Organization and function of a type II secretion complex

通过II型分泌（T2 S）系统的细胞外分泌和靶向递送被认为是革兰氏阴性病原体中的主要毒力机制，因为通过T2 S途径分泌的许多蛋白质构成重要的毒力因子，包括毒素和降解酶。T2 S装置由至少13种不同的蛋白质组成，EpsC-EpsN和PilD，它们组装成跨越霍乱弧菌整个细胞包膜的复合物。这种复合物的动态和可能的瞬时性质可能是其组装和分解可能驱动细胞外分泌的功能的先决条件。这个过程所需的能量被认为是由EpsE的ATP水解产生的，EpsE是一种细胞质蛋白，通过与膜蛋白EpsL的相互作用与细胞质膜结合。EPSM和EPSF。EpsE与这些组分的相互作用调节其ATP酶活性并促进其定位于胆总管弧菌细胞包膜内的不同位点。 本提案中描述的实验旨在检验以下假设：特定的蛋白质-蛋白质相互作用和酸性磷脂在V. cho/v.的细胞包膜中的离散位点以ATP依赖性过程驱动T2 S。具体地，该提议将i）确定EpsE的酶活性由细胞质膜的组分（包括磷脂）控制的机制。EpsL和EpsF; ii）研究Eps组分的有序组装并确定EpsD和EpsC驱动T2 S复合物的焦点组装的机制; iii）绘制当EpsM组装时形成的裂缝并鉴定结合裂缝的细胞因子。 解决T2 S系统的调节组装和空间定位的机制将进一步加深我们对T2 S的理解，并可能确定用于预防、治疗和/或生物技术用途的操纵分泌过程的方法。

Extracellular secretion and targeted delivery by the type II secretion (T2S) system is considered a major virulence mechanism in gram negative pathogens, as many of the proteins secreted via the T2S pathway constitute important virulence factors, including toxins and degradative enzymes. The T2S apparatus is comprised of at least 13 different proteins, EpsC-EpsN and PilD, that assemble into a complex that spans the entire cell envelope of Vibrio cholerae. The dynamic and perhaps transient nature of this complex may be a prerequisite for function as its assembly and disassembly may drive extracellular secretion. The energy required for this process is thought to be generated from ATP hydrolysis by EpsE, a cytoplasmic protein that is associated with the cytoplasmic membrane via interaction with the membrane proteins EpsL. EpsM and EpsF. EpsE's interactions with these components modulate its ATPase activity and promote its localization to distinct sites within the V. cholerae cell envelope. The experiments described in this proposal are designed to test the hypothesis that specific protein-protein interactions and acidic phospholipids drive T2S in an ATP-dependent process at discrete sites In the cell envelope of V. cho/erae. Specffically, this proposal will i) determine the mechanism by which the enzymatic activity of EpsE is controlled by components of the cytoplasmic membrane including phospholipids. EpsL and EpsF; ii) investigate the ordered assembly of Eps components and determine the mechanism by which EpsD and EpsC drive focal assembly of the T2S complex; iii) map the cleft that forms when EpsM assembles and identify the cellular factor that binds to the cleft. Resolving the mechanisms of regulated assembly and spatial localization of the T2S system will further our understanding of T2S and may identify ways to manipulate the secretion process for preventative, therapeutic and/or biotechnological use.