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

PI姓名：微生物生物膜：解读IV型菌毛蛋白表达和N-糖基化的差异性作用微生物包括细菌、古细菌、藻类和真菌可以形成包埋在保护性聚合物基质中的群落，称为生物膜。这些生物膜可能是有益的，例如在制作醋时。 但生物膜也可能造成巨大的危害，因为它们可能很难根除，导致例如水管和污水管道以及用于运输石油和天然气的管道的严重腐蚀和堵塞，以及农作物损害和食品腐败;因此它们对经济产生重大负面影响。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，称为菌毛蛋白，和糖修饰的蛋白质在生物膜形成中发挥的作用。 我们将使用最先进的质谱（MS）技术，在分子水平上表征古生菌模式生物Haloferax volcanii生物膜的形成。 我们的研究不仅将推进科学知识在这个令人兴奋的和及时的研究领域，但也将提供博士后助理，研究生，本科生和高中学生与宝贵的培训机会，研究微生物系统与新的，国家的最先进的技术。此外，Pohlschroder实验室将参加科学教育项目，希望激发年轻学生对科学的热情，特别是那些就读于费城西部服务不足的学校的学生。PI将继续开发供资源有限的学校使用的微生物学实验，实验室将参与课堂教学以及宾夕法尼亚大学主办的教师培训研讨会。最近，敏感的定量蛋白质组学分析已经导致确定细菌和古细菌生物膜形成所需的关键组成部分。然而，到目前为止，很少有人尝试表征差异蛋白质表达模式的细胞，相比，从生物膜发育的各个早期阶段的细胞，已经取得。此外，由于有限的技术手段，以前的质谱（MS）研究差异糖基化在这些转换没有进行，留下的作用所发挥的翻译后修饰在生物膜形成知之甚少。然而，最近的技术进步在MS现在允许最完整的原核糖蛋白质组的生成可用，在两个反硝化细胞和生物膜，澄清在生物膜形成的各个阶段所涉及的机制。使用遗传和生物化学方法与定量糖蛋白组学相结合，实验室将专注于确定IV型菌毛蛋白的差异表达和翻译后修饰如何影响生物膜形成，IV型菌毛蛋白是进化上保守的细胞表面结构IV型皮利的亚基。这些方法也可能导致在生物膜发育中发挥作用的其他蛋白质的鉴定和表征。由于古菌和细菌有许多共同的特征，这些研究不仅将促进对古菌生物膜形成机制的更好理解，还将为细菌生物膜研究提供见解。该奖项反映了NSF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.