Mechanisms of Bacterial Toxin Action

描述(申请人提供)：细菌病原体经常通过毒素的作用损害宿主。细菌毒素通过共价和非共价机制修饰宿主靶标，主要是蛋白质，并具有几种结构-功能组织，包括外毒素和III型分泌细胞毒素。虽然外毒素通常在距离感染部位很远的地方起作用，但III型细胞毒素由细菌直接输送到中毒的宿主细胞。这项应用的目的是表征细菌毒素的底物识别和细胞内运输。在此之前，对铜绿假单胞菌III型细胞毒素的研究确定了EXOS和ExoT的底物识别机制，并表明宿主细胞内的胞内转运增加了EXOS对Ras和Rho GTP酶的效力。还开发了一个翻译项目来检测人类最初的PA感染。这一更新将继续表征EXOS的细胞内转运和底物识别，并启动破伤风神经毒素(TANT)的结构与功能研究。拟议的研究将表征EXOS和TANT的特定性质，这些性质定义了细菌毒素作用的独特方面。分析利用生化和定量细胞生物学方法，允许从试管到细胞进行确证分析。对EXOS的研究将表征宿主细胞内的转运如何提高EXOS的效力，并确定ADP核糖化抑制Ras和Rab5等小GTP酶信号传递的机制。最近的研究证实神经节苷脂是神经元中的功能性受体。这种鉴定可以解决神经毒素病理学的一个基本问题，即帐篷是如何在宿主中引起痉挛瘫痪的？对TANET的研究将表征TANET在神经元中的细胞内运输，并确定TIFET如何裂解SNARE蛋白VAMP2。这些研究定义了毒素的机械特性，这些特性可能导致用于检测、预防和干预细菌感染的翻译产品。

DESCRIPTION (provided by applicant): Bacterial pathogens often damage their host through the action of toxins. Bacterial toxins modify host targets, primarily proteins, through covalent and non-covalent mechanisms and have several structure-function organizations, including exotoxins and type III secreted cytotoxins. While exotoxins often act at a distance from the site of infection, type III cytotoxins are delivered directly into the intoxicated host cell by the bacterium. The aims of this application have been to characterize the substrate recognition and intracellular trafficking of bacterial toxins. During the previous period, studies on the type III cytotoxins of Pseudomonas aeruginosa determined substrate recognition mechanisms of ExoS and ExoT and showed that intracellular trafficking within host cells increased the potency of ExoS for Ras and Rho GTPases. A translational project was also developed to detect initial PA infections in humans. This renewal will continue to characterize the intracellular trafficking and substrate recognition on ExoS and initiate structure-function studies on tetanus neurotoxin (TeNT). The proposed studies will characterize specific properties of ExoS and TeNT that define unique aspects of bacterial toxin action. Analyses utilize biochemical- and quantitative cell biological- approaches that allow corroborative analyses from the test tube to the cell. Studies on ExoS will characterize how intracellular trafficking within host cells increases the potency of ExoS and determine the mechanism that ADP-ribosylation inhibits signaling by small GTPases, like Ras and Rab5. Recent studies determined that gangliosides are the functional receptors of TeNT in neurons. This identification allows a fundamental question of neurotoxin pathology to be addressed, how does TeNT elicit spastic paralysis in the host? Studies on TeNT will characterize the intracellular trafficking of TeNT in neurons and determine how TeNT cleaves the SNARE protein, VAMP2. These studies define mechanistic properties of toxins that may lead to translational products for the detection, prevention, and intervention of bacterial infections.