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Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei , the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1 -null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1 -null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.

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.

The synthesis and biological evaluation of three series of 6-phosphogluconate (6PG) analogues is described. (2R)-2-Methyl-4,5-dideoxy, (2R)-2-methyl-4-deoxy and 2,4-dideoxy analogues of 6PG were tested as inhibitors of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver and also Trypanosoma brucei where the enzyme is a validated drug target. Among the three series of analogues, seven compounds were found to competitively inhibit 6PGDH from T. brucei and sheep liver enzymes at micromolar concentrations. Six inhibitors belong to the (2R)-2-methyl-4-deoxy series (6, 8, 10, 12, 21, 24) and one is a (2R)-2-methyl-4,5-dideoxy analogue (29b). The 2,4-dideoxy analogues of 6PG did not inhibit both enzymes. The trypanocidal effect of the compounds was also evaluated in vitro against T. brucei rhodesiense as well as other related trypanosomatid parasites (i.e., Trypanosoma cruzi and Leishmania donovani).

While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters of metazoa, parazoa, or protozoa. We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily. The gene was expressed in both the procyclic and bloodstream forms of the organism. Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity transport activity for adenine and hypoxanthine, with Km values of 2.1 +/- 0.6 and 0.66 +/- 0.22 microm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine. A transporter with an indistinguishable kinetic profile was identified in T. b. brucei procyclics and designated H4. RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by approximately 80% and H4 transport activity by approximately 90%. Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals to be identified at the molecular level.

Both insect and mammalian life cycle stages ofLeishmania mexicanatake up glucose and express all three isoforms encoded by theLmGTglucose transporter gene family. To evaluate glucose transporter function in intact parasites, a null mutant line has been created by targeted disruption of theLmGTlocus that encompasses theLmGT1,LmGT2, andLmGT3genes. This Δlmgtnull mutant exhibited no detectable glucose transport activity. The growth rate of the Δlmgtknockout in the promastigote stage was reduced to a rate comparable with that of WT cells grown in the absence of glucose. Δlmgtcells also exhibited dramatically reduced infectivity to macrophages, demonstrating that expression of LmGT isoforms is essential for viability of amastigotes. Furthermore, WTL. mexicanawere 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 eitherLmGT2orLmGT3, but not ofLmGT1, in Δlmgtnull mutants significantly restored growth as promastigotes, but onlyLmGT3expression substantially rescued amastigote growth in macrophages. Subcellular localization of the three isoforms was investigated in Δlmgtcells 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.

Recent development of benzoxaborole-based chemistry gave rise to a collection of compounds with great potential in targeting diverse infectious diseases, including human African Trypanosomiasis (HAT), a devastating neglected tropical disease. However, further medicinal development is largely restricted by a lack of insight into mechanism of action (MoA) in pathogenic kinetoplastids. We adopted a multidisciplinary approach, combining a high-throughput forward genetic screen with functional group focused chemical biological, structural biology and biochemical analyses, to tackle the complex MoAs of benzoxaboroles in Trypanosoma brucei. We describe an oxidative enzymatic pathway composed of host semicarbazide-sensitive amine oxidase and a trypanosomal aldehyde dehydrogenase TbALDH3. Two sequential reactions through this pathway serve as the key underlying mechanism for activating a series of 4-aminomethylphenoxy-benzoxaboroles as potent trypanocides; the methylamine parental compounds as pro-drugs are transformed first into intermediate aldehyde metabolites, and further into the carboxylate metabolites as effective forms. Moreover, comparative biochemical and crystallographic analyses elucidated the catalytic specificity of TbALDH3 towards the benzaldehyde benzoxaborole metabolites as xenogeneic substrates. Overall, this work proposes a novel drug activation mechanism dependent on both host and parasite metabolism of primary amine containing molecules, which contributes a new perspective to our understanding of the benzoxaborole MoA, and could be further exploited to improve the therapeutic index of antimicrobial compounds., Author summary Human African Trypanomiasis (HAT) is among a list of Neglected Tropical Diseases (NTDs) that impose devastating burdens on both public health and economy of some of the most unprivileged societies across the world. To secure the long-term global control of the disease, it is critical to understand the mechanisms underlying the interactions of drugs and drug candidates with the causative agents as well as resistance potentially arising from use of the compounds. We demonstrated here a metabolic enzymatic cascade dependent on a host-pathogen interaction that determines potency against T. brucei of a series of benzoxaborole compounds. More importantly, this pathway represents a metabolic interaction network between host and pathogen, illuminating an important perspective on understanding mechanism of action.

