The Experts below are selected from a list of 1359 Experts worldwide ranked by ideXlab platform
Pascal Mäser - One of the best experts on this subject based on the ideXlab platform.
-
Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity.
PLoS pathogens, 2017Co-Authors: Laura Jeacock, Nicola Baker, Pascal Mäser, Natalie Wiedemar, David HornAbstract:Aquaglyceroporins (AQPs) transport water and glycerol and play important roles in drug-uptake in pathogenic trypanosomatids. For example, AQP2 in the human-infectious African trypanosome, Trypanosoma brucei gambiense, is responsible for Melarsoprol and pentamidine-uptake, and Melarsoprol treatment-failure has been found to be due to AQP2-defects in these parasites. To further probe the roles of these transporters, we assembled a T. b. brucei strain lacking all three AQP-genes. Triple-null aqp1-2-3 T. b. brucei displayed only a very moderate growth defect in vitro, established infections in mice and recovered effectively from hypotonic-shock. The aqp1-2-3 trypanosomes did, however, display glycerol uptake and efflux defects. They failed to accumulate glycerol or to utilise glycerol as a carbon-source and displayed increased sensitivity to salicylhydroxamic acid (SHAM), octyl gallate or propyl gallate; these inhibitors of trypanosome alternative oxidase (TAO) can increase intracellular glycerol to toxic levels. Notably, disruption of AQP2 alone generated cells with glycerol transport defects. Consistent with these findings, AQP2-defective, Melarsoprol-resistant clinical isolates were sensitive to the TAO inhibitors, SHAM, propyl gallate and ascofuranone, relative to Melarsoprol-sensitive reference strains. We conclude that African trypanosome AQPs are dispensable for viability and osmoregulation but they make important contributions to drug-uptake, glycerol-transport and respiratory-inhibitor sensitivity. We also discuss how the AQP-dependent inverse sensitivity to Melarsoprol and respiratory inhibitors described here might be exploited.
-
Chimerization at the AQP2-AQP3 locus is the genetic basis of Melarsoprol-pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates
International journal for parasitology. Drugs and drug resistance, 2015Co-Authors: Fabrice E. Graf, Jane C. Munday, Nicola Baker, David Horn, Harry P. De Koning, Pascal MäserAbstract:Aquaglyceroporin-2 is a known determinant of Melarsoprol–pentamidine cross-resistance in Trypanosoma brucei brucei laboratory strains. Recently, chimerization at the AQP2–AQP3 tandem locus was described from Melarsoprol–pentamidine cross-resistant Trypanosoma brucei gambiense isolates from sleeping sickness patients in the Democratic Republic of the Congo. Here, we demonstrate that reintroduction of wild-type AQP2 into one of these isolates fully restores drug susceptibility while expression of the chimeric AQP2/3 gene in aqp2–aqp3 null T. b. brucei does not. This proves that AQP2–AQP3 chimerization is the cause of Melarsoprol–pentamidine cross-resistance in the T. b. gambiense isolates.
-
Aquaporin 2 Mutations in Trypanosoma brucei gambiense Field Isolates Correlate with Decreased Susceptibility to Pentamidine and Melarsoprol
PLoS neglected tropical diseases, 2013Co-Authors: Fabrice E. Graf, Reto Brun, Philipp Ludin, David Horn, Philippe Buscher, Harry P. De Koning, Patient Pati Pyana, Tanja Wenzler, Marcel Kaiser, Pascal MäserAbstract:The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to Melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including Melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in Melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to Melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and Melarsoprol.
-
Drug resistance in African trypanosomiasis: the Melarsoprol and pentamidine story
Trends in Parasitology, 2013Co-Authors: Nicola Baker, Pascal Mäser, Harry P. De Koning, David HornAbstract:Melarsoprol and pentamidine represent the two main classes of drugs, the arsenicals and diamidines, historically used to treat the diseases caused by African trypanosomes: sleeping sickness in humans and Nagana in livestock. Cross-resistance to these drugs was first observed over 60 years ago and remains the only example of cross-resistance among sleeping sickness therapies. A Trypanosoma brucei adenosine transporter is well known for its role in the uptake of both drugs. More recently, aquaglyceroporin 2 (AQP2) loss of function was linked to Melarsoprol–pentamidine cross-resistance. AQP2, a channel that appears to facilitate drug accumulation, may also be linked to clinical cases of resistance. Here, we review these findings and consider some new questions as well as future prospects for tackling the devastating diseases caused by these parasites.
