Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum.
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.
Dharia
Neekesh V
NV
Department of Cell Biology, ICND 202, The Scripps Research Institute, North Torrey Pines Road, La Jolla, CA 92037, USA.
Sidhu
Amar Bir Singh
AB
Cassera
María Belén
MB
Westenberger
Scott J
SJ
Bopp
Selina Er
SE
Eastman
Rich T
RT
Plouffe
David
D
Batalov
Serge
S
Park
Daniel J
DJ
Volkman
Sarah K
SK
Wirth
Dyann F
DF
Zhou
Yingyao
Y
Fidock
David A
DA
Winzeler
Elizabeth A
EA
D004351:Drug Resistance / Q000235:genetics*; D005578:Fosfomycin / Q000031:analogs & derivatives / Q000494:pharmacology; D005784:Gene Amplification; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics; D020641:Polymorphism, Single Nucleotide
D005578:Fosfomycin; C024640:fosmidomycin