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Drug resistance in African trypanosomes - a deep sequencing approach

English title Drug resistance in African trypanosomes - a deep sequencing approach
Applicant Mäser Pascal
Number 135746
Funding scheme Project funding (Div. I-III)
Research institution Swiss Tropical and Public Health Institute Medical Services and Diagnostic Universität Basel
Institution of higher education University of Basel - BS
Main discipline Molecular Biology
Start/End 01.07.2011 - 28.02.2015
Approved amount 412'054.00
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All Disciplines (2)

Discipline
Molecular Biology
Experimental Microbiology

Keywords (7)

Drug resistance; Trypanosoma brucei; Genomics; Molecular mechanism; Diamidine; Arsenical; Oxaborole

Lay Summary (English)

Lead
Lay summary
Drug resistance is a serious threat to the successful therapy of infectious diseases. An understanding of the underlying mechanisms is pivotal for the management of drug resistance, aiming to develop DNA-based tests for early detection of resistance genes. We are studying the molecular mechanisms of drug resistance in African trypanosomes. Trypanosoma brucei rhodesiense and T. b. gambiense are the causative agents of human sleeping sickness, a fatal disease of sub-Saharan Africa that is transmitted by the Tsetse fly (Glossina species). The treatment of sleeping sickness relies on the drugs pentamidine and suramin for the first, haemolymphatic stage of the disease, and on melarsoprol, eflornithine and nifurtimox-eflornithine combination therapy for the second stage, when the trypanosomes have invaded the cerebrospinal fluid. These drugs are outdated and suffer from severe side-effects. Yet even the most optimistic scenario admits several years until better drugs such as fexinidazole or oxaboroles will be available for the treatment of sleeping sickness. Sustainable use of the current drugs requires an understanding of the molecular mechanisms of drug resistance. We have been selecting T. b. rhodesiense in vitro with sublethal doses of melarsoprol and pentamidine. The resultant lines exhibit stable cross resistance to the two drugs. Now we are trying to identify the underlying mutations by comparative genomics and transcriptomics using deep sequencing technologies. The genomes of the resistant T. b. rhodesiense lines and their drug-sensitive parent will be sequenced on the Roche 454 system (possible thanks to a Roche 10Gb Award), and comparative transcriptomics will be performed on the Illumina reader. Candidate mutations for drug resistance will be validated by reverse genetics in T. b. brucei. The results of the proposed research have the potential to be directly translated to the improvement of sleeping sickness therapy and the confinement of drug resistance.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Comparative genomics of drug resistance in Trypanosoma brucei rhodesiense.
Graf Fabrice E, Ludin Philipp, Arquint Christian, Schmidt Remo S, Schaub Nadia, Kunz Renggli Christina, Munday Jane C, Krezdorn Jessica, Baker Nicola, Horn David, Balmer Oliver, Caccone Adalgisa, de Koning Harry P, Mäser Pascal (2016), Comparative genomics of drug resistance in Trypanosoma brucei rhodesiense., in Cellular and molecular life sciences : CMLS, 73(17), 3387-400.
Comparative genomics reveals multiple genetic backgrounds of human pathogenicity in the Trypanosoma brucei complex
Sistrom Mark (2014), Comparative genomics reveals multiple genetic backgrounds of human pathogenicity in the Trypanosoma brucei complex, in Genome Biol Evol, 6, 2811.
TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei
Shameer S (2014), TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei, in Nuc Acids Res, 43, D637.
Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs
Munday Jane (2014), Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs, in J Antimicrob Chemother, 69, 651.
Aquaporin 2 mutations in Trypanosoma brucei gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol
Graf F (2013), Aquaporin 2 mutations in Trypanosoma brucei gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol, in PLoS - NTD, 7, e2475.
Drug resistant African trypanosomiasis: the arsenical and diamidine story
Baker Nicola (2013), Drug resistant African trypanosomiasis: the arsenical and diamidine story, in Trends in Parasitology, 29, 110.
Trypanosoma brucei adenine-phosphoribosyltransferases mediate adenine salvage and aminopurinol susceptibility but not adenine toxicity
Lüscher Alexandra (2013), Trypanosoma brucei adenine-phosphoribosyltransferases mediate adenine salvage and aminopurinol susceptibility but not adenine toxicity, in Int J Parasitol - Drugs Drug Res, 4, 55.
Chimerization at the AQP2-AQP3 locus is the genetic basis of melarsoprol-pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates
Graf Fabrice, Chimerization at the AQP2-AQP3 locus is the genetic basis of melarsoprol-pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates, in Int J Parasitol - Drugs Drug Res.

