Intermembrane Transfer and Assembly of the Type II Secretion System of Gram-Negative Bacteria

我们实验室的研究项目是关于革兰氏阴性菌双膜被膜的结构和功能。我们研究内膜和外膜之间的相互作用以及物质通过外膜的运输。这种运输是一个基本的生命过程，因为膜在保护细胞方面起到了屏障的作用，但这个屏障必须被克服，这样生命所需的营养和蛋白质才能越过它们。 我们目前的研究重点是第二型分泌系统(T2SS)的结构和功能，它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，我们发现分泌素的运输和组装需要一个由两个膜蛋白组成的复合体ExeAB，分泌素是分泌T2SS蛋白的外膜通道。我们已经证明了ExEA与细胞被膜的主要结构成分肽聚糖结合，并确定了肽聚糖结合部位的关键氨基酸。 在我们目前的实验中，一个主要的焦点是ExeAB和肽之间的相互作用如何允许分泌素组装到外膜的通道中。我们假设ExeAB本身形成了一个多聚体支架，促胰液素与其结合以进行组装。我们正在使用生化和遗传学的方法来研究在大肠杆菌和霍乱弧菌的分泌素组装过程中涉及的多种因素。在大肠杆菌中，我们假设这种细菌中两个T2SS系统的分泌素是独立组装的，而在霍乱弧菌中，我们有证据表明，至少有两种不同的机制参与同一分泌素的组装，并确定了第二种机制的候选者，但尚未确定。 这些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。

The research programme of our laboratory is concerned with the structure and function of the double membrane envelope of Gram-negative bacteria. 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 since membranes act as a barrier in protecting the cell, but that barrier must be overcome so that nutrients and proteins required for life can cross them. The major focus of our current 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, ExeAB, are required for the transport and assembly of the secretin, the outer membrane channel through which T2SS proteins are secreted. We have demonstrated that ExeA binds to peptidoglycan, a major structural component of the cell envelope, and identified critical amino acids of the peptidoglycan binding site. In our current experiments, a major focus is how interactions between ExeAB and peptidoglycan allow the secretin to assemble into a channel in the outer membrane. We hypothesize that ExeAB itself forms a multimeric scaffold to which the secretin binds in order to assemble. We are using biochemical and genetic approaches to study the multiple factors involved during secretin assembly in E. coli and V. cholerae. In E. coli we hypothesize that the secretins of the two T2SS systems in this bacterium are assembled independently, whereas in V. cholerae we have evidence that there are at least two different mechanisms involved in assembly of the same secretin, and have identified a candidate for the second, as yet unidentified mechanism. These studies 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.