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

粘附蛋白：结构-功能关系以及在细菌定植和生物膜中的作用。

• 批准号: RGPIN-2016-04810
• 负责人: Davies, Peter
• 金额: $ 4.66万
• 依托单位: Queen's University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=691195
• 关键词: Adhesion; proteins; structure; function; relationships

Bacteria have evolved sophisticated adaptations to colonize specific niches. They can behave as multicellular masses that cooperate with each other to maximize their success. As biofilms they are remarkably resistant to eradication. Here we are studying large outer membrane proteins (adhesins) that function in both adhesion to an extracellular matrix and self-adhesion to form biofilms.****One of several example we are studying is a 1.5-MDa adhesin from a marine bacterium (Marinomonas primoryensis) found under sea ice. At its C-terminal end it has a Type I secretion sequence common to RTX proteins and next to it an ice-binding domain. The other end attaches to the bacterial outer membrane. In between are ~120 identical repeats of an IgG-like domain that project the ice-binding domain away from the bacterial surface. Tandem arrays of the IgG-repeats bind to each other in an antiparallel fashion. Thus the adhesin not only binds the bacteria to ice but also links bacteria together in clusters to strengthen their adhesion to ice. Equally exciting is the role of a sugar-binding domain adjacent to the ice-binding domain that may serve to help bind the adhesin to its neighbours in the growing biofilm.****These RTX adhesins are widely distributed in bacteria. One we will study is from the magnetotactic bacterium, Magnetospirillum magnetotacticum. These bacteria are well known for containing magnetic crystals that help the host to find their optimal niche of low (but essential) oxygen levels. We suggest that the magnetotactic bacteria self-adhere through their adhesins and cooperate in moving to the optimal niche. Another target bacterium is Marinobacter hydrocarbonoclasticus, which is able to form biofilms at the interface between oil and water to degrade the oil.*****Here we will use these bacteria as model organisms to develop strategies for disrupting self-association in the early phases of biofilm formation. We will follow bacterial association in custom designed microfluidics chambers monitored by microscopy. The microfluidics apparatus will allow us to change media, form gradients of attractants and repellents, and observe bacterial associations with substrate like ice, oil droplets, and cellulose.*****An integral part of this proposal will be the structural analysis of each domain and domain combinations used in adhesion. Recombinant proteins will be produced for structural and biophysical analysis. Functional analysis will involve mutagenesis, domain swap and deletion experiments, and transfer of adhesin genes to other hosts.*These are all techniques and procedures that our lab excels in and will be the basis for the training of our three graduate students (T.Vance, C. Stevens and J. Arora) in our bacterial adhesin program. By the time they graduate they will have command of these biochemistry and structural biology methodologies and the ability to apply them to solve complex biological questions. *** *** **

细菌已经进化出复杂的适应能力，以定居特定的利基环境。它们可以表现为相互协作的多细胞团块，以最大限度地提高它们的成功。作为生物膜，它们对根除具有显著的抵抗力。在这里，我们正在研究大的外膜蛋白(粘附素)，它在与细胞外基质的黏附和自我黏附形成生物膜中发挥作用。*我们正在研究的几个例子之一是来自海冰下发现的海洋细菌(Marinomonas Primoryensis)的1.5-丙二醛黏附素。在它的C末端，它有一个RTX蛋白共有的I型分泌序列，旁边是一个冰结合结构域。另一端附着在细菌外膜上。在两者之间是大约120个相同的免疫球蛋白样域的重复，它们将冰结合结构域投影到远离细菌表面的地方。串联排列的免疫球蛋白重复序列以反平行的方式相互结合。因此，粘附素不仅将细菌与冰结合在一起，还将细菌以簇的形式连接在一起，以加强它们与冰的粘附性。同样令人兴奋的是与冰结合结构域相邻的糖结合结构域的作用，这可能有助于在不断增长的生物膜中将粘附素与其邻居结合。*这些RTX粘附素广泛分布于细菌中。我们要研究的一种细菌来自趋磁细菌--磁性螺杆菌。这些细菌以含有磁性晶体而闻名，这些晶体帮助宿主找到低(但必要的)氧气水平的最佳生态位。我们建议趋磁细菌通过它们的粘附素自我黏附，并合作移动到最佳的生态位。另一个目标细菌是海洋细菌，它能够在石油和水的界面上形成生物膜来降解石油。在这里，我们将以这些细菌作为模式生物来开发在生物膜形成的早期阶段破坏自结合的策略。我们将在显微镜监控的定制设计的微流体室中跟踪细菌的相互作用。微流体装置将允许我们更换介质，形成引诱剂和驱避剂的梯度，并观察细菌与冰、油滴和纤维素等底物的联系。*这项建议的一个组成部分将是对粘合中使用的每个结构域和结构域组合的结构分析。重组蛋白将用于结构和生物物理分析。功能分析将包括突变、结构域交换和缺失实验，以及将粘附素基因转移到其他宿主。*这些都是我们实验室擅长的技术和程序，并将成为我们细菌粘附素计划中三名研究生(T·万斯、C·史蒂文斯和J·阿罗拉)培训的基础。到他们毕业时，他们将掌握这些生物化学和结构生物学方法，并有能力应用这些方法来解决复杂的生物学问题。*