Abstract
This dissertation is focused on how metabolism of the malaria-causing parasite, Plasmodium falciparum, changes in response to stressful conditions and how these metabolic adjustments cause increased parasite tolerance to antimalarial drug treatment.
There is intricate interplay between parasites and their environment as pathogens respond to nutrient availability, physical forces, and immune pressure. Mounting evidence indicates non-genetic, metabolic changes in response to these environmental factors can modulate pathogen sensitivity to drug treatment. Chapter 2 reviews, in detail, what is known about changes in growth, survival, and virulence as a consequence of parasite environment. Yet, there are still open questions about the extent of environmental impact on drug uptake, activation, and other effects relevant to parasite drug sensitivity. One technical barrier to answering these research questions is access to high quality in vitro ring stage parasites and non-traditional parasite forms (quiescent rings and ex vivo clinical rings). Ring stage parasites have become a focus of interest in the malaria community because, relative to other more metabolically active stages of the malaria replication cycle, rings demonstrate decreased susceptibility to the World Health Organization frontline antimalarial drug, artemisinin. Further, a small proportion of rings enter a growth arrested quiescent state upon drug administration allowing survival of artemisinin treatment. The exact mechanisms by which rings stage P. falciparum survive drug treatment, such as how parasites enter and exit from quiescence, is poorly understood; however, current evidence heavily suggests survival can be mediated through non-genetic, metabolic changes.
In Chapter 3, I present the “SLOPE” method for enrichment of in vitro, ex vivo, and quiescent ring stage parasites. Prior to the publication of Chapter 3, there was no effective method for the enrichment of these populations leading to considerable host-contributed noise in samples, which limited success of sensitive downstream analyses, particularly of non-genetic, metabolic changes through methods such as proteomics and metabolomics. The development of this effective enrichment method will dramatically improve our ability to study ring stage parasites, as well as ring-derived forms.
While developing the SLOPE enrichment method, which relies on a cholesterol dependent lytic agent, we incidentally noted a reduction of cholesterol on in vitro parasite infected erythrocyte membranes that was not present ex vivo. It was discovered this discrepancy is due to the considerably lower level of cholesterol provided by in vitro media formulations compared to levels in human host plasma. This, paired with other examples of impacts from the non-physiological nutritional environment in vitro detailed in Chapter 2, led us to explore the effect of physiologically relevant nutrient limitation on parasite drug sensitivity.
In Chapter 4, parasites were subjected to metabolic nutrient stress by either 1) decreasing a single metabolite type that has been previously proposed as drug target (i.e., purines or thiamine) or 2) using a media formulation with reductions in many metabolites that simulates the physiological human plasma environment. The adaptive benefits of these mild nutrient stress conditions overshadowed any adverse effects leading increased ring stage parasite survival of artemisinin treatment. While these nutrient stress levels were developed to mimic conditions parasites may encounter in the context of a clinical infection, the extent to which drug survival is truly impacted in vivo merits further investigation. The potential for nutrient stress to be inducing quiescent ring stage parasites as a mechanism to facilitate increased artemisinin survival in this context also remains unknown. We presume that metabolic stress occurs more frequently in vivo compared to the stable, nutrient-rich in vitro environment; therefore, the observation that more a physiological environment increases artemisinin tolerance has ramifications for how in vitro antimalarial drug treatment results translate to in vivo studies.
The availability of effective antimalarial drugs is critical to prevent malaria mortality numbers from rising and work toward malaria eradication. Yet, clinical malaria resistance has now been reported to both the recommended frontline antimalarial treatment and all available partner drugs. My goal to understand how Plasmodium falciparum modulate metabolism to cope with stress will lead to a more accurate understanding of how malaria parasites will respond to drug treatment in a human infection. Understanding this stress responsive ability of parasites to cope with antimalarial pressures will provide foundations for the development of powerful targeted adjuvants to combat the emergence and spread of future malaria drug resistance.
