Protein-protein and protein-peptidoglycan interactions leading to assembly of the Type II secretion system

蛋白质-蛋白质和蛋白质-肽聚糖相互作用导致 II 型分泌系统的组装

• 批准号: RGPIN-2017-04777
• 负责人: Howard, Stephen
• 金额: $ 1.89万
• 依托单位: University of Saskatchewan
• 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=681634
• 关键词: Protein; protein; peptidoglycan; interactions; leading

The research of our laboratory is concerned with the structure and function of the two membranes that surround the cells of a major class of bacteria known as the Gram-negatives. We study interactions between the inner and outer membrane as well as the transport of materials across the outer membrane. This transport is a fundamental life process because membranes act as a barrier in protecting all living cells from the environment, but that barrier must be overcome so that nutrients and proteins required for life can cross them.*** The major focus of our research is the structure and function of the type two secretion system (T2SS) that allows Gram-negative bacteria to secrete degradative enzymes and toxins. In our previous studies we showed that a complex of two membrane proteins, GspAB, is required for the transport and assembly of the secretin (a complex of 12 GspD proteins), which forms the outer membrane channel through which T2SS proteins are secreted. We have demonstrated that in the bacterium Aeromonas hydrophila, GspA binds to peptidoglycan, a major structural component of the cell envelope, and that GspB binds to GspD. In addition we found that in the bacterium Vibrio cholerae a second mechanism involving the protein GspS also functions in the assembly of the secretin.*** The objectives of our current experiments are to determine how the interactions of the assembly factors GspAB and GspS with both GspD and peptidoglycan result in the formation of the secretin in association with both peptidoglycan and the outer membrane to form the export channel. We hypothesize that ExeAB itself forms a multimeric scaffold in the peptidoglycan and recruits GspD to also bind there. We are using biochemical and genetic approaches to study the molecular structures of the complexes that these proteins form. We are also studying the relationship between the GspAB and GspS secretin assembly pathways in Vibrio cholerae, the only known bacterium in which both of these pathways operate in the assembly of the same secretin. Again biochemical and genetic as well as physiological approaches are being used to determine the interactions between these proteins and where in the bacterial cell the two assembly pathways operate. These experiments form part of our long-term objective of understanding the mechanisms by which bacteria transport proteins out of the cell.*** This research will provide new information concerning how Gram negative bacteria secrete enzymes and protein toxins, and lead to new strategies for harnessing bacterial protein synthesis and for interfering with toxin secretion.*It will also contribute to the broader field of biological sciences through the elucidation of fundamental mechanisms of protein trafficking that take place in all living cells.**

我们实验室的研究关注的是围绕一个主要类别的细菌称为革兰氏阴性菌的细胞的两个膜的结构和功能。 我们研究了内外膜之间的相互作用以及物质穿过外膜的运输。 这种运输是一个基本的生命过程，因为膜作为一个屏障，保护所有活细胞免受环境的影响，但必须克服这一屏障，使生命所需的营养物质和蛋白质可以穿过它们。 我们研究的主要焦点是第二型分泌系统（T2SS）的结构和功能，该系统允许革兰氏阴性菌分泌降解酶和毒素。 在我们以前的研究中，我们表明，两个膜蛋白，GspAB的复合物，是所需的分泌素（12 GspD蛋白的复合物）的运输和组装，形成外膜通道，通过它T2SS蛋白的分泌。我们已经证明，在细菌气单胞菌，GspA结合肽聚糖，细胞包膜的主要结构组成部分，和GspB结合GspD。 此外，我们还发现，在霍乱弧菌中，涉及GspS蛋白的第二种机制也在分泌素的组装中发挥作用。 我们当前实验的目的是确定组装因子GspAB和GspS与GspD和肽聚糖两者的相互作用如何导致与肽聚糖和外膜两者相关联的分泌素的形成以形成输出通道。 我们假设ExeAB本身在肽聚糖中形成多聚体支架，并招募GspD也在那里结合。 我们正在使用生物化学和遗传学方法来研究这些蛋白质形成的复合物的分子结构。 我们也在研究霍乱弧菌中GspAB和GspS分泌素组装途径之间的关系，霍乱弧菌是唯一一种已知的细菌，其中这两种途径都在同一分泌素的组装中起作用。 生物化学和遗传学以及生理学方法再次被用于确定这些蛋白质之间的相互作用以及细菌细胞中两种组装途径的运作位置。 这些实验是我们长期目标的一部分，即了解细菌将蛋白质运输出细胞的机制。 这项研究将提供有关革兰氏阴性菌如何分泌酶和蛋白质毒素的新信息，并导致利用细菌蛋白质合成和干扰毒素分泌的新策略。它还将通过阐明所有活细胞中发生的蛋白质运输的基本机制，为更广泛的生物科学领域做出贡献。