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Fitness of P. berghei carrying atovaquone-resistance mutations in the mitochondrial DNA
thesisposted on 15.02.2017, 05:01 authored by Murdiyarso, Lydia Sihanti
The emergence of drug-resistant parasites is a major problem for malaria treatment and control. The resistance phenomenon has been shown to be associated with mutations in the parasite genome, affecting in many cases enzyme of essential metabolic pathways. Evidence regarding the effect of these mutations on the fitness of resistant mutants is important to the understanding of the emergence and spread of antimalarial drugs resistance in populations. This thesis examines the effects of the mutations underlying atovaquone-resistance in the cytochrome b gene of the malaria parasite, on parasite fitness, and the contribution of differences in fitness between mutant and wildtype strains to the stability of the resistance phenomenon. Atovaquone binds competitively with coenzyme Q (CoQ) to Respiratory Complex III, which is essential for pyrimidine biosynthesis in the malaria pc:rasites. Mutations in cytochrome b gene could lead to conformational changes of CoQ binding sites, and thus affect Respiratory Complex III activity, pyrimidine biosynthesis, and subsequently fitness of the mutants. Our laboratory has isolated a range of atovaquone-resistant strains of P. berghei, with well-defined underlying mutations, allowing investigation in a murine malaria model. These include mutations in quinone binding domain 1 (Q01; M133I), binding domain 2 (Q02; Y268C/N/S, L271V/K272R, V284F), and both (M133I/L271V, L144S/V284F, M133I/V284F). Molecular modeling of apocytochrome b with Y268C/N/S (common in P. falciparum) in the present study suggests that these mutations are associated with decreased binding affinity to stigmatellin, a Respiratory Complex III inhibitor with structure similar to atovaquone. A quantitative Real Time PCR procedure was developed for more precise measurement of growth fitness of selected mutants (Y268C/N/S, L271V/K272R, M133I/L271V, and M133I). Growth in Balb/c mice of resistant strains with Y268N (2.05 ± 0.54). Y268C (2.92 ± 0.61), Y268S (5.26 ± 0.42), M133I (3.21 ± 0.30), and M133I/L271V (5.77 ± 1.45) was significantly lower than that of the wildtype (9.46 ± 1.10 ug mtDNA/ml blood/24 hours). The contribution of differences in growth fitness to the stability of the resistance phenomenon was examined for Y268C (most common in P. falciparum), employing mutation-specific qRT-PCR (TaqMan® probe) developed for growth measurement of resistant and wildtype strains in mixed infection in Balb/c mice. The survival advantage of the wildtype strain was clearly observed. The contribution of this survival advantage to the stability of atovaquone-resistant mutations associated with Y268C/N and L271V/K272R was investigated. PCR-RFLP detection methods showed that the mutations are very stable, and no wildtype revertants were detectable after four passages of growth. The results of this study support the argument that differences in fitness between wildtype and resistant mutants are important to the eventual disappearance of resistance in the field upon the discontinuation of antimalarial drugs. The use of atovaquone in particular, alone or in combination with proguanil (Malarone™), leads to rapid development of resistance and consequently to treatment failure, but removal of atovaquone from populations results in rapid disappearance of atovaquone resistance.