Adhesion proteins: structure-function relationships and role in bacterial colonization.

该研究计划将探讨以下假设：粘附蛋白（粘附素）是细菌与其定植的基质之间的第一个接触点，并且它们可能在其上形成生物膜;粘附素的配体结合结构域决定细菌所居住的壁龛;粘附素在粘附到表面和彼此或所得生物膜的凝聚力中发挥作用。在确定了配体结合结构域的3-D结构并确定了它们的配体之后，我们将探索中断或修改这些相互作用的方法，并对粘附素进行工程改造以改变它们的结合位置和伴侣。在这项研究中，我们将重点关注广泛存在于革兰氏阴性细菌中的“毒素重复序列”（RTX）粘附素，包括许多环境上有趣和重要的物种。我们目前正在研究的细菌粘附系统包括荧光假单胞菌，一种有益的植物根部定殖者; Marinomonasprimoryensis，结合冰和硅藻;石油降解剂，Marinomonashydrocarbonoclasticus，当它代谢石油时，在水和石油之间的界面形成短暂的生物膜;和Aeromonaserma，这是一种鱼类病原体。它们的粘附素是巨大的单一蛋白质，通常由细菌中最大的开放阅读框架编码。在过去5年多的时间里，我在NSERC DG上进行的研究表明，位于粘附素末端之间的两个可变区对其功能至关重要。一个是决定粘附素作用范围的延伸区。另一个是靠近粘附素远端的配体结合区，其可以容纳一个或多个负责表面附着的配体结合结构域。我们估计有十几种不同类型的这些结构域在不同的细菌中以各种组合使用，其中只有一些在目前的数据库中具有同源物。我们将继续发现和表征新的粘附素配体结合结构域，解决它们的晶体结构，鉴定它们的配体，并研究它们如何合作来定义宿主细菌抑制的特定生态位。为了使用粘附工程来解决我们的假设，我们有两个系统在发挥作用，我们可以交换配体结合域并改变细菌结合的表面。一个是M. primoryensis在E.杆菌另一个是在大肠杆菌中广泛使用的北方假单胞菌冰核蛋白的18-kDa N-末端结构域。大肠杆菌作为表面展示系统，将酶和抗原放置在细菌表面。21名HQP（6名硕士/博士+15名本科生）将在这个为期5年的计划中接受加拿大经济工作的培训。我们发现的关于如何促进有益细菌定植和阻止有害细菌结合的知识将有利于农业，水产养殖和环境修复，并为微生物学研究提供信息。

This research program will explore the hypotheses that adhesion proteins (adhesins) are the first points of contact between bacteria and substrates they colonize and on which they may form biofilms; that the ligand-binding domains of the adhesins determine the niches inhabited by the bacteria; and that adhesins have a role in both adhesion to a surface and cohesion to each other or to the resulting biofilm. Having determined the 3-D structures of the ligand-binding domains and identified their ligands, we will explore ways to interrupt or modify these interactions, and to engineer changes to adhesins to alter their binding locations and partners. For this study we will focus on the "Repeats-in-Toxin" (RTX) adhesins that are widespread in Gram-negative bacteria, including many environmentally interesting and important species. The bacterial adhesion systems we are currently investigating include Pseudomonas fluorescens, a beneficial colonizer of plant roots; Marinomonas primoryensis that binds to ice and diatoms; the oil degrader, Marinobacter hydrocarbonoclasticus, which forms transient biofilms at the interface between water and oil as it metabolizes the latter; and Aeromonas hydrophila that is a fish pathogen. Their adhesins are gigantic single proteins, often coded by the largest open reading frame in the bacteria. Work funded during the last 5+ years on my NSERC DG revealed that two variable regions lying between the adhesin termini are crucial for their function. One is the extender region that determines the reach of the adhesin. The other is the ligand-binding region near the far end of the adhesin that can house one or more ligand-binding domains responsible for surface attachment. We estimate there are well over a dozen different types of these domains used in various combinations in different bacteria, only some of which have homologues in the present databases. We will continue to discover and characterize new adhesin ligand-binding domains, solve their crystal structures, identify their ligands and research how they cooperate to define the specific niche inhibited by the host bacterium. To address our hypotheses using adhesion engineering, we have two systems in play where we can swap ligand-binding domains and alter the surfaces bound by bacteria. One is a scaled down version of the ice-binding adhesin of M. primoryensis expressed from a plasmid in E. coli. The other is the 18-kDa N-terminal domain of Pseudomonas borealis ice nucleation protein that has been used extensively in E. coli as a surface display system to place enzymes and antigens on the bacterial surface. Twenty-one HQPs (6 MSc/PhD + 15 undergraduates) will be trained in this 5-yr program for work in the Canadian economy. The knowledge we discover on how to promote colonization by beneficial bacteria and block binding by harmful bacteria will benefit agriculture, aquaculture, and environmental remediation, and inform microbiological research.