Isabel M. Vincent

A Molecular Mechanism for Eflornithine Resistance in African Trypanosomes

Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drugs target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered.

Untargeted Metabolomics Reveals a Lack Of Synergy between Nifurtimox and Eflornithine against Trypanosoma brucei

A non-targeted metabolomics-based approach is presented that enables the study of pathways in response to drug action with the aim of defining the mode of action of trypanocides. Eflornithine, a polyamine pathway inhibitor, and nifurtimox, whose mode of action involves its metabolic activation, are currently used in combination as first line treatment against stage 2, CNS-involved, human African trypanosomiasis (HAT). Drug action was assessed using an LC-MS based non-targeted metabolomics approach. Eflornithine revealed the expected changes to the polyamine pathway as well as several unexpected changes that point to pathways and metabolites not previously described in bloodstream form trypanosomes, including a lack of arginase activity and N-acetylated ornithine and putrescine. Nifurtimox was shown to be converted to a trinitrile metabolite indicative of metabolic activation, as well as inducing changes in levels of metabolites involved in carbohydrate and nucleotide metabolism. However, eflornithine and nifurtimox failed to synergise anti-trypanosomal activity in vitro, and the metabolomic changes associated with the combination are the sum of those found in each monotherapy with no indication of additional effects. The study reveals how untargeted metabolomics can yield rapid information on drug targets that could be adapted to any pharmacological situation.

Untargeted metabolomic analysis of miltefosine action in Leishmania infantum reveals changes to the internal lipid metabolism

Graphical abstract

The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control

The Trypanosoma brucei procyclic form resides within the digestive tract of its insect vector, where it exploits amino acids as carbon sources. Threonine is the amino acid most rapidly consumed by this parasite, however its role is poorly understood. Here, we show that the procyclic trypanosomes grown in rich medium only use glucose and threonine for lipid biosynthesis, with threonines contribution being ∼ 2.5 times higher than that of glucose. A combination of reverse genetics and NMR analysis of excreted end‐products from threonine and glucose metabolism, shows that acetate, which feeds lipid biosynthesis, is also produced primarily from threonine. Interestingly, the first enzymatic step of the threonine degradation pathway, threonine dehydrogenase (TDH, EC 1.1.1.103), is under metabolic control and plays a key role in the rate of catabolism. Indeed, a trypanosome mutant deleted for the phosphoenolpyruvate decarboxylase gene (PEPCK, EC 4.1.1.49) shows a 1.7‐fold and twofold decrease of TDH protein level and activity, respectively, associated with a 1.8‐fold reduction in threonine‐derived acetate production. We conclude that TDH expression is under control and can be downregulated in response to metabolic perturbations, such as in the PEPCK mutant in which the glycolytic metabolic flux was redirected towards acetate production.

Metabolomic-based strategies for anti-parasite drug discovery

Metabolomics-based studies are proving of great utility in the analysis of modes of action (MOAs) and resistance mechanisms of drugs in parasitic protozoa. They have helped to determine the MOA of eflornithine, half of the gold standard combination therapy in use against human African trypanosomiasis (HAT), as well as the mechanism of resistance to this drug. In Leishmania, metabolomics has also given insight into the MOA of miltefosine, an alkylphospholipid. Several studies on antimony resistance in Leishmania have been conducted, analyzing the metabolic content of resistant lines, offering clues as to the MOA of this class of drugs. A study of chloroquine resistance in Plasmodium falciparum combined metabolomics techniques with other genetic and proteomic techniques to offer new insight into the role of the PfCRT protein. The MOA and mechanism of resistance to a group of halogenated pyrimidines in Trypanosoma brucei have also recently been elucidated. Effective as metabolomics techniques are, care must be taken in the design and implementation of these experiments, to ensure the resulting data are meaningful. This review outlines the steps required to conduct a metabolomics experiment as well as provide an overview of metabolomics-based drug research in protozoa to date.

TrypanoCyc : a community-led biochemical pathways database for Trypanosoma brucei

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.