A 55 year old woman presented to her general practitioner with a six month history of pain over the lateral aspect of her left hip in the trochanteric region. She described the pain as “a niggling pain that seems to be progressively getting worse.” The pain was exacerbated on weight bearing and by activity, but did not prevent her from being active. The pain woke her at night. She had a medical history of gastro-oesophageal reflux disease, essential hypertension, and early stage breast cancer. Her breast cancer was diagnosed and treated eight years ago, and she had since been discharged from follow-up. Her only regular medication was omeprazole. She lived at home with her husband and worked as a teaching assistant. On examination, she had a marked antalgic gait. She had a good range of pain-free movement but had discomfort over the lateral aspect of the left hip during the extremes of rotation. A radiograph of the pelvis was performed (fig 1). Fig 1 Radiograph of the pelvis (anteroposterior view) ### 1. What are the differential diagnoses? #### Short answer Secondary malignancy, primary bone malignancy, such as multiple myeloma, osteomyelitis, benign neoplasm such as giant cell tumour, brown tumour. #### Discussion The pelvic radiograph showed at least two lytic lesions in the left proximal femur and greater trochanter. Secondary bone malignancy is the most …

Human malaria parasites proliferate in different erythroid cell types during infection. Whilst Plasmodium vivax exhibits a strong preference for immature reticulocytes, the more pathogenic P. falciparum primarily infects mature erythrocytes. In order to assess if these two cell types offer different growth conditions and relate them to parasite preference, we compared the metabolomes of human and rodent reticulocytes with those of their mature erythrocyte counterparts. Reticulocytes were found to have a more complex, enriched metabolic profile than mature erythrocytes and a higher level of metabolic overlap between reticulocyte resident parasite stages and their host cell. This redundancy was assessed by generating a panel of mutants of the rodent malaria parasite P. berghei with defects in intermediary carbon metabolism (ICM) and pyrimidine biosynthesis known to be important for P. falciparum growth and survival in vitro in mature erythrocytes. P. berghei ICM mutants (pbpepc-, phosphoenolpyruvate carboxylase and pbmdh-, malate dehydrogenase) multiplied in reticulocytes and committed to sexual development like wild type parasites. However, P. berghei pyrimidine biosynthesis mutants (pboprt-, orotate phosphoribosyltransferase and pbompdc-, orotidine 5′-monophosphate decarboxylase) were restricted to growth in the youngest forms of reticulocytes and had a severe slow growth phenotype in part resulting from reduced merozoite production. The pbpepc-, pboprt- and pbompdc- mutants retained virulence in mice implying that malaria parasites can partially salvage pyrimidines but failed to complete differentiation to various stages in mosquitoes. These findings suggest that species-specific differences in Plasmodium host cell tropism result in marked differences in the necessity for parasite intrinsic metabolism. These data have implications for drug design when targeting mature erythrocyte or reticulocyte resident parasites., Author Summary Malaria, caused by the Apicomplexan parasites Plasmodium spp., is a deadly disease which poses a huge health and economic burden over many populations in the world, mostly in sub-Saharan Africa and Asia. To design new intervention strategies and to improve upon existing drugs against malaria, it is useful to understand the biochemistry of the Plasmodium parasite and its metabolic interplay with the host. Some species of Plasmodium such as P. vivax grow exclusively in reticulocytes (immature erythrocytes) whereas others e.g. P. falciparum will also readily multiply in mature erythrocytes. We asked the questions, do these two classes of host cell offer different resources for parasite survival and could these resources influence antimalarial drug efficacy? We used metabolomics to compare rodent reticulocytes and mature erythrocytes and identified that the metabolome of the former is more diverse and enriched. Gene disruption in the reticulocyte preferring rodent malaria parasite P. berghei was used to demonstrate that Plasmodium can utilise the elements of the metabolic reserves of reticulocytes that mature erythrocytes cannot provide. Our data suggests that the availability of the reticulocyte metabolome might reduce or block the efficacy of antimalarial drugs that target parasite metabolism and drugs tested against P. falciparum might have significantly reduced activity against P. vivax.

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.

Diseases caused by pathogenic trypanosomatids cause great suffering throughout the developing world. New drugs for these diseases are urgently needed. Recent technological advances have permitted the identification and validation of numerous drug targets in these organisms. However, efforts to develop inhibitors of these targets, that may then be taken forward for development into new drugs, have been comparatively scarce. In this review we discuss the design, synthesis and evaluation of inhibitors of two drug targets in trypanosomatids, 6-phosphogluconate dehydrogenase, the third enzyme of the pentose phosphate pathway, and dihydrofolate reductase, a key enzyme involved in DNA synthesis. Enzyme inhibitors can only be useful as drugs if they can enter cells and bind to their targets. Therefore we also discuss approaches to designing molecules that can specifically cross the plasma membrane of African trypanosomes via unusual nutrient transporters.

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., Research reported in this publication was supported by the Canadian Institutes of Health Research operating grant MOP-12182 awarded to BP; the Spanish Grant Proyecto de Excelencia, Junta de Andalucía Ref. CTS-7282 awarded to FG; the Spanish Grant SAF2015-68042-R awarded to FG; the Wellcome Trust (104111/Z/14/Z) awarded to MPB; and the Alexander von Humboldt Foundation, Germany awarded to APS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

A major mechanism whereby malaria parasites evade the host immune response to give chronic infections in patients' blood for months, or even years, is antigenic variation. In order to generate variant antigens, parasites require large multigene families. Although several gene families involved in these phenomena have been identified in the human malaria Plasmodium falciparum, to date no variant antigen gene families have been identified in malaria species that will infect widely used rodent laboratory hosts. Here we present, for the first time, to our knowledge, a large multigene family conserved in both rodent and human malarias, which is a strong candidate as a major variant antigen gene family. In each of four species of Plasmodium, three rodent malarias and the human pathogen P. vivax, homologues of the gene family were found to have a conserved three-exon structure. In the rodent malaria P. chabaudi, transcription of members of the gene family was developmentally regulated with maximum expression in late trophozoite stages, which is the developmental stage known to express variant antigen proteins.

