The genetics of drug resistance in malaria parasites

This thesis describes work performed on the genetics of resistance to the drugs sulphadiazine and pyrimethamine in the rodent 'malaria parasite Plasmodium chabaudi.A number of lines resistant to pyrimethamine were obtained by direct exposure to the drug in a single step. Characteristically these were some 20 times resistant to pyrimethamine and had an increased dependence on FABA. Subsequent genetic analyses showed that this class of mutant was genetically homogeneous and that the primary resistance to pyrimethamine and the concomitant FABA dependence were inseparable by recombination — both consistent with a single gene model of resistance.a number of lines resistant to sulphadiazine were obtained in a single step either by direct exposure to the drug or following FABA restriction. Characteristically these lines were some 4 times resistant to sulphddiazine and required a reduced FABA growth supplement but exhibited a variable spectrum of crossresistance to pyrimethamine uncorrelated with their mode of selection. A phenotypically indistinguishable example of this type of line was also obtained following exposure to pyrimethamine. Evidence from subsequent selection experiments was taken to show that at least two types of separate heritable change might result in the acquisition of sulphadiazine 1' G kj 1 £> ticlaiC G 0 ai-Xl GUxcLmple of one of these was later shown to segregate in a simple mendelian fashion when crossed with a wild line.A number of lines were obtained by selection in two discrete steps: either following the FABA restriction of a pyrimethamine resistant line; or by the direct exposure of a sulphadiazine resistant line to pyrimethamine. While these lines uere some 40 times resistant to pyrimethamine they exhibited wild type responses to. sulphuuiazine and PABA.One such line was crossed with a wild line and two types of non-parental clone were detected among the-products of the cross. These resembled either the pyrimethamine resistant or sulphadiazine resistant lines obtained by selection in a single step. This result was consistent with a genetic recombination event having taken place between, the genetic elements proposed to confer resistance to either of these two drugs. Evidence from a reconstruction experiment suggested that the linkage between these elements was either loose or non¬ existent. \An unsuccessful attempt was mq.de to analyse this cross in the greater precision afforded by the characterisation of clones derived from single oocysts obtained by micro¬ dissection. ) In another cross between two single step lines resistant to pyrimethamine and to sulphadiazine respectively both wild type and double mutant parasites were detected among the cross—products - thus confirming that recombination could link these mutants to produce multiple drug resistance.during the development of a rat-adapted line of P.chabaudi (which was later shown to be unsuitable for use as a selective marker) it was discovered that garnetocytogenesis was enhanced in unadapted parasites during subsistence in rats. This led to the substitution of rats for mice as rodent carriers of the foi-parental mixed-blood infection prior to cyclical passage in crosses.Estimates of the rates of acquisition are presented for each type of resistance in the Discussion together with a critic appreciation of the extent to which these represent true mutation rates