Identification of putative Plasmodium falciparum mefloquine resistance genes

Mefloquine is an effective antimalarial drug; however, resistant strains of the human malarial pathogen, Plasmodium falciparum, are beginning to arise. The yeast Saccharomyces cerevisiae is sensitive to mefloquine, enabling a screen for P. falciparum genes involved in resistance. Yeast were transformed with a P. falciparum expression library, followed by selection on mefloquine plates and sequencing of plasmids that conferred resistance. We characterized the four genes that conferred the strongest mefloquine-resistant phenotype in yeast. All four (PFD0090c, PFI0195c, PF10_0372 and PF14_0649) are uncharacterized P. falciparum genes from distinct chromosomes (4, 9, 10 and 14, respectively). The mefloquine-resistant phenotype was dependent on induction of the P. falciparum gene and independent of vector context. PFI0195c, which likely encodes a small GTPase activator (GAP), also conferred resistance to cycloheximide and halofantrine in yeast. Immunolocalization of the encoded protein to the Golgi complex in yeast is consistent with potential GAP function. The other three candidate proteins localized to the cytoplasm and plasma membrane (PF14_0649), nuclear envelope/ER (PF10_0372) and Golgi (PFD0090c) of yeast. Analysis of mefloquine-resistant P. falciparum strains and the mefloquine-sensitive strain, W2, by sequencing and semi-quantitative RT-PCR identified no relevant mutations in the resistant strains but showed that PFI0195c was upregulated in two out of three resistant strains and PF14_0649 was upregulated in all resistant strains tested.