-
Genotypic Status of the TbAT1/P2 Adenosine Transporter of Trypanosoma brucei gambiense Isolates from Northwestern Uganda following Melarsoprol Withdrawal
PLoS neglected tropical diseases, 2009Co-Authors: Anne J. N. Kazibwe, Michael P. Barrett, Barbara Nerima, Pascal Mäser, Harry P. De Koning, Enock MatovuAbstract:Human African trypanosomiasis (HAT) manifests as a chronic infection caused by Trypanosoma brucei gambiense, or as a more acute form due to T. b. rhodesiense. Both manifestations occur in Uganda and Melarsoprol use against the former was jeopardised in the 1990s as reports of reduced efficacy increased to the point where it was dismissed as first-line treatment at some treatment centers. Previous work to elucidate possible mechanisms leading to Melarsoprol resistance pointed to a P2 type adenosine transporter known to mediate Melarsoprol uptake and previously shown to be mutated in significant numbers of patients not responding to the drug. Our present findings indicate that there is a low prevalence of mutants in foci where Melarsoprol relapses are infrequent. In addition we observe that at the Omugo focus where the drug was withdrawn as first line over 6 years ago, the mutant alleles have disappeared, suggesting that drug pressure is responsible for fuelling their spread. Thus constant monitoring for mutants could play a key role in cost-effective HAT management by identifying which foci can still use the less logistically demanding Melarsoprol as opposed to the alternative drug eflornithine. What is required now is a simple method for identifying such mutants at the point of care, enabling practitioners to make informed prescriptions at first diagnosis.
Enock Matovu - One of the best experts on this subject based on the ideXlab platform.
-
Genotypic Status of the TbAT1/P2 Adenosine Transporter of Trypanosoma brucei gambiense Isolates from Northwestern Uganda following Melarsoprol Withdrawal
2016Co-Authors: Anne J. N. Kazibwe, Michael P. Barrett, Barbara Nerima, Harry P. De Koning, Enock MatovuAbstract:Background: The development of arsenical and diamidine resistance in Trypanosoma brucei is associated with loss of drug uptake by the P2 purine transporter as a result of alterations in the corresponding T. brucei adenosine transporter 1 gene (TbAT1). Previously, specific TbAT1 mutant type alleles linked to Melarsoprol treatment failure were significantly more prevalent in T. b. gambiense from relapse patients at Omugo health centre in Arua district. Relapse rates of up to 30% prompted a shift from Melarsoprol to eflornithine (a-difluoromethylornithine, DFMO) as first-line treatment at this centre. The aim of this study was to determine the status of TbAT1 in recent isolates collected from T. b. gambiense sleeping sickness patients from Arua and Moyo districts in Northwestern Uganda after this shift in first-line drug choice. Methodology and results: Blood and cerebrospinal fluids of consenting patients were collected for DNA preparation and subsequent amplification. All of the 105 isolates from Omugo that we successfully analysed by PCR-RFLP possessed the TbAT1 wild type allele. In addition, PCR/RFLP analysis was performed for 74 samples from Moyo, where Melarsoprol is still the first line drug; 61 samples displayed the wild genotype while six were mutant and seven had a mixed pattern of both mutant and wild-type TbAT1. The Melarsoprol treatment failure rate at Moyo over the same period was nine out of 101 stage II cases that were followed up at least once. Five of the relapse cases harboured mutant TbAT1, one had the wild type, while no amplification was achieved from the remaining three samples
-
Drug resistance in human African trypanosomiasis
