Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response.

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana.

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not virulent in mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both.

Drug resistance and treatment failure in leishmaniasis: A 21st century challenge.

Reevaluation of treatment guidelines for Old and New World leishmaniasis is urgently needed on a global basis because treatment failure is an increasing problem. Drug resistance is a fundamental determinant of treatment failure, although other factors also contribute to this phenomenon, including the global HIV/AIDS epidemic with its accompanying impact on the immune system. Pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis since the first half of the 20th century, but the last 10 to 20 years have witnessed an increase in clinical resistance, e.g., in North Bihar in India. In this review, we discuss the meaning of "resistance" related to leishmaniasis and discuss its molecular epidemiology, particularly for Leishmania donovani that causes visceral leishmaniasis. We also discuss how resistance can affect drug combination therapies. Molecular mechanisms known to contribute to resistance to antimonials, amphotericin B, and miltefosine are also outlined.

Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana.

Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites' genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme's active site, consistent with the fact that the resistant line continues to produce the enzyme's product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself.

The animal trypanosomiases and their chemotherapy: a review.

Pathogenic animal trypanosomes affecting livestock have represented a major constraint to agricultural development in Africa for centuries, and their negative economic impact is increasing in South America and Asia. Chemotherapy and chemoprophylaxis represent the main means of control. However, research into new trypanocides has remained inadequate for decades, leading to a situation where the few compounds available are losing efficacy due to the emergence of drug-resistant parasites. In this review, we provide a comprehensive overview of the current options available for the treatment and prophylaxis of the animal trypanosomiases, with a special focus on the problem of resistance. The key issues surrounding the main economically important animal trypanosome species and the diseases they cause are also presented. As new investment becomes available to develop improved tools to control the animal trypanosomiases, we stress that efforts should be directed towards a better understanding of the biology of the relevant parasite species and strains, to identify new drug targets and interrogate resistance mechanisms.

Benznidazole biotransformation and multiple targets in Trypanosoma cruzi revealed by metabolomics.

BACKGROUND: The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. In the present work we used a non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn. METHODOLOGY/PRINCIPAL FINDINGS: Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols trypanothione, homotrypanothione and cysteine were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment. These metabolites included reduction products, fragments and covalent adducts of reduced Bzn linked to each of the major low molecular weight thiols: trypanothione, glutathione, γ-glutamylcysteine, glutathionylspermidine, cysteine and ovothiol A. Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI, were found within the parasites, but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected. CONCLUSIONS/SIGNIFICANCE: Our data is indicative of a major role of the thiol binding capacity of Bzn reduction products in the mechanism of Bzn toxicity against T. cruzi.

The 6-phosphogluconate dehydrogenase of Leishmania (Leishmania) mexicana: gene characterization and protein structure prediction.

6-Phosphogluconate dehydrogenase (6PGDH) is a key enzyme of the oxidative branch involved in the generation of NADPH and ribulose 5-phosphate. In the present work, we describe the cloning, sequencing and characterization of a 6PGDH gene from Leishmania (Leishmania) mexicana. The gene encodes a polypeptide chain of 479 amino acid residues with a predicted molecular mass of 52 kDa and a pI of 5.77. The recombinant protein possesses a dimeric quaternary structure and displays kinetic parameter values intermediate between those reported for Trypanosoma brucei and T. cruzi with apparent K(m) values of 6.93 and 5.2 µM for 6PG and NADP(+), respectively. The three-dimensional structure of the enzymes of Leishmania and T. cruzi were modelled from their amino acid sequence using the crystal structure of the enzyme of T. brucei as template. The amino acid residues located in the 6PGDH C-terminal region, which are known to participate in the salt bridges maintaining the protein dimeric structure, differed significantly among the enzymes of Leishmania, T. cruzi, and T. brucei. Our results strongly suggest that 6PGDH can be selected as a potential target for the development of new therapeutic drugs in order to improve existing chemotherapeutic treatments against these parasites.

Transketolase from Leishmania mexicana has a dual subcellular localization.

