OUTER MEMBRANE SECRETION MECHANISM FOR AUTOTRANSPORTER PROTEINS

描述（由申请人提供）：本研究项目的长期目标是了解革兰氏阴性细菌病原体自转运蛋白（AT）毒力蛋白的外膜（OM）分泌机制。AT蛋白代表了包括Y. pestis，H. pylori、B.百日咳、肠出血性E. coli、志贺菌、A. baumannii，K. pneumoniae和N.脑膜炎总的来说，这些病原体每年杀死超过500万人，并且使用现有的抗生素可能具有挑战性。控制AT分泌机制将为控制革兰氏阴性病原体开辟新的途径。但要获得这种控制，需要进行基础研究，以确定用于将AT毒力蛋白传递到细胞表面的特定机制。特别是，目前还不清楚这些“一次性”分子机器是如何在没有ATP或质子梯度的情况下穿过OM运输的。为了最好地解决这个问题，我们提出的研究结合了生物化学，生物物理学，遗传学和结构学方法，以建立AT OM分泌的能量和动力学原理。本研究有三个具体目标：（1）我们将确定成熟AT毒力蛋白（中央“乘客结构域”）折叠后释放的能量如何影响OM分泌，特别是AT乘客结构域折叠稳定性和动力学之间的相互作用，以及C-与N-末端稳定性。(2)我们将确定AT乘客是否通过其自身的C-末端孔蛋白结构域或通过另一个OM孔蛋白（如BamA）退出细胞。(3)我们将测试的假设，即AT乘客结构域折叠的能量景观具有功能，禁用从N-末端折叠的起始。尽管有广泛的序列和功能多样性，但最近的高分辨率结构已经确定了AT乘客中的共同结构特征，我们自己的实验室也确定了共同的折叠特征。而且，我们的实验室开创了新的生物物理测定，直接询问体内AT OM分泌机制的特征，建立了OM分泌通过乘客结构域的C-至N-末端转运进行的当前范例。最近，我们已经确定OM分泌效率可以通过乘客域内的区域化稳定性来控制。这些初步研究确立了这些目标的概念框架和可行性。在这里，我们将利用我们的测定定量和可逆地阻止OM分泌中间体，以解决以前无法回答的问题，关于AT OM分泌机制。总之，这些研究将确定这种最常见的毒力蛋白分泌机制的关键特征和最容易获得的攻击点。由于AT蛋白在细菌毒力中的关键作用，耐药菌株的迅速增加，以及革兰氏阴性菌感染导致的大量死亡，我们提出的研究将对分子医学和人类健康产生重大影响。

DESCRIPTION (provided by applicant): The long-term goal of this research project is to understand the outer membrane (OM) secretion mechanism for autotransporter (AT) virulence proteins from Gram-negative bacterial pathogens. AT proteins represent the largest class of secreted virulence proteins from pathogens including Y. pestis, H. pylori, B. pertussis, enterohemorrhagic E. coli, Shigella, A. baumannii, K. pneumoniae, and N. meningitidis. Collectively, these pathogens kill >5 million people annually, and can be challenging to defeat using current antibiotics. Controlling the AT secretion mechanism would open up new avenues to control Gram-negative pathogens. But to gain such control, basic research is needed to define the specific mechanism used to deliver AT virulence proteins to the cell surface. In particular, it is unclear how these "one-shot" molecular machines are transported across the OM in the absence of ATP or a proton gradient. To best address this question, our proposed research combines biochemical, biophysical, genetic and structural methods to establish the energetic and kinetic principles underlying AT OM secretion. This research has three specific aims: (1) We will determine how the energy released upon folding of the mature AT virulence protein (the central "passenger domain") affects OM secretion, specifically the interplay between AT passenger domain folding stability and kinetics, and C- versus N-terminal stability. (2) We will determine whether the AT passenger exits the cell through its own C-terminal porin domain, or through another OM porin such as BamA. (3) We will test the hypothesis that the energy landscape for AT passenger domain folding possesses features that disable initiation of folding from the N-terminus. Despite broad sequence and functional diversity, recent high- resolution structures have identified common structural features in AT passengers, and our own lab has identified common folding features. And, our laboratory has pioneered new biophysical assays to directly interrogate features of the AT OM secretion mechanism in vivo, establishing the current paradigm that OM secretion proceeds via C- to N-terminal transport of the passenger domain. Recently, we have determined that OM secretion efficiency can be controlled by regionalized stability within the passenger domain. These preliminary studies have established the conceptual framework and feasibility of these aims. Here, we will exploit our assay to quantitatively and reversibly arrest OM secretion intermediates to address previously unanswerable questions regarding the AT OM secretion mechanism. Together, these studies will identify the crucial features and most-easily-accessed attack points for this most common mechanism for virulence protein secretion. Due to the crucial role of AT proteins in bacterial virulence, the rapid rise of drug-resistant bacterial strains, and large number of fatalities due to Gram-negative bacterial infections, our proposed studies will have a large impact on molecular medicine and human health.