Future Microbiology, 2011Co-Authors: Michael P. Barrett, Anne J. N. Kazibwe, Isabel May Vincent, Richard Burchmore, Enock MatovuAbstract:Human African trypanosomiasis or 'sleeping sickness' is a neglected tropical disease caused by the parasite Trypanosoma brucei. A decade of intense international cooperation has brought the incidence to fewer than 10,000 reported cases per annum with anti-trypanosomal drugs, particularly against stage 2 disease where the CNS is involved, being central to control. Treatment failures with Melarsoprol started to appear in the 1990s and their incidence has risen sharply in many foci. Loss of plasma membrane transporters involved in drug uptake, particularly the P2 aminopurine transporter and also a transporter termed the high affinity pentamidine transporter, relate to Melarsoprol resistance selected in the laboratory. The same two transporters are also responsible for the uptake of the stage 1 drug pentamidine and, to varying extents, other diamidines. However, reports of treatment failures with pentamidine have been rare from the field. Eflornithine (difluoromethylornithine) has replaced Melarsoprol as first-line treatment in many regions. However, a need for protracted and complicated drug dosing regimens slowed widespread implementation of eflornithine monotherapy. A combination of eflornithine with nifurtimox substantially decreases the required dose and duration of eflornithine administration and this nifurtimox-eflornithine combination therapy has enjoyed rapid implementation. Unfortunately, selection of resistance to eflornithine in the laboratory is relatively easy (through loss of an amino acid transporter believed to be involved in its uptake), as is selection of resistance to nifurtimox. The first anecdotal reports of treatment failures with eflornithine monotherapy are emerging from some foci. The possibility that parasites resistant to Melarsoprol on the one hand, and eflornithine on the other, are present in the field indicates that genes capable of conferring drug resistance to both drugs are in circulation. If new drugs, that act in ways that will not render them susceptible to resistance mechanisms already in circulation do not appear soon, there is also a risk that the current downward trend in Human African trypanosomiasis prevalence will be reversed and, as has happened in the past, the disease will become resurgent, only this time in a form that resists available drugs.
-
Genotypic Status of the TbAT1/P2 Adenosine Transporter of Trypanosoma brucei gambiense Isolates from Northwestern Uganda following Melarsoprol Withdrawal
PLoS neglected tropical diseases, 2009Co-Authors: Anne J. N. Kazibwe, Michael P. Barrett, Barbara Nerima, Pascal Mäser, Harry P. De Koning, Enock MatovuAbstract:Human African trypanosomiasis (HAT) manifests as a chronic infection caused by Trypanosoma brucei gambiense, or as a more acute form due to T. b. rhodesiense. Both manifestations occur in Uganda and Melarsoprol use against the former was jeopardised in the 1990s as reports of reduced efficacy increased to the point where it was dismissed as first-line treatment at some treatment centers. Previous work to elucidate possible mechanisms leading to Melarsoprol resistance pointed to a P2 type adenosine transporter known to mediate Melarsoprol uptake and previously shown to be mutated in significant numbers of patients not responding to the drug. Our present findings indicate that there is a low prevalence of mutants in foci where Melarsoprol relapses are infrequent. In addition we observe that at the Omugo focus where the drug was withdrawn as first line over 6 years ago, the mutant alleles have disappeared, suggesting that drug pressure is responsible for fuelling their spread. Thus constant monitoring for mutants could play a key role in cost-effective HAT management by identifying which foci can still use the less logistically demanding Melarsoprol as opposed to the alternative drug eflornithine. What is required now is a simple method for identifying such mutants at the point of care, enabling practitioners to make informed prescriptions at first diagnosis.
