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

这项研究计划将探索以下假设：黏附蛋白(粘附素)是细菌和它们定居的底物之间的第一个接触点，它们可能在上面形成生物膜；粘附素的配体结合结构域决定细菌栖息的生态位；粘附素在附着到表面和相互粘合或最终形成的生物膜中都有作用。在确定了配体结合域的三维结构并确定了它们的配体后，我们将探索中断或修改这些相互作用的方法，并设计粘附素的变化来改变它们的结合位置和伙伴。在这项研究中，我们将重点研究在革兰氏阴性细菌中广泛存在的“毒素重复序列”(RTX)粘附素，包括许多环境感兴趣和重要的物种。我们目前正在研究的细菌附着系统包括：荧光假单胞菌，一种有益的植物根部定殖者；原始海洋单胞菌，它与冰和硅藻结合；石油降解菌，海洋杆菌，它在代谢油和水的过程中，在水和油的界面形成短暂的生物膜；以及嗜水气单胞菌，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。在过去的5年多里，我在NSERC DG上资助的工作表明，粘附素末端之间的两个可变区对它们的功能至关重要。一个是决定粘附素到达范围的延伸区。另一个是粘附素远端附近的配体结合区，它可以容纳一个或多个负责表面附着的配体结合域。我们估计，在不同的细菌中，有十几种不同类型的结构域用于不同的组合，其中只有一些在目前的数据库中有同源性。我们将继续发现和表征新的粘附素配体结合域，解决它们的晶体结构，确定它们的配体，并研究它们如何合作来定义被宿主细菌抑制的特定生态位。为了利用黏附工程解决我们的假设，我们有两个正在发挥作用的系统，在这两个系统中，我们可以交换配体结合结构域，并改变细菌结合的表面。一种是从大肠杆菌中的一个质粒表达的原始分枝杆菌的冰结合粘附素的缩小版本。另一个是北极假单胞菌冰核蛋白的18 kDa N末端结构域，它在大肠杆菌中被广泛用作表面展示系统，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)将接受这个为期5年的计划的培训，以便在加拿大经济中工作。我们发现的关于如何促进有益细菌的定植和阻止有害细菌的结合的知识将有助于农业、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.