Natural product discovery and biosynthetic pathway elucidation from the human pathogen Legionella

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Project Summary: Natural products play essential biological roles in producing organisms and have been a historically important source of medicines. Microbial natural products research has focused largely on environmental organisms; natural products, especially the potential small molecule virulent factor produced by pathogenic microbes, are much less understood. Legionella infection causes Legionellosis, which can be present in its non-pneumonic form as Pontiac fever or acute pneumonic form as Legionnaires’ disease. The fatality rate of legionnaires disease is about 10% due to complications and about 25% for those infected in the healthcare facility. While how the protein effectors of Legionella affect the host have been intensively studied, identifying the secondary metabolites Legionella produce and the roles these natural products play in Legionella infection are understudied. The proposed work will identify and characterize the potential bioactive nature products from the human pathogen Legionella. Using a gene-targeting approach to identify a potential novel metalloenzyme that catalyzes unusual oxidative rearrangement to generate the N-nitroso product, one homolog conserved in over 170 Legionella pneumophila subspecies and another homolog conserved in Legionella drozanskii was discovered. Activity assay shows this homolog catalyzes similar reactions but utilizes different substrates. Bioinformatic analysis of the gene neighborhood identifies the biosynthetic gene cluster (BGC) encodes resistance enzyme and a prodrug activating enzyme, possibly indicating the biosynthesis of potential bioactive metabolites. Aim 1 will identify the natural product and bioactivity from the biosynthetic gene cluster from Legionella to uncover the potential virulent factor. Aim 2 will initiate the characterization and mechanistic study of two enzymes in the BGC involving the biosynthesis of the potential pharmacophore to facilitate the understanding and pave the way for the inhibitor design of this new class of pharmacophore-producing enzyme pair. Successful completion of these aims will identify the chemical structure of the potential bioactive natural products from the human pathogen Legionella. In addition, two key enzymes involving the potential pharmacophore biosynthesis will also be characterized. Identification of those natural products and their biosynthesis will possibly reveal new virulence pathways that can be targeted to combat Legionella infection. Ultimately, the overall workflow will be generalized to investigate other novel natural products or potential virulent factors from the human pathogen Legionella to fight the emerging infectious disease by expanding the pool of 1) new classes of cytotoxic drug candidates/virulent factors and 2) new inhibition targets from the biosynthetic pathway.