-
genotypic status of the tbat1 p2 adenosine transporter of trypanosoma brucei gambiense isolates from northwestern uganda following Melarsoprol withdrawal
PLOS Neglected Tropical Diseases, 2009Co-Authors: Anne J. N. Kazibwe, Michael P. Barrett, Barbara Nerima, Pascal Mäser, Harry P. De Koning, Enock MatovuAbstract:Human African trypanosomiasis (HAT) manifests as a chronic infection caused by Trypanosoma brucei gambiense, or as a more acute form due to T. b. rhodesiense. Both manifestations occur in Uganda and Melarsoprol use against the former was jeopardised in the 1990s as reports of reduced efficacy increased to the point where it was dismissed as first-line treatment at some treatment centers. Previous work to elucidate possible mechanisms leading to Melarsoprol resistance pointed to a P2 type adenosine transporter known to mediate Melarsoprol uptake and previously shown to be mutated in significant numbers of patients not responding to the drug. Our present findings indicate that there is a low prevalence of mutants in foci where Melarsoprol relapses are infrequent. In addition we observe that at the Omugo focus where the drug was withdrawn as first line over 6 years ago, the mutant alleles have disappeared, suggesting that drug pressure is responsible for fuelling their spread. Thus constant monitoring for mutants could play a key role in cost-effective HAT management by identifying which foci can still use the less logistically demanding Melarsoprol as opposed to the alternative drug eflornithine. What is required now is a simple method for identifying such mutants at the point of care, enabling practitioners to make informed prescriptions at first diagnosis.
-
mechanisms of arsenical and diamidine uptake and resistance in trypanosoma brucei
Eukaryotic Cell, 2003Co-Authors: Enock Matovu, Michael P. Barrett, Richard Burchmore, Reto Brun, Pascal Mäser, John Enyaru, Mhairi Stewart, Federico Geiser, Lynsey J M Wallace, Ronald KaminskyAbstract: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.
Reto Brun - One of the best experts on this subject based on the ideXlab platform.
-
Aquaporin 2 Mutations in Trypanosoma brucei gambiense Field Isolates Correlate with Decreased
2016Co-Authors: Susceptibility To Pentamidine, Reto Brun, Fabrice E. Graf, Harry P. De Koning, Patient Pati Pyana, Tanja Wenzler, Marcel Kaiser, David HornAbstract:The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to Melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including Melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in Melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense ar
-
Aquaporin 2 Mutations in Trypanosoma brucei gambiense Field Isolates Correlate with Decreased Susceptibility to Pentamidine and Melarsoprol
PLoS neglected tropical diseases, 2013Co-Authors: Fabrice E. Graf, Reto Brun, Philipp Ludin, David Horn, Philippe Buscher, Harry P. De Koning, Patient Pati Pyana, Tanja Wenzler, Marcel Kaiser, Pascal MäserAbstract:The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to Melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including Melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in Melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to Melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and Melarsoprol.
-
Genotypic and phenotypic characterization of Trypanosoma brucei gambiense isolates from Ibba, South Sudan, an area of high Melarsoprol treatment failure rate.
Acta tropica, 2007Co-Authors: Naomi Maina, Pascal Mäser, Kagira Joseph Maina, Reto BrunAbstract:Resistance of trypanosomes to Melarsoprol is ascribed to reduced uptake of the drug via the P2 nucleoside transporter. The aim of this study was to look for evidence of drug resistance in Trypanosoma brucei gambiense isolates from sleeping sickness patients in Ibba, South Sudan, an area of high Melarsoprol failure rate. Eighteen T. b. gambiense stocks were phenotypically and only 10 strains genotypically characterized. In vitro, all isolates were sensitive to Melarsoprol, melarsen oxide, and diminazene. Infected mice were cured with a 4 day treatment of 2.5mg/kg bwt Melarsoprol, confirming that the isolates were sensitive. The gene that codes for the P2 transporter, TbATI, was amplified by PCR and sequenced. The sequences were almost identical to the TbAT1(sensitive) reference, except for one point mutation, C1384T resulting in the amino acid change proline-462 to serine. None of the described TbAT1(resistant)-type mutations were detected. In a T. b. gambiense sleeping sickness focus where Melarsoprol had to be abandoned due to the high incidence of treatment failures, no evidence for drug resistant trypanosomes or for TbAT1(resistant)-type alleles of the P2 transporter could be found. These findings indicate that factors other than drug resistance contribute to Melarsoprol treatment failures.
-
Melarsoprol- and pentamidine-resistant Trypanosoma brucei rhodesiense populations and their cross-resistance.
