Leishmania are protozoan parasites that cause a spectrum of diseases in humans and are a significant human health burden. Current treatment paradigms are based exclusively on a handful of drugs that can be toxic, costly, and difficult to administer, and whose usefulness is undermined by the emergence of drug resistance. Since successful parasitism is contingent upon an ability to subsist even when environmental conditions, including nutrient availability, are unfavorable, nutrient-stress response pathways in Leishmania offer an attractive target for chemotherapeutic exploration. However, little is known about the signaling pathways or molecular mechanisms that facilitate adaptation to nutrient-stress in these parasites. The long-term goal of this research program is to elucidate nutrient stress response pathways in Leishmania. To probe nutrient stress response we have used purine starvation as a paradigm. Purine acquisition in Leishmania is an essential process and starvation for purines is readily induced in culture and provokes a response that is both robust and tractable. Our previous studies indicate that purine scarcity leads to profound morphological and metabolic changes that stem from an overall restructuring of the parasite proteome and metabolome. To comprehend the molecular mechanisms underlying proteome transformation in response to purine starvation, we have profiled changes in the global transcriptome of purine-starved Leishmania by the method of RNA-seq. These studies indicated that a significant discordance exists between changes observed at the mRNA level and those manifested within the proteome of purine-starved cells, implying that translational and post-translational mechanisms are predominant in proteome remodeling during purine stress. The goal of this developmental and exploratory application is to establish the relative contributions of translation and post-translational mechanisms on proteome dynamics during purine starvation. The approach that we will use involves adapting two innovative metabolic labeling and proteomic technologies for use in Leishmania, and will provide a direct measure of global protein synthesis in purine-starved and purine-replete Leishmania (Aim 1), as well as affording an in-depth study of protein half-life on a proteome-wide scale (Aim 2). This is the first documented use of these techniques in Leishmania or other related pathogens, and the studies address an exigency within the field, to provide both a direct and quantitative assessment of translation and post-translational stability, two key areas of gene regulation that have defied study on a global scale in these parasites. The successful implementation of these approaches also lays the foundation for future studies in these and other parasites that promise a much broader interrogation of translational regulation and protein stability than has traditionally been afforded. Finally, by th integration of these systems-level results with our previous proteome and RNA-seq data, we will offer one of the most inclusive views of the molecular mechanisms governing adaptation to micro-environmental stress in Leishmania and related pathogens.
Successful parasitism by Leishmania, the cause of a deadly spectrum of diseases in humans, is dependent on an ability to prosper even when environmental conditions, including nutrient availability are unfavorable. The long-term goal of this research is to elucidate how Leishmania respond to a particular type of nutrient stress, purine starvation, and consequently, identify key stress response pathways and proteins for future therapeutic exploration. In this proposal, we will adapt two novel metabolic labeling and proteomic technologies for use in Leishmania that will be used to measure changes at the level of protein synthesis and protein turnover, which are a preface to the metabolic adjustments needed to survive purine stress.