Deconstructing Artemisinin Resistance in Plasmodium falciparum Using Genetic Crosses and Systems Biology Approaches

In this thesis, I aim to understand the genetic and transcriptomic causes of artemisinin resistance and predict what drugs may prove effective in combination with artemisinin. Artemisinin resistance raises alarming concerns for the eradication efforts of malaria: if the resistance were to spread from Southeast Asia to Africa, as has previous anti-malarial resistances, the consequences are predicted to be dire. Further exacerbating the situation is the reports of resistance to artemisinin combination therapies, the front-line treatment as recommended by the World Health Organization, combining artemisinin with a long-lasting partner drug. To combat the spread of artemisinin and artemisinin combination therapy resistances, an understanding of artemisinin’s mechanism of action, the resistance mechanisms against artemisinin, and more rationally designed combination therapies are required.

In the first section of my thesis, I take a genetic approach understand artemisinin resistance. Using a recently generated genetic cross whose parents have diverging artemisinin resistance phenotypes, I aim to determine the genetic loci controlling artemisinin resistance. What makes this approach unique is that the resistant parent harbors no K13 mutations, the genetic marker of resistance in the field. Although I find no suggestive or significant peak associated with artemisinin response, I do find two weaker peaks on chromosome 11 and 12, one which corresponds to pfmrp2, a gene involved in multitude of drug responses. I also attempt to develop a high throughput alternative to measure artemisinin response.

In the second section of my thesis, I take a transcriptomic approach to understand artemisinin resistance. I collect transcription samples from 55 Southeast Asian isolates across two timepoints, two perturbations, and in replicate to understand how the biology of artemisinin resistant parasites has changed. Using gene co-expression networks and applying systems biology approaches, I find that gene co-expression networks illuminate known biology about artemisinin, including putative functions of K13. Additionally, I discover two genes, a putative DNA helicase MCM9 and an osmiophilic body protein G377 are two strong candidates whose functions are important for artemisinin response and resistance, with support of previously published data. I show how the functions of these two genes may have changed under differing conditions, highlighting DNA damage as a potentially important pathway in understanding artemisinin and its effect on malaria.

In the third and final chapter of my dissertation I perturb three Southeast Asian isolates with thirty drugs/compounds of diverse targets and mechanisms, in addition to dihydroartemisinin, to determine what drugs may work best in combination with artemisinin. Transcriptional responses to drugs/compounds have previously proven to be a strong predictor of drug synergy in other models, and here I apply the same logic to malaria. I find that two drugs/compounds, namely epoxomicin and cisplatin seem to show high promise for more synergy than the currently used anti-malarials. Epoxomicin and cisplatin also seem to perturb G377 and MCM9 strongly and in the same manner as dihydroartemisinin, further encouraging their targets/mechanisms as potential combination therapy targets for future use.