Analysis of resistance to inhibitors of Plasmodium falciparum dihydrofolate reductase in yeast

Abstract

Resistance to the malaria drugs is an increasing problem for treating the 200-300 million people infected yearly with falciparum malaria. Detailed analysis of the development of drug resistance has been complicated by difficulties in defining differences between P. falciparum isolates and by the difficult culture conditions required to grow parasites. The inability to distinguish P. falciparum isolates has complicated interpretation of drug resistance data from field isolates and hampered genetic analysis of drug resistance. A method was developed based on the polymerase chain reaction (PCR) to quickly genotype P. falciparum strains using polymorphisms in genes containing variable repeats (RESA, MSA-1, MSA-2, and CSP). The technique requires very small samples (100 $\mu1$ of infected blood) and is suitable for field use. Additionally, the technique is sensitive enough to detect a 1% contamination of one strain with another.Examination of populations of P. falciparum sensitive or resistant to the malaria drugs pyrimethamine (pyr) and cycloguanil showed that point mutations within the dihydrofolate reductase (DHFR) gene correlated with the drug resistance. To study the frequency of these changes and the effect of particular mutations on resistance, we have replaced the DHFR gene from S. cerevisiae with the DHFR domain of the DHFR-TS gene from a drug sensitive P. falciparum strain. The P. falciparum DHFR gene complements all normal functions of the DHFR gene from S. cerevisiae. Furthermore, the sensitivity of this S. cerevisiae to the malarial antifolates is comparable to the sensitivity seen for the original P. falciparum strain. A selection experiment was performed to test the ability of a plasmid-based expression system to generate DHFR point mutants. When ${\approx}10\sp8$ drug sensitive yeast cells were plated on media containing pyrimethamine and the experimental drug WR99210 (WR), resistant colonies were recovered at frequencies of 1.3 $\times$ 10$\sp{-6}$ and 9.5 $\times$ 10$\sp{-7}$ for pyr and WR respectively. Subsequent analysis indicated that a large amount of the resistant colonies most likely arose from DHFR copy # increases via plasmid missegregation errors. Transferring the construct into a chromosomal location will reduce this background and facilitate the efficient isolation of DHFR point mutants.