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

细菌对抗生素耐药性的日益增加对人类健康的威胁是真实而重大的。令人震惊的是，已经确定了对目前临床使用的所有主要抗生素具有耐药性的微生物菌株，并且开始出现泛耐药病原体。目前的预测表明，大规模的抗菌素无效迫在眉睫，我们可能很快就会再次面临“前抗生素”时代挑战医学的问题。尽管细菌耐药性迅速增加，但在过去的50年里，只有两种新型抗生素进入了临床，因此，开发具有新作用模式和新药效团的新型抗生素药物来对抗传染病显然是迫切需要的。天然产物化学通常提供前所未有的铅结构，具有理想的生物活性和效力，这是通过合理和组合合成策略无法获得的。以天然产物为定义的化学空间与以药物靶点和疾病调节基因为代表的生物空间之间也存在高度重叠和互补性。天然产物化学已经发现了大量具有生物活性的有机化合物，它们具有成为或激发新疗法的真正潜力，并将在未来继续这样做。我的研究项目侧重于分离具有生物活性的天然产物，这些天然产物对病原微生物的耐药菌株具有活性，因此具有开发感染性疾病新疗法的先导化合物的巨大潜力。我们专注于陆地、海洋和淡水植物内生真菌产生的天然产物，目前正在研究250个真菌内生菌培养文库。其他内生菌株将被分离、培养和提取以增加我们的收集，我们也将在与其他真菌和细菌共培养的条件下发酵真菌，以触发隐藏的生物合成基因，并诱导抗生素天然产物的生产。真菌提取物将在一系列生物测定中进行筛选，以评估其对病原微生物的抑制作用及其调节这些病原体内外排泵的能力。这将使我们能够编制抗生素成分的生物活性概况，这些成分可以与其作用方式相关，以努力发现新的抗生素类别，并确定可用于通过纠正耐药性影响来提高我们现有抗生素有效性的天然产物。我们还将制定一项测定方案，使我们能够对通过共同外排泵从致病细胞中转运出来的提取物的活性成分的易感性进行分析，并分离出对耐药病原体菌株更有效的天然产物。结合生物活性谱的编制，基于NMR和LC-MS的代谢组学方法将用于重复含有已知抗生素的提取物，并优先考虑进一步研究的项目。具有生物活性特征和独特代谢组学特征的提取物将进行生物测定指导分离，以分离和鉴定活性成分。分离出来的新型天然产物的作用模式将通过基因组学、蛋白质组学和化学遗传学进行探测。我的研究最终将导致从真菌内生菌提供的广泛生物多样性中发现先导化合物，用于开发对抗传染病的新疗法。

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.