A 28 year old man was brought to hospital after a road traffic incident. He described hearing a “pop” as his right knee impacted against the dashboard at the time of collision, and he subsequently experienced extreme pain. On examination in the emergency department, his knee was swollen and tender, and range of movement was severely limited. After initial management in the emergency department, he was admitted under the orthopaedic team. Once the swelling was resolved, his knee was re-examined, and was found to be grossly unstable (fig 1⇓). Fig 1 Clinical photograph of the patient’s right knee ### 1. What sign is shown in the clinical photograph? #### Short answer Posterior sag sign suggesting injury to the posterior cruciate ligament. #### Discussion The posterior cruciate ligament serves as the main restraint to posterior displacement of the tibia relative to the femur. Secondary functions include resisting excessive valgus, varus, and external rotation movements. Disruption of the posterior cruciate ligament can be seen via posterior draw testing or by assessing for posterior sag (fig 2⇓). Normally, the anterior surface of the tibia lies around 1 cm anterior to the femoral surface when the knee is held in 90° of flexion. Any …

What strategies can leaders deploy to facilitate the early stage development of IT innovation ecosystems? Using our longitudinal in- depth case study of the ‘Internet of Things’ (IoT) we sought to ...

We have previously reported that curcumin analogs with a C7 linker bearing a C4-C5 olefinic linker with a single keto group at C3 (enone linker) display midnanomolar activity against the bloodstream form of Trypanosoma brucei. However, no clear indication of their mechanism of action or superior antiparasitic activity relative to analogs with the original di-ketone curcumin linker was apparent. To further investigate their utility as antiparasitic agents, we compare the cellular effects of curcumin and the enone linker lead compound 1,7-bis(4-hydroxy-3-methoxyphenyl)hept-4-en-3-one (AS-HK014) here. An AS-HK014–resitant line, trypanosomes adapted to AS-HK014 (TA014), was developed by in vitro exposure to the drug. Metabolomic analysis revealed that exposure to AS-HK014, but not curcumin, rapidly depleted glutathione and trypanothione in the wild-type line, although almost all other metabolites were unchanged relative to control. In TA014 cells, thiol levels were similar to untreated wild-type cells and not significantly depleted by AS-HK014. Adducts of AS-HK014 with both glutathione and trypanothione were identified in AS-HK014–exposed wild-type cells and reproduced by chemical reaction. However, adduct accumulation in sensitive cells was much lower than in resistant cells. TA014 cells did not exhibit any changes in sequence or protein levels of glutathione synthetase and γ-glutamylcysteine synthetase relative to wild-type cells. We conclude that monoenone curcuminoids have a different mode of action than curcumin, rapidly and specifically depleting thiol levels in trypanosomes by forming an adduct. This adduct may ultimately be responsible for the highly potent trypanocidal and antiparasitic activity of the monoenone curcuminoids.

Peripheral infection by Trypanosoma brucei, the protozoan responsible for sleeping sickness, activates lymphocytes, and, at later stages, causes meningoencephalitis. We have videoed the cortical meninges and superficial parenchyma of C56BL/6 reporter mice infected with T.b.brucei. By use of a two-photon microscope to image through the thinned skull, the integrity of the tissues was maintained. We observed a 47-fold increase in CD2+ T cells in the meninges by 12 days post infection (dpi). CD11c+ dendritic cells also increased, and extravascular trypanosomes, made visible either by expression of a fluorescent protein, or by intravenous injection of furamidine, appeared. The likelihood that invasion will spread from the meninges to the parenchyma will depend strongly on whether the trypanosomes are below the arachnoid membrane, or above it, in the dura. Making use of optical signals from the skull bone, blood vessels and dural cells, we conclude that up to 40 dpi, the extravascular trypanosomes were essentially confined to the dura, as were the great majority of the T cells. Inhibition of T cell activation by intraperitoneal injection of abatacept reduced the numbers of meningeal T cells at 12 dpi and their mean speed fell from 11.64 ± 0.34 μm/min (mean ± SEM) to 5.2 ± 1.2 μm/min (p = 0.007). The T cells occasionally made contact lasting tens of minutes with dendritic cells, indicative of antigen presentation. The population and motility of the trypanosomes tended to decline after about 30 dpi. We suggest that the lymphocyte infiltration of the meninges may later contribute to encephalitis, but have no evidence that the dural trypanosomes invade the parenchyma., Author Summary African trypanosomes are motile parasites that cause sleeping sickness. They multiply first in the blood then cause death mainly by effects on the brain: immune system cells, including T cells and dendritic cells, play major roles in this. Thinking we might see the attack on the brain, we infected mice with trypanosomes and used a two-photon microscope, which allowed us to image the superficial brain and the delicate tissue between the skull and the brain called the meninges without making a hole in the skull. The mice (which were anesthetized) had been genetically modified so that T cells and dendritic cells were fluorescent, as were the trypanosomes. We did not notice much happening in the brain itself, but in the meninges, in a compartment called the dura, huge numbers of T cells and dendritic cells appeared. Trypanosomes also moved from the blood into this compartment. Since T cells, dendritic cells and trypanosomes had not been videoed in the meninges before, we began by observing them carefully: their numbers, their movements and their interactions. The accumulation of lymphocytes is a sign of meningitis, a feature of infection by a wide range of pathogens and our results suggest interesting future work.

