Advances in methods to characterize the initiation of drug resistance and measure rates of mutation in Plasmodium falciparum

Resistance to commonly-used antimalarials consistently arose in some Plasmodium falciparum strains common to Southeast Asia, Papua New Guinea and South America. Studies formally showed that geographically-distinct strains had varying capacities to acquire new antimalarial resistance. Parasites from S.E. Asia acquired resistance to atovaquone and 5-fluoorotate at significantly higher frequencies than other common strains. The S.E. Asian clone was labeled with the Accelerated Resistance to Multiple Drugs (ARMD) phenotype. It was hypothesized that the higher frequencies of resistance displayed by ARMD parasites were due to higher rates of mutation.;Systematic approaches to quantify mutagenesis and characterize the initiation of resistance are lacking in the malaria field. Selection experiments established that mutations in protein farnesyltransferase accumulated at rates two to three orders of magnitude higher than in other eukaryotes. Tools were developed to address whether ARMD parasites were mutating at a high constitutive rate or utilizing inducible diversification strategies. To study the initiation of resistance Luria-Delbruck fluctuation tests and Lederberg replica plating were adapted for malaria parasite culture. Such methods where capable of deciphering the timing of resistance events in ARMD populations. Stress-induced resistance was identified in fluctuation tests with ARMD populations selected with 6-mercaptopurine (6-MP). Replica plating experiments highlighted an epigenetic component to 6-MP resistance inheritance. The propensity to mutate was transient and eventually lost in the absence of selection pressure. Stress-induced mutagenesis may limit collateral DNA damage to only periods of stress. Adaptive amplification may be an additional mechanism that reduces selection pressure and limits damage to the P. falciparum haploid genome while providing additional sites for mutagenesis. The methods presented in this thesis can be used to further explore rates of mutation and characterize the initiation of resistance to extant and novel antimalarials.