Untargeted Metabolomics To Ascertain Antibiotic Modes of Action

ABSTRACT Deciphering the mode of action (MOA) of new antibiotics discovered through phenotypic screening is of increasing importance. Metabolomics offers a potentially rapid and cost-effective means of identifying modes of action of drugs whose effects are mediated through changes in metabolism. Metabolomics techniques also collect data on off-target effects and drug modifications. Here, we present data from an untargeted liquid chromatography-mass spectrometry approach to identify the modes of action of eight compounds: 1-[3-fluoro-4-(5-methyl-2,4-dioxo-pyrimidin-1-yl)phenyl]-3-[2-(trifluoromethyl)phenyl]urea (AZ1), 2-(cyclobutylmethoxy)-5′-deoxyadenosine, triclosan, fosmidomycin, CHIR-090, carbonyl cyanide m-chlorophenylhydrazone (CCCP), 5-chloro-2-(methylsulfonyl)-N-(1,3-thiazol-2-yl)-4-pyrimidinecarboxamide (AZ7), and ceftazidime. Data analysts were blind to the compound identities but managed to identify the target as thymidylate kinase for AZ1, isoprenoid biosynthesis for fosmidomycin, acyl-transferase for CHIR-090, and DNA metabolism for 2-(cyclobutylmethoxy)-5′-deoxyadenosine. Changes to cell wall metabolites were seen in ceftazidime treatments, although other changes, presumably relating to off-target effects, dominated spectral outputs in the untargeted approach. Drugs which do not work through metabolic pathways, such as the proton carrier CCCP, have no discernible impact on the metabolome. The untargeted metabolomics approach also revealed modifications to two compounds, namely, fosmidomycin and AZ7. An untreated control was also analyzed, and changes to the metabolome were seen over 4 h, highlighting the necessity for careful controls in these types of studies. Metabolomics is a useful tool in the analysis of drug modes of action and can complement other technologies already in use.

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.

Metabolomics Identifies Multiple Candidate Biomarkers to Diagnose and Stage Human African Trypanosomiasis

Treatment for human African trypanosomiasis is dependent on the species of trypanosome causing the disease and the stage of the disease (stage 1 defined by parasites being present in blood and lymphatics whilst for stage 2, parasites are found beyond the blood-brain barrier in the cerebrospinal fluid (CSF)). Currently, staging relies upon detecting the very low number of parasites or elevated white blood cell numbers in CSF. Improved staging is desirable, as is the elimination of the need for lumbar puncture. Here we use metabolomics to probe samples of CSF, plasma and urine from 40 Angolan patients infected with Trypanosoma brucei gambiense, at different disease stages. Urine samples provided no robust markers indicative of infection or stage of infection due to inherent variability in urine concentrations. Biomarkers in CSF were able to distinguish patients at stage 1 or advanced stage 2 with absolute specificity. Eleven metabolites clearly distinguished the stage in most patients and two of these (neopterin and 5-hydroxytryptophan) showed 100% specificity and sensitivity between our stage 1 and advanced stage 2 samples. Neopterin is an inflammatory biomarker previously shown in CSF of stage 2 but not stage 1 patients. 5-hydroxytryptophan is an important metabolite in the serotonin synthetic pathway, the key pathway in determining somnolence, thus offering a possible link to the eponymous symptoms of “sleeping sickness”. Plasma also yielded several biomarkers clearly indicative of the presence (87% sensitivity and 95% specificity) and stage of disease (92% sensitivity and 81% specificity). A logistic regression model including these metabolites showed clear separation of patients being either at stage 1 or advanced stage 2 or indeed diseased (both stages) versus control.

Different mutations in a p-type ATPase transporter in Leishmania parasites are associated with cross-resistance to two leading drugs by distinct mechanisms

Leishmania infantum is an etiological agent of the life-threatening visceral form of leishmaniasis. Liposomal amphotericin B (AmB) followed by a short administration of miltefosine (MF) is a drug combination effective for treating visceral leishmaniasis in endemic regions of India. Resistance to MF can be due to point mutations in the miltefosine transporter (MT). Here we show that mutations in MT are also observed in Leishmania AmB-resistant mutants. The MF-induced MT mutations, but not the AmB induced mutations in MT, alter the translocation/uptake of MF. Moreover, mutations in the MT selected by AmB or MF have a major impact on lipid species that is linked to cross-resistance between both drugs. These alterations include changes of specific phospholipids, some of which are enriched with cyclopropanated fatty acids, as well as an increase in inositolphosphoceramide species. Collectively these results provide evidence of the risk of cross-resistance emergence derived from current AmB-MF sequential or co-treatments for visceral leishmaniasis.