Combining bioactivity and metabolomic profiling in the discovery of antibiotic natural products from endophytic fungi

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The threat to human health as a result of the growing emergence of bacterial resistance to antibiotic agents is both real and significant. It is alarming to realise that microbial strains resistant to all of the major classes of antibiotics currently in clinical use have been identified, and pan-resistant pathogens are beginning to emerge. Current forecasts predict that broad-scale antimicrobial ineffectiveness is imminent and we may soon once again face the problems that challenged medicine in the "pre-antibiotic" era. Despite this rapid increase in bacterial resistance, only two new classes of antibiotics have reached clinics in the last fifty years, and there is an obvious and urgent need to develop new antibiotic agents with novel modes of action and new pharmacophores to combat infectious disease. Natural products chemistry often provides access to unprecedented lead structures with desirable bioactivities and potencies that are unavailable through rational and combinatorial synthetic strategies. There is also a high degree of overlap and complementarity between the chemical space defined by natural products and the biological space represented by drug-able targets and disease-modulating genes. Natural products chemistry has uncovered a vast number of biologically active organic compounds with genuine potential to become or inspire new therapeutics and will continue to do so in the future. My research program focuses on the isolation of biologically active natural products that exhibit activity against drug resistant strains of pathogenic microbes, and consequently have significant potential as lead compounds for the development of new therapeutics for infectious disease. We focus on natural products produced by endophytic fungi of terrestrial, marine and freshwater plants and a library of 250 fungal endophyte cultures is currently under investigation. Additional endophytic strains will be isolated, cultured and extracted to augment our collection, and we will also ferment fungi under co-culture conditions with other fungi and bacteria to trigger cryptic biosynthetic genes and induce the production of antibiotic natural products. Fungal extracts will be screened in a suite of bioassays to evaluate their inhibitory effects against pathogenic microorganisms and their ability to modulate efflux pumps within these pathogens. This will allow us to compile a bioactivity profile for the antibiotic constituents that can be related to their mode-of-action in an effort to discover new antibiotic classes and also identify natural products that can be used to increase the effectiveness of our current antibiotics by remediating for the effects of drug resistance. We will also develop an assay protocol that will allow us to profile the susceptibility of the active constituents of the extracts to be transported out of pathogenic cells through common efflux pumps and isolate natural products that will be more effective against resistant strains of pathogens. In conjunction with the compilation of the bioactivity profiles, NMR and LC-MS based metabolomics approaches will be used to dereplicate extracts that contain known antibiotics and prioritise projects for further investigation. Extracts that exhibit promising bioactivity profiles and unique metabolomics signatures will be subjected to bioassay-guided fractionation to isolate and identify the active components. The modes-of-action of the novel natural products that are isolated will then be probed by genomics, proteomics and chemical genetics. My research will ultimately lead to the discovery of lead compounds from the extensive biodiversity provided by fungal endophytes for the development of new therapeutics to fight infectious disease.