sudanese isolate of the human pathogen Leishmania donovani. This parasite secretory enzyme is involved in the salvage of host-derived purines and is constitutively expressed by both developmental forms of the parasite. Currently, we assessed whether an LdNuc(s)-like nuclease was conserved among other geographically disparate isolates of L. donovani and whether this enzyme was produced by intracellular amastigotes during human infections. Using RT-PCR and Southern blotting, we showed that LdNuc(s) gene homologs were present in each of the viscerotropic Leishmania tested (i.e., L. donovani isolates from the Sudan, Ethiopia and India as well as L. infantum). Further results of in situ enzyme activity gel analyses showed that each of these parasite isolates also expressed a released/secreted LdNuc(s)-like nuclease activity. In Western blots, our anti-LdNuc(s) (Sudan) peptide-specific antibody reacted with only a single ~35 kDa protein in each of the viscerotropic Leishmania isolates. Further, the ~35 kDa nuclease secreted by each of these isolates was specifically immunoprecipitated by the anti-LdNuc(s) antibody above. In situ gel analyses showed that each of these immunoprecipitates had LdNuc(s)-like nuclease activity. Moreover, sera from acute visceral leishmaniasis patients from India, Sudan and Brazil all immunoprecipitated an LdNuc(s)-HA expressed nuclease demonstrating, that these patients possessed antibodies against this parasite secretory enzyme. Cumulatively, these results showed that the LdNuc(s) homologs were functionally conserved among geographically disparate visceral Leishmania spp. and that amastigotes of these parasites must produce this nuclease enzyme during the course of human disease.     

Metabolic adaptations of Leishmania donovani in relation to differentiation,drug resistance,and drug pressure

Antimonial (sodium stibogluconate, SSG) resistance and differentiation have been shown to be closely linked in Leishmania donovani, with SSG‐resistant strains showing an increased capacity to generate infectious (metacyclic) forms. This is the first untargeted LC‐MS metabolomics study which integrated both phenomena in one experimental design and provided insights into metabolic differences between three clinical L. donovani strains with a similar genetic background but different SSG‐susceptibilities. We performed this analysis at different stages during promastigote growth and in the absence or presence of drug pressure. When comparing SSG‐resistant and SSG‐sensitive strains, a number of metabolic changes appeared to be constitutively present in all growth stages, pointing towards a clear link with SSG‐resistance, whereas most metabolic changes were only detected in the stationary stage. These changes reflect the close intertwinement between SSG‐resistance and an increased metacyclogenesis in resistant parasites. The metabolic changes suggest that SSG‐resistant parasites have (i) an increased capacity for protection against oxidative stress; (ii) a higher fluidity of the plasma membrane; and (iii) a metabolic survival kit to better endure infection. These changes were even more pronounced in a resistant strain kept under Sb^III drug pressure.     

Leishmania donovani: CD2 biased immune response skews the SAG mediated therapy for a predominant Th1 response in experimental infection

We have evaluated the effect of combining CD2 with conventional antimonial (sb) therapy in protection in BALB/c mice infected with either drug sensitive or resistant strain of Leishmania donovani with 3×10(7) parasites via-intra-cardiac route. Mice were treated with anti CD2 adjunct SAG sub-cutaneously twice a week for 4 weeks. Assessment for measurement of weight, spleen size, anti-Leishmania antibody titer, T cell and anti-leishmanial macrophage function was carried out day 0, 10, 22 and 34 post treatments. The combination therapy was shown boosting significant proportion of T cells to express CD25 compared to SAG monotherapy. Although, the level of IFN-γ was not statistically different between combination vs monotherapy (p=0.298) but CD2 treatment even alone significantly influenced IFN-γ production than either SAG treatment (p=0.045) or with CD2 adjunct SAG treatment (p=0.005) in Ld-S strain as well as in Ld-R strain. The influence of CD2 adjunct treatment was also documented in anti-leishmanial functions in macrophages. As shown, the super-oxide generation began enhancing very early on day 10 after SAG treatment with CD2 during which SAG action was at minimum. Interestingly, the super-oxide generation ability remained intact in macrophage after treatment with immuno-chemotherapy even in mice infected with Leishmania resistant strain. Unlike SAG treatment, treatment of SAG with CD2 also led to production of nitric oxide and TNF-α, resulting in resulting in most effective clearance of L. donovani from infected macrophages. Our results indicate that CD2, which can boost up a protective Th1 response, might also be beneficial to enable SAG to induce Macrophages to produce Leishmanicidal molecules and hence control the infection in clinical situation like Kala-azar. Drug resistance is the major impedance for disease control but the encouraging results obtained after infecting mice with resistant strain of the parasite strongly imply that this drug can be effective even in treating resistant cases of Kala-azar.     