International Journal for Parasitology, 2007Co-Authors: Sonja C. Bernhard, Barbara Nerima, Pascal Mäser, Reto BrunAbstract:Resistance to Melarsoprol and pentamidine was induced in bloodstream-form Trypanosoma brucei rhodesiense STIB 900 in vitro, and drug sensitivity was determined for Melarsoprol, pentamidine and furamidine. The resistant populations were also inoculated into immunosuppressed mice to verify infectivity and to monitor whether rodent passage selects for clones with altered drug sensitivity. After proliferation in the mouse, trypanosomes were isolated and their IC(50) values to the three drugs were determined. To assess the stability of drug-induced resistance, drug pressure was ceased for 2 months and the drug sensitivity was determined again. Resistance was stable, with a few exceptions that are discussed. Drug IC(50)s indicated cross-resistance among all drugs, but to varying extents: resistance of the Melarsoprol-selected and pentamidine-selected trypanosomes to pentamidine was the same, but the pentamidine-selected trypanosome population showed lower resistance to Melarsoprol than the Melarsoprol-selected trypanosomes. Interestingly, both resistant populations revealed the same intermediate cross-resistance to furamidine. Resistant trypanosome populations were characterised by molecular means, referring to the status of the TbAT1 gene. The Melarsoprol-selected population apparently had lost TbAT1, whereas in the pentamidine-selected trypanosome population it was still present.
-
The role of Trypanosoma brucei MRPA in Melarsoprol susceptibility
Molecular and biochemical parasitology, 2005Co-Authors: Vincent P. Alibu, Reto Brun, Christina Richter, Frank Voncken, Gabriela Marti, Sanjay K. Shahi, Christina Kunz Renggli, Thomas Seebeck, Christine ClaytonAbstract:We previously showed that over-expression of Trypanosoma brucei MRPA, a member of the multidrug resistance protein family in T. brucei, reproducibly resulted in resistance to the anti-trypanosomal drug Melarsoprol in vitro. MRPA is predicted to mediate efflux of Melarsoprol as a conjugate with trypanothione, a glutathione–spermidine conjugate which is the major small thiol in trypanosomes. Here, we show that depletion of MRPA by RNA interference resulted in moderate hypersensitivity to both Melarsoprol and melarsen oxide. Over-expression of MRPA alone is not sufficient to cause Melarsoprol resistance in vivo, although it is sufficient in vitro. This discrepancy is not an effect of drug metabolism since over-expression of MRPA alone conferred resistance to Melarsoprol and its principle metabolite, melarsen oxide, in vitro. Over-expression of MRPA was not detected in four Melarsoprol-resistant trypanosome isolates from sleeping sickness patients. © 2005 Elsevier B.V. All rights reserved.
David Horn - One of the best experts on this subject based on the ideXlab platform.
-
Melarsoprol Resistance in African Trypanosomiasis.
Trends in parasitology, 2018Co-Authors: Alan H. Fairlamb, David HornAbstract:Arsenicals were introduced as monotherapies for the treatment of human African trypanosomiasis, or sleeping sickness, over 100 years ago. Toxicity has always been an issue but these drugs have proven to be both effective and quite durable. Unfortunately, Melarsoprol-resistant parasites emerged as early as the 1970s and were widespread by the late 1990s. Resistance was due to mutations affecting an aquaglyceroporin (AQP2), a parasite solute and drug transporter. This is the only example of widespread drug resistance in trypanosomiasis patients for which the genetic basis is known. This link between Melarsoprol and AQP2 illustrates how a drug transporter can improve drug selectivity but, at the same time, highlights the risk of resistance when the drug uptake mechanism is dispensable for parasite viability and virulence.
-
Quantitative analysis of the Melarsoprol-induced mitotic defect.
2018Co-Authors: James A. Thomas, Nicola Baker, Sebastian Hutchinson, Caia Dominicus, Anna Trenaman, Lucy Glover, Sam Alsford, David HornAbstract:(A) ImageJ analysis. Raw images (left hand panels) and ImageJ outputs (right hand panels) demonstrating conjoined nuclei (red arrowheads) after Melarsoprol treatment. (B) DNA content of nuclei in Melarsoprol (Mel) treated and non-treated (NT) cells is plotted. Ratio of area and signal (area multiplied by average intensity) of nuclei to average area and signal of kinetoplasts in ‘1n2k’ cells. Error bars, SD; red lines, averages; ***, P < 0.001 from Students t-test.
