Deciphering the roles of differential type IV pilin expression and N-glycosylation in biofilm formation

项目名称：Pohlschroder， MechthildPI机构：宾夕法尼亚大学，费城，美国提案标题：微生物生物膜：破译IV型pilin表达和n -糖基化的差异微生物包括细菌，古生菌，藻类和真菌可以形成嵌入保护性聚合物基质的群落，称为生物膜。这些生物膜是有益的，例如在制作醋的时候。但生物膜也会造成巨大的危害，因为它们非常坚硬，极难根除，例如造成严重的腐蚀和污水管道以及用于运输石油和天然气的管道堵塞，以及作物受损和食物变质；因此，它们对经济产生了重大的负面影响。该项目将解决调节和促进生物膜的建立和传播的生物过程，从而将大大提高对生物膜形成的理解。特别是，我们的目标是了解细胞表面结构，称为柱状结构，和蛋白质的糖修饰在生物膜形成中发挥的作用。我们将使用尖端的质谱（MS）技术在分子水平上表征古细菌模式生物的生物膜形成，火山盐藻。我们的研究不仅将在这个令人兴奋和及时的研究领域推进科学知识，而且还将为博士后、研究生、本科生和高中生提供宝贵的培训机会，用最新的、最先进的技术研究微生物系统。此外，波尔施罗德实验室将参与科学教育项目，希望能激发年轻学生对科学的热情，尤其是那些就读于西费城服务不足学校的学生。PI将继续开发微生物实验，供资源有限的学校使用，该实验室将参加由宾夕法尼亚大学主办的课堂教学和教师培训讲习班。最近，敏感的定量蛋白质组学分析已经导致鉴定了细菌和古细菌生物膜形成所需的关键组成部分。然而，迄今为止，很少有人尝试将浮游细胞与生物膜发育不同早期阶段的细胞进行比较，以表征浮游细胞中不同的蛋白质表达模式。此外，由于技术手段有限，以前的质谱（MS）研究在这些转变过程中没有进行差异糖基化，使得这种翻译后修饰在生物膜形成中的作用知之甚少。然而，最近在质谱方面的技术进步现在允许在浮游细胞和生物膜中产生最完整的原核糖蛋白组，阐明了生物膜形成的各个阶段所涉及的机制。利用遗传和生化方法与定量糖蛋白组学相结合，该实验室将专注于确定IV型菌毛的差异表达和翻译后修饰如何影响生物膜的形成。IV型菌毛是进化上保守的细胞表面结构的亚基。这些方法也可能导致鉴定和表征在生物膜发育中起作用的其他蛋白质。由于古细菌和细菌有许多共同的特征，这些研究不仅将促进对古细菌生物膜形成机制的更好理解，而且还将为研究细菌生物膜提供见解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

PI Name: Pohlschroder, MechthildPI Institution: University of Pennsylvania, Philadelphia, PAProposal title: Microbial biofilms: Deciphering the roles of differential type IV pilin expression and N-glycosylationMicrobes including bacteria, archaea, algae, and fungi can form communities that are embedded in protective polymeric matrices, called biofilms. These biofilms can be beneficial, for example when making vinegar. But biofilms can also cause great harm as they can be tough and extremely difficult to eradicate, causing for example substantial corrosion and clogging of water and sewage pipes and of the pipelines used to transport oil and gas, as well as crop damage and food spoilage; thus they have significant negative impacts on the economy. This project will address the biological processes that regulate and facilitate the establishment and spread of biofilms and thus will result in a greatly improved understanding of biofilm formation. In particular, we aim to understand the role cell surface structures, termed pilins, and sugar-modifications of proteins play in biofilm formation. We will use cutting edge mass spectrometric (MS) technology to characterize at the molecular level biofilm formation in an archaea model organism, Haloferax volcanii. Our research will not only advance the scientific knowledge in this exciting and timely field of research, but will also provide post-doctoral associates, graduate, undergraduate, and high school students with invaluable training opportunities to study microbiological systems with novel, state-of-the-art technologies. Furthermore, the Pohlschroder lab will take part in science education programs in hopes of stimulating an enthusiasm for science among young students, particularly those who attend the underserved schools of West Philadelphia. The PI will continue to develop microbiology experiments for use by schools having limited resources, and the lab will participate in in-class instruction as well as teacher-training workshops hosted by the University of Pennsylvania.Recently, sensitive quantitative proteomics analyses have led to the identification of crucial components underpinning processes that are required for biofilm formation in bacteria and archaea. Thus far, however, few attempts to characterize differential protein expression patterns in planktonic cells, as compared to cells from various early stages of biofilm development, have been made. Moreover, given limited technological means, previous mass spectrometric (MS) studies of differential glycosylation during these transitions had not been performed, leaving the role played by this post-translational modification in biofilm formation poorly understood. However, recent technological advances in MS now allow generation of the most complete prokaryotic glycoproteome available, in both planktonic cells and biofilms, clarifying the mechanisms involved at various stages of biofilm formation. Using genetic and biochemical approaches in concert with quantitative glycoproteomics, the lab will focus on determining how differential expression and post-translational modifications of type IV pilins, which are subunits of the evolutionarily conserved cell surface structures, type IV pili, affect biofilm formation. These methods will likely also lead to the identification and characterization of other proteins that play roles in biofilm development. Because archaea and bacteria share many features in common, these studies will not only promote a better understanding of the mechanisms underlying archaeal biofilm formation but will also provide insights for those studying bacterial biofilms.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.