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.

The treatment of Human African trypanosomiasis remains a major unmet health need in sub-Saharan Africa. Approaches involving new molecular targets are important; pteridine reductase 1 (PTR1), an enzyme that reduces dihydrobiopterin in Trypanosoma spp., has been identified as a candidate target, and it has been shown previously that substituted pyrrolo[2,3-d]pyrimidines are inhibitors of PTR1 from Trypanosoma brucei (J. Med. Chem. 2010, 53, 221-229). In this study, 61 new pyrrolo[2,3-d]pyrimidines have been prepared, designed with input from new crystal structures of 23 of these compounds complexed with PTR1, and evaluated in screens for enzyme inhibitory activity against PTR1 and in vitro antitrypanosomal activity. Eight compounds were sufficiently active in both screens to take forward to in vivo evaluation. Thus, although evidence for trypanocidal activity in a stage I disease model in mice was obtained, the compounds were too toxic to mice for further development.

One significant challenge in medical diagnostics lies in the development of label-free methods to separate different cells within complex biological samples. Here we demonstrate a generic, low-power ultrasonic separation technique, able to enrich different cell types based upon their physical properties. For malaria, we differentiate between infected and non-infected red blood cells in a fingerprick-sized drop of blood. We are able to achieve an enrichment of circulating cells infected by the ring stage of the parasite over nonparasitized red blood cells by between two and three orders of magnitude in less than 3 seconds (enabling detection at parasitemia levels as low as 0.0005 %). In a second example, we also show that our methods can be used to enrich different cell types, concentrating Trypanosoma in blood at very low levels of infection, on disposable, low-cost chips.

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., Author Summary The unicellular parasite Trypanosoma cruzi infects humans, leading to Chagas disease, endemic in Central and South America and responsible for 13,000 annual deaths. Only two drugs have proven effective against Chagas, nifurtimox and benznidazole (Bzn). Bzn has the best safety and efficacy profiles and is thus used as first line treatment. Bzn is a pro-drug, and possesses a nitro group which needs to be enzymatically reduced within the parasite to become active. We have investigated for the first time, by means of mass spectrometry based metabolomics, the global changes to small metabolites that occur once Bzn enters the parasite. A decrease in the levels of several thiols, including cysteine and trypanothione, and an increase in gamma-glutamyl containing dipeptides were observed after treatment. Reduced metabolites of Bzn were also detected, together with numerous covalent conjugates of the drug combined with low molecular weight thiols and some non-thiol metabolites. Overall, Bzn treatment primarily affects thiol containing molecules in T. cruzi, and this interference with thiol metabolism contributes to the drug's mode of action.

The first genome survey sequencing of the rodent malaria parasite Plasmodium chabaudi is presented here. In 766 sequences, 131 putative gene sequences have been identified by sequence similarity database searches. Further, 7 potential gene families, four of which have not previously been described, were discovered. These genes may be important in understanding the biology of malaria, as well as offering potential new drug targets. We have also identified a number of candidate minisatellite sequences that could be helpful in genetic studies. Genome survey sequencing in P. chabaudi is a productive strategy in further developing this in vivo model of malaria, in the context of the malaria genome projects.

Sleeping sickness is resurgent in Africa. Adverse side-effects and drug-resistance are undermining the few drugs currently licensed for use against this disease, which is caused by parasitic protozoa of the Trypanosoma brucei group. Pentamidine and suramin are used before parasites become manifest in the central nervous system, after which the organic arsenical melarsoprol is used. Eflornithine is also useful in late-stage disease. A mode of action has been elucidated only for the ornithine decarboxylase inhibitor eflornithine. Both uptake and potential intracellular targets need to be considered when contemplating modes of action. The melaminophenyl arsenicals are accumulated via an unusual amino-purine transporter termed P2, which also seems to have a role in the uptake of the diamidine class of drugs to which pentamidine belongs. Since loss of this transporter leads to drug-resistance, other uptake mechanisms also need to be considered in generating novel trypanocides. Some nitroheterocyclic drugs have prolific activity against trypanosomes, although the fact that they are mutagenic in Ames' tests is acting as a barrier to further development. New drugs are urgently needed and the advent of genome sequencing and target validation using genetic modification will hopefully accelerate this process.

A number of triphenylmethane derivatives have been screened against 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Some of these compounds show good inhibition of the enzymes and also selectivity towards the parasite enzyme. Modelling was undertaken to dock the compounds into the active sites of both enzymes. Using a combination of DOCK 3.5 and FLEXIDOCK a correlation was obtained between docking score and both activity for the enzymes and selectivity. Visualisation of the docked structures of the inhibitors in the active sites of the enzymes yielded a possible explanation of the selectivity for the parasite enzyme.