Collaboration

Group / person Country
Types of collaboration
Prof. I. Roditi, Uni Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Prof. D. Horn Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Dr. H.P. de Koning Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. A. Caccone, Yale U. United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. P. Büscher Belgium (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Irseer Naturstofftage Talk given at a conference Medikamentenresistenz bei parasitischen Protozoen 25.02.2015 Irsee, Germany Mäser Pascal;
Frontiers of Parasitology Talk given at a conference Human African trypanosomiasis - from neglect to control to elimination? 29.06.2014 Berlin, Germany Mäser Pascal;
BSP Trypanosomiasis & Leishmaniasis Seminar Poster Molecular mechanisms of melarsoprol-pentamidine cross-resistance in Trypanosoma brucei 06.04.2014 Cambridge, Great Britain and Northern Ireland Graf Fabrice;
Swiss Trypanosomatid Meeting Talk given at a conference Molecular mechanisms of melarsoprol-pentamidine cross-resistance in Trypanosoma brucei 22.01.2014 Leysin, Switzerland Graf Fabrice;
Molecular Parasitology Poster Molecular mechanisms of melarsoprol-pentamidine cross-resistance in Trypanosoma brucei 08.09.2013 Woods Hole, United States of America Graf Fabrice;
Emerging paradigms in anti-infective drug design Talk given at a conference From genome to antiparasitic drug target 17.09.2012 London, Great Britain and Northern Ireland Mäser Pascal;
Indo-Swiss Joint Symposium Talk given at a conference Parasite comparative genomics 03.11.2011 New Delhi, India Mäser Pascal;


Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
ONE HEALTH - gemeinsame Weiterbildung für Human- und VeterinärmedizinerInnen Talk 08.10.2014 Basel, Switzerland Mäser Pascal;


Self-organised

Title Date Place
Swiss TPH Open Day - Medikamente gegen die Schlafkrankheit 30.06.2014 Basel, Switzerland

Communication with the public

Communication Title Media Place Year
Other activities Parasitologische Führungen am Naturhist. Museum BS German-speaking Switzerland 2015
Talks/events/exhibitions GenSuisse - Gentechnologie als Werkzeug zur Entwicklung von Medikamenten gegen die Schlafkrankheit Western Switzerland 2014
Other activities KidsLab Basel German-speaking Switzerland 2014

Awards

Title Year
Award for best talk, Swiss Trypanosomatid Meeting 2014
Award for best poster, Swiss Trypanosomatid Meeting 2013

Associated projects

Number Title Start Funding scheme
114819 Molecular mechanisms of nutrient acquisition in parasites 01.05.2007 SNSF Professorships
156264 Drug resistance in African trypanosomes - from the lab to the field and back 01.07.2015 Project funding (Div. I-III)
66150 Molecular mechanisms of nutrient transport and drug resistance in Trypanosoma brucei 01.05.2003 SNSF Professorships
156264 Drug resistance in African trypanosomes - from the lab to the field and back 01.07.2015 Project funding (Div. I-III)
141913 Transporters of Trypanosoma brucei: Phylogeny - Physiology - Pharmacology 01.06.2013 Sinergia
127300 Functional genomics of nutrient transporters in Trypanosoma brucei: From physiology to pharmacology 01.01.2010 Sinergia
120566 Genetic and chemical validation of Trypanosoma brucei rhodesiense adenosine kinase, the intracellular target of CD12001 01.01.2009 Project funding (Div. I-III)

Abstract

Sleeping sickness is a fatal disease of sub-Saharan Africa, caused by Trypanosoma brucei ssp. and transmitted by the Tsetse fly (Glossina spp.). The treatment of sleeping sickness relies on the drugs pentamidine and suramin for the first, haemolymphatic stage of the disease, respectively melarsoprol, eflornithine and nifurtimox-eflornithine combination therapy for the second stage, when the trypanosomes have invaded the cerebrospinal fluid. These drugs are outdated and suffer from severe side-effects. Yet even the most optimistic scenario admits several years until better drugs such as fexinidazole or oxaboroles will be available for the treatment of sleeping sickness. Sustainable use of the current drugs requires an understanding of the molecular mechanisms of drug resistance, allowing for DNA-based diagnostic tests to monitor the spread of resistant parasites. Cross-resistance between pentamidine and melarsoprol is a well known phenomenon and has been attributed to loss of the gene TbAT1, which encodes an adenosine transporter that also mediates cellular uptake of diamidines and melamine-based arsenicals. However, we have selected cross-resistant T. b. rhodesiense lines in vitro with resistance factors to pentamidine and melarsoprol an order of magnitude above those of tbat1 null trypanosomes, demonstrating that additional mutations must be involved. Here we propose to identify these mutations by comparative genomics and transcriptomics using deep sequencing technologies. The genomes of the two resistant T. b. rhodesiense lines and their drug-sensitive parent will be sequenced on the Roche 454 system (possible thanks to a Roche 10Gb Award), and comparative transcriptomics will be performed on the Illumina reader. Candidate mutations for drug resistance will be validated by reverse genetics in wildtype as well as tbat1 null T. b. brucei. In addition, we are selecting for oxaborole-resistant T. b. rhodesiense lines which, once obtained, will be characterized using the same approach. The results of the proposed research have the potential to be directly translated to the improvement of sleeping sickness therapy and the confinement of drug resistance.
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