-
Melarsoprol inhibits mitosis by a kinase-dependent mechanism.
2018Co-Authors: James A. Thomas, Nicola Baker, Sebastian Hutchinson, Caia Dominicus, Anna Trenaman, Lucy Glover, Sam Alsford, David HornAbstract:(A) The microscopy images show the localisation of three C-terminal epitope (myc) tagged kinases. Scale bar, 5 μm. (B) Protein blotting indicates tetracycline (Tet)-inducible RNAi-mediated knockdown of the indicated kinases. Coomassie-stained gels serve as loading-controls. (C) Dose-response curves for Melarsoprol following knockdown (+Tet) of the indicated kinases. EC50 values are shown +/- SD. Data are shown for pairs of biological replicates; similar data were also obtained for a third biological replicate in each case. (D) The Melarsoprol-induced conjoined nuclei phenotype is diminished following knockdown of the indicated kinases. *, P < 0.05 from Students t-test.
-
Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity.
PLoS pathogens, 2017Co-Authors: Laura Jeacock, Nicola Baker, Pascal Mäser, Natalie Wiedemar, David HornAbstract:Aquaglyceroporins (AQPs) transport water and glycerol and play important roles in drug-uptake in pathogenic trypanosomatids. For example, AQP2 in the human-infectious African trypanosome, Trypanosoma brucei gambiense, is responsible for Melarsoprol and pentamidine-uptake, and Melarsoprol treatment-failure has been found to be due to AQP2-defects in these parasites. To further probe the roles of these transporters, we assembled a T. b. brucei strain lacking all three AQP-genes. Triple-null aqp1-2-3 T. b. brucei displayed only a very moderate growth defect in vitro, established infections in mice and recovered effectively from hypotonic-shock. The aqp1-2-3 trypanosomes did, however, display glycerol uptake and efflux defects. They failed to accumulate glycerol or to utilise glycerol as a carbon-source and displayed increased sensitivity to salicylhydroxamic acid (SHAM), octyl gallate or propyl gallate; these inhibitors of trypanosome alternative oxidase (TAO) can increase intracellular glycerol to toxic levels. Notably, disruption of AQP2 alone generated cells with glycerol transport defects. Consistent with these findings, AQP2-defective, Melarsoprol-resistant clinical isolates were sensitive to the TAO inhibitors, SHAM, propyl gallate and ascofuranone, relative to Melarsoprol-sensitive reference strains. We conclude that African trypanosome AQPs are dispensable for viability and osmoregulation but they make important contributions to drug-uptake, glycerol-transport and respiratory-inhibitor sensitivity. We also discuss how the AQP-dependent inverse sensitivity to Melarsoprol and respiratory inhibitors described here might be exploited.
-
Aquaporin 2 Mutations in Trypanosoma brucei gambiense Field Isolates Correlate with Decreased
2016Co-Authors: Susceptibility To Pentamidine, Reto Brun, Fabrice E. Graf, Harry P. De Koning, Patient Pati Pyana, Tanja Wenzler, Marcel Kaiser, David HornAbstract:The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to Melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including Melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in Melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense ar
Michael P. Barrett - One of the best experts on this subject based on the ideXlab platform.
