Organization and function of a type II secretion complex

在革兰氏阴性病原菌中，通过T2S途径分泌的许多蛋白质构成了重要的毒力因子，包括毒素和降解酶，因此T2S系统的胞外分泌和靶向递送被认为是革兰氏阴性菌的主要毒力机制。T2S装置由至少13种不同的蛋白质组成，EPSC-EPSN和PilD，它们组装成一个跨越霍乱弧菌整个细胞膜的复合体。这种复合体的动态和可能是瞬时的性质可能是发挥功能的先决条件，因为它的组装和拆解可能会驱动细胞外分泌。这一过程所需的能量被认为是由EPSE产生的，EPSE是一种细胞质蛋白，通过与膜蛋白EPSL的相互作用与细胞膜相关。EPSM和EPSF。EPSE与这些组分的相互作用调节其ATPase活性，并促进其在霍乱弧菌细胞膜内的不同位置定位。 这项建议中描述的实验旨在测试这一假设，即特定的蛋白质-蛋白质相互作用和酸性磷脂在V.cho/erae细胞被膜的离散位置上以依赖于ATP的过程驱动T2S。具体地说，这项建议将确定EPSE的酶活性受包括磷脂在内的细胞膜成分控制的机制。EPSL和EPSF；ii)研究EPS组分的有序组装，并确定EpsD和EPSC驱动T2S复合体的焦点组装的机制；iii)定位EpsM组装时形成的裂隙，并确定与裂隙结合的细胞因子。 解决T2S系统的调控组装和空间定位的机制将加深我们对T2S的理解，并可能找到控制分泌过程的方法，用于预防、治疗和/或生物技术应用。

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.