Nutrient Limitation Mimics Artemisinin Tolerance in Malaria

Mounting evidence demonstrates that nutritional environment can alter pathogen drug sensitivity. While the rich media used for in vitro culture contains supraphysiological nutrient concentrations, pathogens encounter a relatively restrictive environment in vivo. We assessed the effect of nutrient limitation on the protozoan parasite that causes malaria and demonstrated that short-term growth under physiologically relevant mild nutrient stress (or "metabolic priming") triggers increased tolerance of a potent antimalarial drug. We observed beneficial effects using both short-term survival assays and longer-term proliferation studies, where metabolic priming increases parasite survival to a level previously defined as resistant (>1% survival). We performed these assessments by either decreasing single nutrients that have distinct roles in metabolism or using a media formulation that simulates the human plasma environment. We determined that priming-induced tolerance was restricted to parasites that had newly invaded the host red blood cell, but the effect was not dependent on genetic background. The molecular mechanisms of this intrinsic effect mimic aspects of genetic tolerance, including translational repression and protein export. This finding suggests that regardless of the impact on survival rates, environmental stress could stimulate changes that ultimately directly contribute to drug tolerance. Because metabolic stress is likely to occur more frequently in vivo compared to the stable in vitro environment, priming-induced drug tolerance has ramifications for how in vitro results translate to in vivo studies. Improving our understanding of how pathogens adjust their metabolism to impact survival of current and future drugs is an important avenue of research to slow the evolution of resistance. IMPORTANCE There is a dire need for effective treatments against microbial pathogens. Yet, the continuing emergence of drug resistance necessitates a deeper knowledge of how pathogens respond to treatments. We have long appreciated the contribution of genetic evolution to drug resistance, but transient metabolic changes that arise in response to environmental factors are less recognized. Here, we demonstrate that short-term growth of malaria parasites in a nutrient-limiting environment triggers cellular changes that lead to better survival of drug treatment. We found that these strategies are similar to those employed by drug-tolerant parasites, which suggests that starvation "primes" parasites to survive and potentially evolve resistance. Since the environment of the human host is relatively nutrient restrictive compared to growth conditions in standard laboratory culture, this discovery highlights the important connections among nutrient levels, protective cellular pathways, and resistance evolution.