UC San Diego

The emergence of drug resistance in an ever-present threat to the successful treatment of infectious diseases. Microorganisms have an almost unparalleled abilityto divide rapidly to achieve large population numbers, introducing a potentially large number of random mutations into the population over time which can confer a distinct advantage (or disadvantage) depending on the environment and selection conditions. In vitro evolution combined with whole genome analysis is a powerful forward genetics tool used to study the development of antimicrobial resistance within a tightly controlled experimental environment (i.e. a tissue culture flask). This dissertation explores the use of this method to begin to collectively understand the genomic drivers of drug resistance across multiple eukaryotic microbes: the human malaria parasite Plasmodium falciparum, the toxoplasmosis-causing parasite Toxoplasma gondii, and the fungus and “model organism” Saccharomyces cerevisiae. We find that multidrug resistance mechanisms are major drivers of resistance for both P. falciparum and S. cerevisiae.

In Chapter 2, in vitro evolution of resistance to the antimalarial clinical candidate DSM265 is shown to mirror the results of in vivo resistance development both in a murine model and in Phase 2a clinical trial data, thus supporting its value as a method toward understanding the mechanics of P. falciparum resistance development. Then in Chapter 3, the method is expanded to 113 different compound selections either performed within the Malaria Drug Accelerator Consortium or by other groups which have made their data publicly available. Here, we show that half of all compounds taken into selection yield parasites with mutations in multidrug resistance mechanisms.

In Chapter 4, we explore how selections performed in T. gondii with the antimalarial drug Artemisinin differ from those performed in the malaria parasite. Both organisms are Apicomplexan parasites but parasitize their hosts in distinctly differentways. While the selections do not yield any homologous resistance genes, both yield mutations that are believed to be involved in each respective parasite’s stress response. Moreover, we find that a key shared feature is the multigenic nature of resistance to Artemisinin, which is likely tied to it mode of action.

Finally, in Chapter 5, experimental evolution is applied at scale in S. cerevisiae to model resistance development in fungi. Selections with 80 different compounds yielded 355 compound-resistant clones and once again we identify a multidrug resistance mechanism that is strongly overrepresented across the dataset. The two Zn2C6 transcription factors YRR1 and YRM1, which are known to induce the pleiotropic drug response, were mutated 100 different times and conferred resistance to 19 structurally distinct compounds.