Drug resistance evolution is an intricate process, requiring a series of epistatic interactions to maintain balance between selective advantage and fitness loss from drug-resistant mutations. The key questions are why treatment with certain antibiotics does not lead to full resistance, and why some drugs can be quickly overcome by drug-resistant mutations. Malaria drug resistance has become an excellent model to study these question since the parasites have been under a plethora of drug regimens for more than a century. Here we used antifolate and artemisinin resistance as models to show that the difference comes from the gain of robustness during the evolutionary process, which allows mutations to be accumulated without fitness loss.
In antifolate resistance, we demonstrated how robustness evolved under selective pressure from an antimalarial drug inhibiting the folate synthesis pathway. A series of four nonsynonymous amino acid substitutions at the targeted enzyme, dihydrofolate reductase (DHFR), render the parasites highly resistant to the antifolate drug pyrimethamine. The stepwise gain of these four dhfr mutations results in tradeoffs between pyrimethamine resistance and parasite fitness. The epistatic interactions between dhfr mutations and amplification of the gene encoding the first upstream enzyme in the folate pathway, GTP cyclohydrolase I (GCH1) promotes pyrimethamine resistance. gch1 amplification confers low level pyrimethamine resistance and would thus be selected for by pyrimethamine treatment. Interestingly, the gch1 amplification can then be co-opted by the parasites because it reduces the cost of acquiring drug-resistant dhfr mutations downstream in the same metabolic pathway.
On the other hand, the resistance level of artemisinin, the drug of choice for severe malaria treatment, is relatively low with only clinical presentation of delayed parasite clearance by a few hours. Convergent evolution, driven by multiple trajectories to overcome artemisinin-induced stress, was observed, but it was precluded to become full blown resistance by high fitness cost as shown by unstable resistant phenotypes. Artemisinin-resistant parasites suffered from fitness loss and produced fewer progenies. Under amino acid starvation, the resistant parasites failed to undergo maturation unlike their sensitive counterparts as a result of reduced hemoglobin consumption. The reduction leads to loss in artemisinin sensitivity, but the defect causes significant fitness trade-off. Without the gain of a new robustness system, long-term drug selection experiment failed to drive artemisinin resistance to surpass therapeutic doses, but the selection toward antifolate resistance was quickly and repeatedly accomplished under increased robustness by extra GCH1.