-
Genotypic Status of the TbAT1/P2 Adenosine Transporter of Trypanosoma brucei gambiense Isolates from Northwestern Uganda following Melarsoprol Withdrawal
2016Co-Authors: Anne J. N. Kazibwe, Michael P. Barrett, Barbara Nerima, Harry P. De Koning, Enock MatovuAbstract:Background: The development of arsenical and diamidine resistance in Trypanosoma brucei is associated with loss of drug uptake by the P2 purine transporter as a result of alterations in the corresponding T. brucei adenosine transporter 1 gene (TbAT1). Previously, specific TbAT1 mutant type alleles linked to Melarsoprol treatment failure were significantly more prevalent in T. b. gambiense from relapse patients at Omugo health centre in Arua district. Relapse rates of up to 30% prompted a shift from Melarsoprol to eflornithine (a-difluoromethylornithine, DFMO) as first-line treatment at this centre. The aim of this study was to determine the status of TbAT1 in recent isolates collected from T. b. gambiense sleeping sickness patients from Arua and Moyo districts in Northwestern Uganda after this shift in first-line drug choice. Methodology and results: Blood and cerebrospinal fluids of consenting patients were collected for DNA preparation and subsequent amplification. All of the 105 isolates from Omugo that we successfully analysed by PCR-RFLP possessed the TbAT1 wild type allele. In addition, PCR/RFLP analysis was performed for 74 samples from Moyo, where Melarsoprol is still the first line drug; 61 samples displayed the wild genotype while six were mutant and seven had a mixed pattern of both mutant and wild-type TbAT1. The Melarsoprol treatment failure rate at Moyo over the same period was nine out of 101 stage II cases that were followed up at least once. Five of the relapse cases harboured mutant TbAT1, one had the wild type, while no amplification was achieved from the remaining three samples
-
Aquaglyceroporin 2 controls susceptibility to Melarsoprol and pentamidine in African trypanosomes.
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Nicola Baker, Michael P. Barrett, Jane C. Munday, Harry P. De Koning, Lucy Glover, David Aguinaga Andrés, David HornAbstract:African trypanosomes cause sleeping sickness in humans, a disease that is typically fatal without chemotherapy. Unfortunately, drug resistance is common and Melarsoprol-resistant trypanosomes often display cross-resistance to pentamidine. Although Melarsoprol/pentamidine cross-resistance (MPXR) has been an area of intense interest for several decades, our understanding of the underlying mechanisms remains incomplete. Recently, a locus encoding two closely related aquaglyceroporins, AQP2 and AQP3, was linked to MPXR in a high-throughput loss-of-function screen. Here, we show that AQP2 has an unconventional “selectivity filter.” AQP2-specific gene knockout generated MPXR trypanosomes but did not affect resistance to a lipophilic arsenical, whereas recombinant AQP2 reversed MPXR in cells lacking native AQP2 and AQP3. AQP2 was also shown to be disrupted in a laboratory-selected MPXR strain. Both AQP2 and AQP3 gained access to the surface plasma membrane in insect life-cycle–stage trypanosomes but, remarkably, AQP2 was specifically restricted to the flagellar pocket in the bloodstream stage. We conclude that the unconventional aquaglyceroporin, AQP2, renders cells sensitive to both Melarsoprol and pentamidine and that loss of AQP2 function could explain cases of innate and acquired MPXR.
-
Drug resistance in human African trypanosomiasis
Future Microbiology, 2011Co-Authors: Michael P. Barrett, Anne J. N. Kazibwe, Isabel May Vincent, Richard Burchmore, Enock MatovuAbstract:Human African trypanosomiasis or 'sleeping sickness' is a neglected tropical disease caused by the parasite Trypanosoma brucei. A decade of intense international cooperation has brought the incidence to fewer than 10,000 reported cases per annum with anti-trypanosomal drugs, particularly against stage 2 disease where the CNS is involved, being central to control. Treatment failures with Melarsoprol started to appear in the 1990s and their incidence has risen sharply in many foci. Loss of plasma membrane transporters involved in drug uptake, particularly the P2 aminopurine transporter and also a transporter termed the high affinity pentamidine transporter, relate to Melarsoprol resistance selected in the laboratory. The same two transporters are also responsible for the uptake of the stage 1 drug pentamidine and, to varying extents, other diamidines. However, reports of treatment failures with pentamidine have been rare from the field. Eflornithine (difluoromethylornithine) has replaced Melarsoprol as first-line treatment in many regions. However, a need for protracted and complicated drug dosing regimens slowed widespread implementation of eflornithine monotherapy. A combination of eflornithine with nifurtimox substantially decreases the required dose and duration of eflornithine administration and this nifurtimox-eflornithine combination therapy has enjoyed rapid implementation. Unfortunately, selection of resistance to eflornithine in the laboratory is relatively easy (through loss of an amino acid transporter believed to be involved in its uptake), as is selection of resistance to nifurtimox. The first anecdotal reports of treatment failures with eflornithine monotherapy are emerging from some foci. The possibility that parasites resistant to Melarsoprol on the one hand, and eflornithine on the other, are present in the field indicates that genes capable of conferring drug resistance to both drugs are in circulation. If new drugs, that act in ways that will not render them susceptible to resistance mechanisms already in circulation do not appear soon, there is also a risk that the current downward trend in Human African trypanosomiasis prevalence will be reversed and, as has happened in the past, the disease will become resurgent, only this time in a form that resists available drugs.
