Open Access Highly Accessed Research

Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum

Neekesh V Dharia1, Amar Bir Singh Sidhu23, María Belén Cassera4, Scott J Westenberger1, Selina ER Bopp1, Rich T Eastman2, David Plouffe5, Serge Batalov5, Daniel J Park6, Sarah K Volkman78, Dyann F Wirth67, Yingyao Zhou5, David A Fidock29 and Elizabeth A Winzeler15*

Author Affiliations

1 Department of Cell Biology, ICND 202, The Scripps Research Institute, North Torrey Pines Road, La Jolla, CA 92037, USA

2 Department of Microbiology, Columbia University College of Physicians and Surgeons, West 186th Street, New York, NY 10032, USA

3 The Broad Institute of MIT and Harvard, Cambridge Center, Cambridge, MA 02142, USA

4 Department of Biochemistry, Albert Einstein College of Medicine at Yeshiva University, Morris Park Avenue, Bronx, NY 10461, USA

5 Genomics Institute of the Novartis Research Foundation, John Jay Hopkins Drive, San Diego, CA 92121, USA

6 The Broad Institute of MIT and Harvard, Cambridge Center, Cambridge, MA 02142, USA

7 Department of Immunology and Infectious Diseases, Harvard School of Public Health, Huntington Avenue, Boston, MA 02115, USA

8 School for Health Studies, Simmons College, The Fenway, Boston, MA 02115, USA

9 Department of Medicine, Columbia University College of Physicians and Surgeons, West 186th Street, New York, NY 10032, USA

For all author emails, please log on.

Genome Biology 2009, 10:R21  doi:10.1186/gb-2009-10-2-r21

Published: 13 February 2009

Abstract

Background

The identification of genetic changes that confer drug resistance or other phenotypic changes in pathogens can help optimize treatment strategies, support the development of new therapeutic agents, and provide information about the likely function of genes. Elucidating mechanisms of phenotypic drug resistance can also assist in identifying the mode of action of uncharacterized but potent antimalarial compounds identified in high-throughput chemical screening campaigns against Plasmodium falciparum.

Results

Here we show that tiling microarrays can detect de novo a large proportion of the genetic changes that differentiate one genome from another. We show that we detect most single nucleotide polymorphisms or small insertion deletion events and all known copy number variations that distinguish three laboratory isolates using readily accessible methods. We used the approach to discover mutations that occur during the selection process after transfection. We also elucidated a mechanism by which parasites acquire resistance to the antimalarial fosmidomycin, which targets the parasite isoprenoid synthesis pathway. Our microarray-based approach allowed us to attribute in vitro derived fosmidomycin resistance to a copy number variation event in the pfdxr gene, which enables the parasite to overcome fosmidomycin-mediated inhibition of isoprenoid biosynthesis.

Conclusions

We show that newly emerged single nucleotide polymorphisms can readily be detected and that malaria parasites can rapidly acquire gene amplifications in response to in vitro drug pressure. The ability to define comprehensively genetic variability in P. falciparum with a single overnight hybridization creates new opportunities to study parasite evolution and improve the treatment and control of malaria.