Graphical abstract, Trypanosoma brucei, the parasite that causes human African trypanosomiasis, is auxotrophic for purines and has specialist nucleoside transporters to import these metabolites. In particular, the P2 aminopurine transporter can also selectively accumulate melamine derivatives. In this Letter, we report the coupling of the melamine moiety to 2-hydroxy APA, a potent ornithine decarboxylase inhibitor, with the aim of selectively delivering this compound to the parasite. The best compound described here shows an increased in vitro trypanocidal activity compared with the parent.

Problems associated with the current therapies of sleeping sickness include toxicity, resistance and a lack of a guaranteed supply. However, no new formulations are close to gaining a licence for clinical use and relatively few compounds have been shown to be effective in experimental systems. Many potentially good biochemical targets for drugs have been identified. Some of these have been validated and lead compounds have been developed. However, the biology of trypanosomes means that various pharmacological demands must be met in developing new trypanocides for clinical use. Foremost among these problems is the blood-brain barrier, across which trypanocides must cross to reach parasites in the cerebrospinal fluid.The principal problem, however, relates not to biological difficulties, which are technically surmountable, but to economics. Put simply, most representatives of the pharmaceutical industry believe that selling drugs to the victims of sleeping sickness will not yield sufficient income to justify expenses needed for the development of novel reagents. Only when this economic barrier can be lowered will new drugs emerge for use against sleeping sickness.

We have previously characterized, in Trypanosoma brucei, a multigene family encoding two developmentally regulated glucose transporters that are 80% identical at the amino-acid level. We report here the characterization of the homologous glucose transporters (TcoHT1 and TcoHT2) in Trypanosoma congolense, an African trypanosome responsible for disease in domestic animals. Both TcoHT isoforms, which are 92.4% identical, are encoded by a single cluster of genes containing two copies of TcoHT1 and three copies of TcoHT2 arranged alternately. Northern blot analysis revealed that TcoHT2 is expressed in all of the adaptive forms, while mRNA encoding TcoHT1 is only present in the metacyclic and bloodstream forms of T. congolense. When transfected with the TcoHT2 gene, Chinese Hamster Ovary cells express a hexose transporter with properties similar to those of the T. congolense procyclic forms (Kmd-glucose = 41 µm versus 64 µm). In contrast to TcoHT2, TcoHT1 expressed in the Chinese hamster ovary cells appeared to be a relatively low affinity glucose transporter (Kid-glucose = 0.8 mm). To determine the region(s) involved in the different apparent affinities for glucose, a chimera analysis was undertaken on the TcoHT isoforms. This study shows that amino-acid residues important for d-glucose recognition are located in the central region (between transmembrane domains 3 and 7) and in the C-terminal intracellular domain of TcoHT2. Site directed mutagenesis identified Ser193 located within transmembrane helix 4 as a key residue in relaxing the apparent affinity of TcoHT1 for glucose.

In the last few years, several reports have linked resistance to melaminophenyl arsenicals or diamidines in African trypanosomes to the loss or alteration of the P2 purine transporter [1–6]. This link was first established by Carter and Fairlamb [1]. They identified two adenosine transporters in Trypanosoma brucei brucei bloodstream forms, designated P1 and P2, that also mediated the uptake of inosine and adenine, respectively [1]. The P2 transporter exhibited a high affinity for melarsoprol, and both adenosine and adenine, but not inosine, protected bloodstream forms of trypanosomes from melarsen oxide-induced lysis in vitro. Interestingly, P2 transporter activity was not detectable in the melarsoprol-resistant clone RU15 [1]. These results suggested that the clinically important melaminophenyl arsenical drugs are substrates for the P2 adenosine/adenine transporter and as such RU15 should be useful in further delineating the role of nucleoside transporters in drug uptake. In order to investigate any additional changes in purine and drug uptake in melarsoprol-resistant clones, stabilates of RU15 were obtained from Professor A.H. Fairlamb (Dundee, UK) and grown in mice (BALB/c). The authenticity of the strain was investigated using PCR-based analysis of a hypervariable, tandemly repeated sequence that can be used as a genetic Abbre6iations: LSHTM, London School of Hygiene and Tropical Medicine; PCR, polymerase chain reaction; tbms, T. brucei minisatellite; UKC, University of Kent at Canterbury. * Corresponding author: Tel.: +44-1227-827581; fax: +441227-763912. E-mail address: s.m.jarvis@ukc.ac.uk (S.M. Jarvis) 1 Present address: Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.

The unique aspects of the biochemistry of trypanosomatids make rational drug design an attractive approach, but targets must be selected carefully. Genetic manipulation provides a valuable means of mimicking loss of function attributable to therapeutic intervention, but caution must be exercised when interpreting such data with respect to target validation.