Defective DNA repair as a potential mechanism for the rapid development of drug resistance in Plasmodium falciparum

The development and spread of highly drug-resistant parasites pose a central problem in the control of malaria.Understanding mechanisms that regulate genomic stability, such as DNA repair, in drug-resistant parasites and during drug treatment may help determine whether this rapid onset of resistance is due to an increase in the rate at which resistance-causing mutations are generated. This is the first report to demonstrate DNA repair activities from the malaria-causing parasite Plasmodium falciparum that are specific for ultraviolet light-induced DNA damage. The efficiency of DNA repair differs dramatically among P. falciparum strains with varying drug sensitivities. Most notable is the markedly reduced level of repair in the highly drug-resistant W2 isolate, which has been shown to develop resistance to novel drugs at an increased rate when compared to drug-sensitive strains. Additionally, the antimalarial drug chloroquine and other quinoline-like compounds interfered with the DNA synthesis step of the repair process, most likely a result of direct binding to repair substrates. We propose that altered DNA repair, either through defective repair mechanisms or drug-mediated inhibition, may contribute to the accelerated development of drug resistance in the parasite.     

Increased metacyclogenesis of antimony-resistant Leishmania donovani clinical lines

Mathematical models predict that the future of epidemics of drug-resistant pathogens depends in part on the competitive fitness of drug-resistant strains. Considering metacyclogenesis (differentiation process essential for infectivity) as a major contributor to the fitness of Leishmania donovani, we tested its relationship with pentavalent antimony (SbV) resistance in clinical lines. Different methods for the assessment of metacyclogenesis were cross-validated: gene expression profiling (META1 and SHERP), morphometry (microscopy and FACS), in vitro infectivity to macrophages and resistance to complement lysis. This was done on a model constituted by 2 pairs of reference strains cloned from a SbV-resistant and -sensitive isolate. We selected the most adequate parameter and extended the analysis of metacyclogenesis diversity to a sample of 20 clinical lines with different in vitro susceptibility to the drug. The capacity of metacyclogenesis, as measured by the complement lysis test, was shown to be significantly higher in SbV-resistant clinical lines of L. donovani than in SbV-sensitive lines. Together with other lines of evidence, it is concluded that L. donovani constitutes a unique example and model of drug-resistant pathogens with traits of increased fitness. These findings raise a fundamental question about the potential risks of selecting more virulent pathogens through massive chemotherapeutic interventions.     

Evolutionary Epidemiology of Drug-Resistance in Space

How can we optimize the use of drugs against parasites to limit the evolution of drug resistance? This question has been addressed by many theoretical studies focusing either on the mixing of various treatments, or their temporal alternation. Here we consider a different treatment strategy where the use of the drug may vary in space to prevent the rise of drug-resistance. We analyze epidemiological models where drug-resistant and drug-sensitive parasites compete in a one-dimensional spatially heterogeneous environment. Two different parasite life-cycles are considered: (i) direct transmission between hosts, and (ii) vector-borne transmission. In both cases we find a critical size of the treated area, under which the drug-resistant strain cannot persist. This critical size depends on the basic reproductive ratios of each strain in each environment, on the ranges of dispersal, and on the duration of an infection with drug-resistant parasites. We discuss optimal treatment strategies that limit disease prevalence and the evolution of drug-resistance.     

Visceral leishmaniasis: resistance to reinfection in the liver following chemotherapy in the BALB/c mouse

The ability of BALB/c mice to resist reinfection with Leishmania donovani following chemotherapy was studied. BALB/c mice, infected with L. donovani, were treated on Days 7 and 8 postinfection with free, niosomal, or liposomal sodium stibogluconate. It was found that all three drug treatments caused a dramatic reduction in liver parasite burdens as measured on Days 6 and 29 post-treatment. On Day 6 postdrug treatment infection with L. donovani amastigotes, of mice from infected, drug-treated groups, along with age- and sex-matched uninfected controls, showed that at 23 days later, significantly fewer parasites were recovered from the livers of reinfected animals compared with controls given their first infection. Treatment of mice with sodium stibogluconate 6 days prior to a primary infection significantly reduced the number of parasites recovered 14 days later, especially using the carrier form of the drug. In vivo macrophage activity in the liver, as measured by the uptake of radiolabeled horseradish peroxidase immune complex, was significantly raised following stibogluconate treatment of infected but not uninfected mice. These results suggest that a state of resistance persists in the liver of infected mice following chemotherapy which may in part be due to local macrophage activation but also to an unsuspected persistance of the drug.     

Mechanisms of drug resistance in Leishmania

The emergence of drug resistance in protozoan parasites is a major obstacle to their control. Since vaccines are not yet in sight for several of these parasites, there is on urgent need to develop new and better drugs. These antimicrobial agents will possibly be more expensive, and will therefore impose on additional burden in health-care costs and in the planning of public health policies of the developing countries. A better understanding of drug resistance, to try to circumvent or overcome it, and the search for new specific cellular targets of parasites are warranted. The development, in vitro, of drug-resistant parasite cell lines has been instrumental in our understanding of the mechanisms of drug resistance in parasitic protozoans. Marc Ouellette and Barbara Popodopoulou here present on overview of the recent progress on the elucidation of mechanisms of drug resistance in the protozoan parasite Leishmania, selected under laboratory conditions.     

A proteomics screen implicates HSP83 and a small kinetoplastid calpain-related protein in drug resistance in Leishmania donovani clinical field isolates by modulating drug-induced programmed cell death

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.