Chemical and biochemical tools to study the functions of exopolysaccharides in bacterial biofilms

项目摘要/摘要： 据估计，美国每年有超过170万的医院感染是由微生物引起的 生物膜。因此，专门针对和破坏细菌生物膜的新方法将产生重大影响 关于人类的医疗保健。胞外多糖如聚N-乙酰氨基葡萄糖(PNAG)是关键的生物膜 促进细胞间相互作用并作为抵抗宿主免疫的保护性屏障的组件 用于治疗细菌感染的系统和常用抗生素疗法。然而，人们对此知之甚少 关于PNAG与其他生物膜EPS组分的分子相互作用。阻止两国之间的互动 细菌细胞和PNAG或使用糖苷酶来分解PNAG和破坏生物膜是有吸引力的 治疗生物被膜感染的方法。 总体而言，我们的目标是表征胞外多糖的分子相互作用网络，如PNAG， 确定它们如何促进生物膜的形成和扩散，并开发处理生物膜的新策略 通过阻断这些相互作用网络或通过催化关键生物膜EPS的分解来感染 组件。为了实现这一总体目标，我们确定了未来5年的两个主要研究领域 几年的时间和未来的计划。第一个领域寻求开发快速鉴定细菌中PNAG的工具 生物膜和开发活细胞邻近标记平台用于鉴定和随后表征 PNAG结合蛋白。这些蛋白质-碳水化合物结合作用有可能成为新的目标 抗生物被膜疗法。第二个领域将开发高吞吐量方法，以识别和随后 通过环境元基因组的功能筛选和迭代来工程PNAG糖苷酶 位点饱和诱变即将到来。为了能够进行这些研究，我们开发了新的比色法和 能够高通量鉴定PNAG糖苷酶活性的荧光PNAG类似物。这 研究将在我的实验室建立一个平台，快速识别和开发用于生物膜的新型生物催化剂 散布活动。总体而言，这些研究工作将为PNAG在生物膜中的作用提供新的独特见解 形成，并将提供工具，将影响我们处理生物被膜感染的方式。

PROJECT SUMMARY/ABSTRACT: It is estimated that over 1.7 million hospital acquired infections per year in the U.S. result from microbial biofilms. New approaches to specifically target and disrupt bacterial biofilms will thus have a significant impact on human healthcare. Exopolysaccharides like poly-N-acetylglucosamine (PNAG) are critical biofilm components that facilitate cell-cell interactions and serve as a protective barrier against the host’s immune system and common antibiotic therapeutics used to treat bacterial infections. However, there is little is known about the molecular interactions of PNAG with other biofilm EPS components. Blocking the interactions between bacterial cells and PNAG or using glycosidase enzymes that break down PNAG and disrupt biofilms are attractive approaches to treat biofilm infections. Overall, our goal is to characterize molecular interaction networks of exopolysaccharides like PNAG, determine how they contribute to biofilm formation and dispersal, and develop novel strategies to treat biofilm infections by blocking these interaction networks or by catalyzing the breakdown of critical biofilm EPS components. To accomplish this overarching goal, we have identified two primary research areas for the next 5 years and plans for beyond. The first area seeks to develop tools for rapid identification of PNAG in bacterial biofilms and develop a live cell proximity labeling platform for identification and subsequent characterization of PNAG-binding proteins. These protein-carbohydrate binding interaction have the potential to be targets for new anti-biofilm therapeutics. A second area will develop high-throughput approaches to identify and subsequently engineer PNAG glycosidase enzymes through functional screening of environmental metagenomes and iterative site saturation mutagenesis approaches. To enable these studies, we have developed new colorimetric and fluorometric PNAG analogs that enable the high throughput identification of PNAG glycosidase activity. This research will establish a platform in my laboratory to rapidly identify and develop novel biocatalysts for biofilm dispersal activity. Overall, these research efforts will provide new unique insight into the role of PNAG in biofilm formation and will provide tools that will impact the way that we approach treating biofilm infections.