A 36-kDa protein was isolated by affinity chromatography using Cymelarsan, an arsenical drug currently used in African trypanosomiasis treatment, as ligand. This protein was identified as glycerol-3-phosphate dehydrogenase. Trypanosomal glycerol-3-phosphate was bound covalently, whereas its counterpart from rabbit muscle bound by ionic interaction. Arsenical drugs inhibit the enzyme in a dose-dependent manner. Oxidation of cysteine residues protects against inactivation without significantly diminishing enzymic activity. Drug concentrations giving 50% inhibition of the dehydrogenase activity were determined for the enzyme from both Trypanosoma brucei and rabbit and indicate a higher sensitivity of the trypanosomal enzyme to arsenical drugs and thiol reagents. MS was used to identify residues of glycerol-3-phosphate dehydrogenase bound by Cymelarsan; they are not conserved in the mammalian enzyme.

Two apparently similar methods for making numerical judgments about a set of objects (point allocation and direct rating) have been shown to yield different profiles of values (here, numerical judgments) attached to Ranks. Direct rating typically leads to a nearly linear relationship of Value with Rank. When using point allocation, people tend to produce a half U-shape of Value with Rank, evidenced by a positive quadratic term in Rank, which is not present for direct rating. We argue that point allocation imposes a fixed-sum frame on the judgment task. Furthermore, certain problems, such as allocating a budget, are intrinsically framed as fixed-sum. These problems also lead to quadratic curvature of numerical judgment with Rank. We discuss the implications of these effects, both in formal and informal contexts.

(1998). Emerging therapeutic targets in parasitic protozoa. Emerging Therapeutic Targets: Vol. 2, No. 2, pp. 57-85.

Sugar transport across the plasma membrane is one of the most important transport processes. The cloning and expression of cDNAs from a superfamily of related sugar transporters that all adopt a 12-membrane-spanning-domain structure has opened new avenues of investigation, including pre-steady-state kinetic analysis. Structure–function analyses of mammalian and bacterial sugar transporters, and comparisons of these transporters with those of parasitic trypanosomatids, indicate that different environmental pressures have tailored the evolution of the various members of the sugar-transporter superfamily. Subtle distinctions in the function of these proteins can be related to particular amino acid residue substitutions.

The three-dimensional structure of 6-phosphogluconate dehydrogenase (6PGDH) from the parasitic protozoan Trypanosoma brucei has been solved at 2.8 A resolution. This pentose phosphate pathway enzyme is NADP-dependent; NADPH generated in the reaction protects against oxidative stress. The enzyme crystallises in the space-group P 3 1 21 with a dimer in the asymmetric unit and cell dimensions a = b =135.13 A, c =116.74 A, α=β=90°, γ=120°. The structure has refined to R =18.6% ( R free =27.3%) with good geometry. The amino acid sequence of T. brucei 6PGDH is only 35% identical to that of the sheep liver enzyme and significant activity differences have been observed. The active dimer assembles with the C-terminal tail of one subunit threaded through the other, forming part of the substrate binding site. The tail of T. brucei 6PGDH is shorter than that of the sheep enzyme and its terminal residues associate tightly with the second monomer. The three-dimensional structure shows this generates additional interactions between the subunits close to the active site; the coenzyme binding domain is thereby associated more tightly with the helical domain. Three residues, conserved in all other known sequences, are important in creating a salt bridge between monomers close to the substrate binding site. The differences could explain the 200-fold enhanced affinity observed for the substrate analogue 6-phospho-2-deoxy- d -gluconate and suggest targets for anti-parasite drug design. The coenzyme binding domain of 6PGDH has a β-α-β fold; while in most species the “fingerprint” sequence is GxAxxG, in the T. brucei enzyme it is GxGxxG. Additional interactions between the enzyme and the coenzyme bis-phosphate are likely in the parasite 6PGDH, accounting for greater inhibition (40-fold) of 2′5′-ADP. While the core of the T . brucei dimer was restrained during refinement, several conformational differences have been found between the monomers; those at the coenzyme binding site suggest the molecule could be asymmetric during the enzyme reaction.

Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (γ-Glu-Cys-Gly) and spermidine to form trypanothione (N 1,N 8-bis(glutathionyl)spermidine), which is involved in maintaining intracellular thiol redox and in defense against oxidants. In this study, the genes from Crithidia fasciculata, Cf-GSS and Cf-TRS, which encode, respectively, glutathionylspermidine synthetase (EC 6.3.1.8) and trypanothione synthetase (EC 6.3.1.9) have been cloned and expressed. The deduced amino acid sequence of both Cf-GSSand Cf-TRS share 50% sequence similarity with theEscherichia coli glutathionylspermidine synthetase/amidase. Both genes are present as single copies in the C. fasciculata genome. When expressed in E. coli andSaccharomyces cerevisiae, neither protein was present in an active soluble form. However, thiol analysis of S. cerevisiae demonstrated that cells transformed with theCf-GSS gene contained substantial amounts of glutathionylspermidine, whereas cells expressing both theCf-GSS and Cf-TRS genes contained glutathionylspermidine and trypanothione, confirming that these genes encode the functional glutathionylspermidine and trypanothione synthetases from C. fasciculata. The translation products of Cf-GSS and Cf-TRS show significant homology to the amidase domain present in E. coliglutathionylspermidine synthetase, which can catalyze both synthesis and degradation of glutathionylspermidine. Glutathionylspermidine synthetase isolated from C. fasciculata was found to possess a similar amidase activity.