Transketolase has been characterized in Leishmania mexicana. A gene encoding this enzyme was identified and cloned. The gene was expressed in Escherichia coli and the protein was purified and characterized. An apparent K(m) of 2.75 mM for ribose 5-phosphate was determined. X-ray crystallography was used to determine the three-dimensional structure of the enzyme to a resolution of 2.2 A (1 A identical with 0.1 nm). The C-terminus of the protein contains a type-1 peroxisome-targeting signal, suggestive of a possible glycosomal subcellular localization. Subcellular localization experiments performed with promastigote forms of the parasite revealed that the protein was predominantly cytosolic, although a significant component of the total activity was associated with the glycosomes. Transketolase is thus the first enzyme of the nonoxidative branch of the pentose phosphate pathway whose presence has been demonstrated in a peroxisome-like organelle.

Pentose phosphate metabolism in Leishmania mexicana.

The metabolism of pentose phosphates was studied in Leishmania mexicana promastigotes. Each of the enzymes of the classical pentose phosphate pathway (PPP) has been identified and specific activities measured. Functioning of the PPP was demonstrated in non-growing cells by measuring the evolution of 14CO2 from [1-14C]D-glucose and [6-14C]D-glucose under normal conditions and also under selective stimulation of the PPP by exposure to methylene blue. The proportion of glucose which passes through the PPP increases in the latter condition, thus suggesting a protective role against oxidant stress. The incorporation into nucleic acids of ribose 5-phosphate provided via either glucose or free ribose was also determined. Results indicate that the PPP enables glucose to serve as a source of ribose 5-phosphate in nucleotide biosynthesis. Moreover, free ribose is incorporated efficiently, implying the presence of a ribose uptake system and also of ribokinase. Ribose was shown to be accumulated by a carrier mediated process in L. mexicana promastigotes and ribokinase activity was also measured in these cells.

Genetic characterization of glucose transporter function in Leishmania mexicana.

Both insect and mammalian life cycle stages of Leishmania mexicana take up glucose and express all three isoforms encoded by the LmGT glucose transporter gene family. To evaluate glucose transporter function in intact parasites, a null mutant line has been created by targeted disruption of the LmGT locus that encompasses the LmGT1, LmGT2, and LmGT3 genes. This deltalmgt null mutant exhibited no detectable glucose transport activity. The growth rate of the deltalmgt knockout in the promastigote stage was reduced to a rate comparable with that of WT cells grown in the absence of glucose. deltalmgt cells also exhibited dramatically reduced infectivity to macrophages, demonstrating that expression of LmGT isoforms is essential for viability of amastigotes. Furthermore, WT L. mexicana were not able to grow as axenic culture form amastigotes if glucose was withdrawn from the medium, implying that glucose is an essential nutrient in this life cycle stage. Expression of either LmGT2 or LmGT3, but not of LmGT1, in deltalmgt null mutants significantly restored growth as promastigotes, but only LmGT3 expression substantially rescued amastigote growth in macrophages. Subcellular localization of the three isoforms was investigated in deltalmgt cells expressing individual LmGT isoforms. Using anti-LmGT antiserum and GFP-tagged LmGT fusion proteins, LmGT2 and LmGT3 were localized to the cell body, whereas LmGT1 was localized specifically to the flagellum. These results establish that each glucose transporter isoform has distinct biological functions in the parasite.

Resistance to pentamidine in Leishmania mexicana involves exclusion of the drug from the mitochondrion.

The uptake of [(3)H]pentamidine into wild-type and drug-resistant strains of Leishmania mexicana was compared. Uptake was carrier mediated. Pentamidine-resistant parasites showed cross-resistance to other toxic diamidine derivatives. A substantial decrease in accumulation of the drug accompanied the resistance phenotype, although the apparent affinity for pentamidine by its carrier was not altered when initial uptake velocity was measured. The apparent V(max), however, was reduced. An efflux of pentamidine could be measured in both wild-type and resistant cells. Only a relatively small proportion of the total accumulated pentamidine was available for efflux in wild-type cells, while in resistant cells the majority of loaded pentamidine was available for release. Pharmacological reagents which diminish the mitochondrial membrane potential reduced pentamidine uptake in wild-type parasites, and the mitochondrial membrane potential was shown to be reduced in resistant cells. A fluorescent analogue of pentamidine, 4',6'-diamidino-2-phenylindole, accumulated in the kinetoplast of wild-type but not resistant parasites. These data together indicate that diamidine drugs accumulate in the Leishmania mitochondrion and that the development of the resistance phenotype is accompanied by lack of mitochondrial accumulation of the drug and its exclusion from the parasites.