Investigation of Polyketide Synthase Secondary Metabolic Pathways in the Apicomplexan Parasite Toxoplasma gondii

Abstract

The apicomplexan parasite Toxoplasma gondii is one of the most highly distributed parasites on earth. Despite this, very little is known about the chemical mechanisms utilized by the parasite during the stages of its complex life cycle. Although protists have not traditionally been studied as natural product producing organisms, T. gondii possess multiple putative polyketide synthase (PKS) biosynthetic gene clusters (BGCs), indicating their potential to be a unique source of undiscovered secondary metabolites. In other infectious agents like bacteria and fungi, well-established culture conditions, tractable genomes, and often predictable bioinformatics have made these organisms the standard systems for natural product discovery. Research in this area has demonstrated that such pathogens use their metabolome to shape their environment and enhance survival through the production of bioactive natural products, many of which have been utilized as, or have inspired, therapeutic compounds. PKS biosynthetic gene clusters illustrate how microorganisms can utilize simple building blocks from primary metabolism to assemble complex scaffolds with evolutionary advantageous activities. Despite the prolific nature of apicomplexan parasites and the presence of PKS biosynthetic gene clusters in several members of this phylum, there have been no characterized enzymes or natural product secondary metabolites from apicomplexan sources; leaving a potentially rich area unexplored. To address this gap of knowledge, we aimed to investigate both the biosynthetic proteins and the resultant polyketide metabolites from the model apicomplexan T. gondii. To this end, Chapters 2 and 3 describes our sequencing and bioinformatic analyses of TgPKS2, which resolves the complete sequence and domain architecture of this megaenzyme. Additionally, these chapters describe our work to biochemically characterize individual domains from these proteins, as well as our work towards the reconstitution of full protein modules. Both studies further strive to illuminate structural components of the polyketide metabolite from TgPKS2. To expand beyond TgPKS2, Chapter 4 focuses on the second T. gondii PKS, TgPKS1 and its unique fatty acid loading mechanism, and represents the first metabolic study investigating metabolite production in a heterologously expressed PKS from an apicomplexan parasite. Finally, Chapter 5 highlights our investigations into the biological role of theses gene clusters in T. gondii and apicomplexan parasites, confirming the upregulation of TgPKS2 in the bradyzoite cysts forming stage of T. gondii development. To complement this, we describe our efforts towards generation of a CRISPR mediated TgPKS2 knockout strain of T. gondii and optimization of mass spectrometry conditions for differential metabolic analysis of these T. gondii life stages. In all, this work has made significant progress in our understanding of polyketide biosynthesis in T. gondii and has laid the groundwork for future biochemical and metabolomic studies into natural product biosynthesis in this phylum of pathogenic organisms.