In this paper we describe an approach to selectively deliver compounds to trypanosomes using an adenosine transporter which is unique to the trypanosome. Various polyamine analogues have been attached to known substrates of this adenosine transporter. The compounds prepared interact specifically with the adenosine transporter, some with a similar efficiency to berenil, a known substrate.

The rapid emergence of metabolomics has enabled system-wide measurements of metabolites in various organisms. However, advances in the mechanistic understanding of metabolic networks remain limited, as most metabolomics studies cannot routinely provide accurate metabolite identification, absolute quantification and flux measurement. Stable isotope labeling offers opportunities to overcome these limitations. Here we describe some current approaches to stable isotope-labeled metabolomics and provide examples of the significant impact that these studies have had on our understanding of cellular metabolism. Furthermore, we discuss recently developed software solutions for the analysis of stable isotope-labeled metabolomics data and propose the bioinformatics solutions that will pave the way for the broader application and optimal interpretation of system-scale labeling studies in metabolomics.

The African trypanosome, Ttypanosoma brucei, is a unicellular parasite causing African Trypanosomiasis (sleeping sickness in humans and nagana in animals). Due to some of its unique properties, it has emerged as a popular model organism in systems biology. A predictive quantitative model of glycolysis in the bloodstream form of the parasite has been constructed and updated several times. The Silicon Trypanosome is a project that brings together modellers and experimentalists to improve and extend this core model with new pathways and additional levels of regulation. These new extensions and analyses use computational methods that explicitly take different levels of uncertainty into account. During this project, numerous tools and techniques have been developed for this purpose, which can now be used for a wide range of different studies in systems biology.

Human African trypanosomiasis (HAT), often called sleeping sickness in its second, central nervous system–involved stage, is a disease of Sub-Saharan Africa. It is caused by subspecies of the protozoan parasite Trypanosoma brucei, which is transmitted by tsetse flies. Two distinctive conditions, classically discriminated by diverging clinical progression, are caused by different trypanosome subspecies. In East and Southern Africa, Trypanosoma brucei rhodesiense causes a rapidly advancing form that within weeks passes from the hemolymphatic first stage to the neurological second stage. In West and Central Africa, Trypanosoma brucei gambiense takes many months to progress to its second-stage form. Other trypanosome subspecies infect animals but not humans, in whom they are killed by several trypanosome-lytic factors. Rare genetic polymorphisms in which one such factor, apolipoprotein L1 (APOL1), is absent can lead to human infection by other trypanosome species (e.g., Trypanosoma evansi). Genetic variants of APOL1, common in African Americans suffering chronic kidney disease, originate in West Africa, where their prevalence is high. These alleles might have been selected during human evolution because of their ability to lyse trypanosomes (e.g., Trypanosoma rhodesiense) that would otherwise bypass the lytic effects of other APOL1 variants. Sleeping sickness treatment is stratified depending on the causative subspecies and whether the disease is diagnosed in the first or the second stage. For a variety of reasons, current drugs are unsatisfactory in the treatment of HAT.

o-phthalaldehyde inactivates homodimeric, NADP+ dependent, 6-phosphogluconate dehydrogenase from sheep liver, upon formation of a single isoindole derivative per enzyme subunit. This indicates that the thiol group of a cysteine residue or the epsilon-amino group of a lysine residue located within 3 A and crosslinked by the reagent is essential for catalysis. Fluorescence analyses of the modified enzyme suggest that the isoindole derivative forms at the binding site of the nicotinamide moiety of NADP+. The enzymes from Trypanosoma brucei and Lactococcus lactis are also inactivated suggesting a similar three-dimensional structure in this domain. The isoindole derivative does not form with two mutants of the T. brucei enzyme (Lys185His and Lys185Leu), this allowing to identify not only the lysine but also the cysteine involved in the cross-linking. The formation of the isoindole derivative inactivates not only the oxidative decarboxylation, but also two partial reactions catalysed by the enzyme.

Light-emitting diode (LED) fluorescence microscopy offers potential benefits in the diagnosis of human African trypanosomiasis and in other aspects of diseases management, such as detection of drug-resistant strains. To advance such approaches, reliable and specific fluorescent markers to stain parasites in human fluids are needed. Here we describe a series of novel green fluorescent diamidines and their suitability as probes with which to stain trypanosomes.

In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

International audience; 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 threonine's contribution being approximate to 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.

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 threonine's 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.

