基因组挖掘铁载体类抗耐药菌化合物

Iron–binding siderophores are key virulence factors for many pathogens, including the human opportunistic bacteria pathogen. Indeed, many studies have shown that ferric iron-chelating agents play important roles in host-pathogen interactions. Given the rapid expansion of microbial genome databases, siderophore-specific genome mining is now possible due to conserved siderophore biosynthetic pathways, including both non-ribosomal peptide synthetase (NRPS) pathways and NRPS independent siderophore (NIS) pathways. In addition, various approaches, such as chemical modification and mutasynthesis, provide powerful tools to diversify the structures of natural siderophores, generating analogs with enhanced bioactivities. Here, we will develop a siderophore-specific genome mining pipeline coupled with mutasynthesis to develop novel semi-synthetic siderophores with anti-microbial activity in a bacteria infection model involving the extensively studied nematode C. elegans. Using this pipeline, we will identify “rare” actinomycete species from our bacterial library with high-level producer of novel siderophores. Then use mutasynthesis to modify the natural siderophores , generating a panel of novel analogs that are not normally found in nature. We will screen the activity of these siderophores which target the interaction of bacteria with its host, clearing the bacteria from the C. elegans intestine and efficiently rescuing the worm from bacteria-mediated killing. Furthermore , we will test novel compounds in the C. elegans- pathogenetic bacteria infection model using multi-drug resistant (MDR) bacteria clinical isolates. Taken together, siderophore-specific genome mining and mutasynthesis, coupled with relatively high throughput pathogenesis screening models, provide powerful tools to discover novel siderophores that may be effective in the treatment of bacteria infections.

致病菌的铁离子运输系统对于自身毒力及抵抗宿主免疫至关重要，铁载体介导的宿主铁平衡在病原菌与宿主免疫系统中也起着重要的作用。铁载体的生物合成不仅提供了调节与控制铁载体产生的靶点，也为利用铁载体的铁螯合功能基团构建新的药物提供帮助，该类化合物分子已经逐渐成为化学与生物学交叉研究的一个热点。本研究基于铁载体在病原与宿主互作中的重要作用，拟采用铁载体特异性基因组挖掘-化合物合成通路改造组合策略，力图发现具有治疗耐药菌感染线虫效果的新型天然铁载体化合物，初步解析活性化合物的作用机制。研究中发现的新化合物可以作为分子探针进一步深入研究病原和宿主的互作关系，同时基因簇挖掘中发现的生物合成途径可以为进一步开发和利用极端环境下的微生物资源提供帮助。本课题对于更好的揭示宿主和微生物之间对铁离子的竞争机制，同时通过提高宿主免疫力阻止细菌感染，以及对未来非抗生素类药物的研发具有指导作用。

1..构建了一种筛选抗P .aeruginosa感染活性的铁螯合剂的组合策略。通过对111株放线菌的铁螯合剂特异性挖掘，获得了300个潜在的基因簇；利用缺铁（M2）和补充铁原子（M2F）的培养基对16株含有目标基因簇的潜力菌株进行发酵并结合检测铁螯合剂的CAS显色方法，发现有13株菌可在缺铁培养基M2中产生铁螯合剂，而相应的M2F产物中则不含此类化合物；将此13株菌的M2产物和M2F产物在C. elegans-P .aeruginosa感染模型下检测活性，发现其中9株菌的M2产物表现出明显增强的拯救C. elegans活性。我们另外的工作，从其中一株阳性菌A. methanolica 239T中成功获得了具有较弱活性的铁螯合剂amychelin，并进一步通过对其合成通路的改造获得了一系列新颖类似物，其中2个新产物表现出强的抗P .aeruginosa感染活性。本研究为抗P. aeruginosa药物发现提供了一种新的思路，大大拓宽了新型抗P. aeruginosa活性化合物的来源，也可能在未来增加发现新药的概率。通过21种不同成分培养基OSMAC发酵，发现菌株XJ31能够产生丰富的次级代谢产物。进一步结合抗BCG活性测试，选择了代谢产物丰富、有抗BCG活性的Noc05培养基进行放大发酵和天然产物分离。通过系统的化学分离工作，共得到14个化合物，采用HRESIMS、MALDI-TOF MS/MS、一维和二维NMR等技术，鉴定了以上化合物的结构，包含12个寡肽类化合物（铁螯合剂，siderophore），其中，10个是新化合物。