Open Access Highly Accessed Research article

Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites

Paul Hunt12*, Axel Martinelli13, Katarzyna Modrzynska1, Sofia Borges4, Alison Creasey1, Louise Rodrigues3, Dario Beraldi105, Laurence Loewe6, Richard Fawcett1, Sujai Kumar7, Marian Thomson7, Urmi Trivedi7, Thomas D Otto8, Arnab Pain89, Mark Blaxter57 and Pedro Cravo3

Author Affiliations

1 Institute for Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

2 Centre for Immunity, Infection and Evolution, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

3 Centro de Malaria e Outras Doenças Tropicais/IHMT/UEI Biologia Molecular, Universidade Nova de Lisboa, Lisbon, Portugal

4 Centro de Malaria e Outras Doenças Tropicais/IHMT/UEI Malaria, Universidade Nova de Lisboa, Lisbon, Portugal

5 Institute for Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

6 Centre for Systems Biology at Edinburgh, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

7 The GenePool, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

8 Pathogen Genomics Group, Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK

9 Computational Bioscience Research Center, Chemical and Life Sciences and Engineering Division, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia

10 Division of Genetics and Genomics, The Roslin Institute and R(D)SVS, University of Edinburgh, Roslin, UK

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BMC Genomics 2010, 11:499  doi:10.1186/1471-2164-11-499

Published: 16 September 2010

Abstract

Background

Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum.

Results

A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina® Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme.

Conclusions

This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.