-
Melarsoprol cyclodextrin inclusion complexes as promising oral candidates for the treatment of human african trypanosomiasis
PLoS neglected tropical diseases, 2011Co-Authors: Jean Rodgers, Michael P. Barrett, Barbara Bradley, George Gettinby, Stéphane Gibaud, Amy J. Jones, Christopher Mccabe, Peter G. E. KennedyAbstract:Human African trypanosomiasis (HAT), or sleeping sickness, results from infection with the protozoan parasites Trypanosoma brucei (T.b.) gambiense or T.b.rhodesiense and is invariably fatal if untreated. There are 60 million people at risk from the disease throughout sub-Saharan Africa. The infection progresses from the haemolymphatic stage where parasites invade the blood, lymphatics and peripheral organs, to the late encephalitic stage where they enter the central nervous system (CNS) to cause serious neurological disease. The trivalent arsenical drug Melarsoprol (Arsobal) is the only currently available treatment for CNS-stage T.b.rhodesiense infection. However, it must be administered intravenously due to the presence of propylene glycol solvent and is associated with numerous adverse reactions. A severe post-treatment reactive encephalopathy occurs in about 10% of treated patients, half of whom die. Thus Melarsoprol kills 5% of all patients receiving it. Cyclodextrins have been used to improve the solubility and reduce the toxicity of a wide variety of drugs. We therefore investigated two Melarsoprol cyclodextrin inclusion complexes; Melarsoprol hydroxypropyl-β-cyclodextrin and Melarsoprol randomly-methylated-β-cyclodextrin. We found that these compounds retain trypanocidal properties in vitro and cure CNS-stage murine infections when delivered orally, once per day for 7-days, at a dosage of 0.05 mmol/kg. No overt signs of toxicity were detected. Parasite load within the brain was rapidly reduced following treatment onset and magnetic resonance imaging showed restoration of normal blood-brain barrier integrity on completion of chemotherapy. These findings strongly suggest that complexed Melarsoprol could be employed as an oral treatment for CNS-stage HAT, delivering considerable improvements over current parenteral chemotherapy.
-
Genotypic Status of the TbAT1/P2 Adenosine Transporter of Trypanosoma brucei gambiense Isolates from Northwestern Uganda following Melarsoprol Withdrawal
PLoS neglected tropical diseases, 2009Co-Authors: Anne J. N. Kazibwe, Michael P. Barrett, Barbara Nerima, Pascal Mäser, Harry P. De Koning, Enock MatovuAbstract:Human African trypanosomiasis (HAT) manifests as a chronic infection caused by Trypanosoma brucei gambiense, or as a more acute form due to T. b. rhodesiense. Both manifestations occur in Uganda and Melarsoprol use against the former was jeopardised in the 1990s as reports of reduced efficacy increased to the point where it was dismissed as first-line treatment at some treatment centers. Previous work to elucidate possible mechanisms leading to Melarsoprol resistance pointed to a P2 type adenosine transporter known to mediate Melarsoprol uptake and previously shown to be mutated in significant numbers of patients not responding to the drug. Our present findings indicate that there is a low prevalence of mutants in foci where Melarsoprol relapses are infrequent. In addition we observe that at the Omugo focus where the drug was withdrawn as first line over 6 years ago, the mutant alleles have disappeared, suggesting that drug pressure is responsible for fuelling their spread. Thus constant monitoring for mutants could play a key role in cost-effective HAT management by identifying which foci can still use the less logistically demanding Melarsoprol as opposed to the alternative drug eflornithine. What is required now is a simple method for identifying such mutants at the point of care, enabling practitioners to make informed prescriptions at first diagnosis.