Human African trypanosomiasis (HAT) manifests in two stages of disease: firstly, haemolymphatic, and secondly, an encephalitic phase involving the central nervous system (CNS). New drugs to treat the second-stage disease are urgently needed, yet testing of novel drug candidates is a slow process because the established animal model relies on detecting parasitemia in the blood as late as 180 days after treatment. To expedite compound screening, we have modified the GVR35 strain of Trypanosoma brucei brucei to express luciferase, and have monitored parasite distribution in infected mice following treatment with trypanocidal compounds using serial, non-invasive, bioluminescence imaging. Parasites were detected in the brains of infected mice following treatment with diminazene, a drug which cures stage 1 but not stage 2 disease. Intravital multi-photon microscopy revealed that trypanosomes enter the brain meninges as early as day 5 post-infection but can be killed by diminazene, whereas those that cross the blood-brain barrier and enter the parenchyma by day 21 survived treatment and later caused bloodstream recrudescence. In contrast, all bioluminescent parasites were permanently eliminated by treatment with melarsoprol and DB829, compounds known to cure stage 2 disease. We show that this use of imaging reduces by two thirds the time taken to assess drug efficacy and provides a dual-modal imaging platform for monitoring trypanosome infection in different areas of the brain., Author Summary Trypanosoma brucei, a parasite transmitted by the bite of tsetse fly, is responsible for the disease human African trypanosomiasis (HAT). In advanced stages of HAT, trypanosomes invade the central nervous system (CNS), resulting in an array of neurological symptoms, and eventually death. Existing drugs for treatment of HAT are highly unsatisfactory and new safe drugs are urgently needed. Currently, potential drugs for HAT are screened in a mouse model that relies on the emergence of trypanosomes from tissues and their detection in blood. This can take up to 200 days, making selection and further development of new drugs slow and costly. Here, we employ in vivo imaging and genetically modified trypanosomes to monitor parasite distribution throughout the body in live infected mice. Our bioluminescence imaging approach provides sensitive detection of trypanosomes at sites of infection, allowing more rapid and more effective in vivo screening of candidate HAT drugs. Higher resolution intra-vital microscopy was used to investigate trypanosome dynamics in the brain and their accessibility to drugs during infection. These approaches allow more sensitive real time tracking of trypanosomes during chronic infections and will provide new insights about trypanosome pathogenesis in future experiments.

In vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra- and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form Trypanosoma brucei . The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (

This study examines small intestinal absorption-permeability, intestinal inflammation and ileal structure and function in HIV-positive male homosexuals.Thirty HIV-seropositive male homosexuals at various stages of disease underwent intestinal absorption permeability and 111indium leukocyte studies (for quantification of intestinal inflammation). Twenty-six men with AIDS had a dual radioisotopic ileal function test (whole body retention of tauro 23-[75Se]-selena 25-homocholic acid and 58cobalt-labelled cyanocobalamine), and 17 underwent ileocolonoscopy with terminal ileal biopsy.Well, HIV-infected, subjects had normal intestinal absorption-permeability, but both functions were impaired upon the development of AIDS. The median faecal excretion of 111indium in well patients (0.66%) did not differ significantly (P0.5) from controls (0.46%), but subjects with AIDS who were well or who had diarrhoea had significant (P0.005) intestinal inflammation (1.33% and 2.18%, respectively). The median 7-day retention of tauro 23-[75Se]-selena 25-homocholic acid in well patients with AIDS (38.9%) did not differ significantly (P0.2) from controls (39.3%), whereas the absorption of 58cobalt-labelled cyanocobalamine was significantly (P0.05) lower than controls (32.1% and 59.4%). Patients with AIDS-diarrhoea had significant (P0.001) malabsorption of both the bile acid (7.7%) and vitamin B12 (8.9%) which was more severe than in Crohn's ileitis (14.2% and 30.3%, respectively). Morphometric analyses of ileal biopsies were unremarkable in AIDS.These studies demonstrate a low-grade enteropathy in patients with AIDS, severe ileal malabsorption in patients with AIDS diarrhoea and relatively minor ileal morphologic changes. Malabsorption of bile acids may play a pathogenic role in patients with AIDS and diarrhoea.

The copy number of the gene encoding the carrier protein responsible for the uptake of glucose in bloodstream form Trypanosoma brucei (THT1) was investigated in the genome of 55 different members of the subgenus Trypanozoon. The gene is present in multiple copies in tandem arrays on two homologous chromosomes in these organisms, and copy number varies both intra- and interspecifically. Variability is also apparent in the number of genes encoding a second hexose transporter (THT2) which is the only isoform expressed in procyclic organisms. Multiple copies of THT2 are conserved in representatives of the non-tsetse transmitted species, Trypanosoma evansi and Trypanosoma equiperdum.

Minor groove binders are small molecules that form strong complexes with the minor groove of DNA. There are several structural types of which distamycin and netropsin analogues, oligoamides built from heterocyclic and aromatic amino acids, and bis-amidines separated by aromatic and heterocyclic rings are of particular pharmaceutical interest. These molecules have helical topology that approximately matches the curvature of DNA in the minor groove. Depending upon the precise structure of the minor groove binder, selectivity can be obtained with respect to the DNA base sequence to which the compound binds. Minor groove binders have found substantial applications in anti-cancer therapy but their significance in anti-infective therapy has also been significant and is growing. For example, compounds of the bis-amidine class have been notable contributors to antiparasitic therapy for many years with examples such as berenil and pentamidine being well-known. A recent growth area has been inreased sophistication in the oligoamide class. High sequence selectivity is now possible and compounds with distinct antibacterial, antifungal, antiviral, and antiparasitic activity have all been identified. Importantly, the structures of the most active compounds attacking the various infective organisms differ significantly but not necessarily predictively. This poses interesting questions of mechanism of action with many different targets involved in DNA processing being candidates. Access of compounds to specific cell types also plays a role and in some cases, can be decisive. Prospects for a range of selective therapeutic agents from this class of compounds are higher now than for some considerable time.