A Small Molecule Approach Toward Understanding Bacterial Toxin Transport

描述（由申请人提供）：志贺毒素（Stx）、霍乱毒素（Ctx）和植物毒素蓖麻毒素是通过复杂的细胞内途径到达细胞内目的地的几种毒素。尽管它们的细胞内靶点不同，但它们的毒力有一个共同而重要的步骤，即到达细胞质的能力，大多数毒素在细胞质中发挥酶的作用。细菌产物Stx和Ctx，以及植物毒素蓖麻毒素，通过高尔基体从内体逆行运输到内质网，而绕过溶酶体降解。越来越明显的是，这种逆行运输途径并不是细菌毒素所独有的。相反，细菌毒素被认为劫持了现有的宿主运输途径。这些毒素利用的顺序逆行过程已经转化为探测宿主内吞机制的独特系统，并增强了我们对逆行运输的理解。为了解剖和抑制Stx的逐步贩运，我们已经开发了一种抑制毒素运输的替代方法。先前旨在确定毒素运输必要成分的遗传方法已被证明是不一致的，并揭示了这些途径的复杂性。因此，我们采用了一种高灵敏度、高通量的基于荧光素酶的检测方法来筛选小分子化合物库，以确定它们阻断蛋白质合成抑制的能力。从最初的筛选中，我们已经确定了各种强有力地防止志贺中毒的化合物。一种新的化合物B06可以产生类似brefeldin a的高尔基体分散，初步证据表明它可以作为阐明arf1介导的高尔基体运输的有用工具。此外，我们希望探索激酶在毒素运输中的作用。虽然有证据表明激酶在毒素内吞作用中的作用，但它们在细胞内毒素运输中的功能仍不清楚。一个新的kinome siRNA文库将通过基于荧光素酶的试验筛选，以确定Stx运输所必需的激酶。更重要的是，确定在Stx转运途径的各个阶段参与的激酶将进一步了解它们在介导细胞内运输中的功能。在毒素运输的不同步骤中所涉及的激酶的鉴定同样将提供治疗毒素介导的疾病的可能性。细菌和植物毒素是人类疾病的重要病原体，也是生物恐怖主义的潜在载体。我们的高通量分析强调了针对毒素介导疾病的小分子方法的治疗效用。

DESCRIPTION (provided by applicant): Shiga toxin (Stx), cholera toxin (Ctx), and the plant toxin ricin are among several toxins that reach their intracellular destinations via complex intracellular routes. Though diverse in their intracellular targets, a common and essential step in their virulence is the ability to reach the cytosol, where most toxins exert their enzymatic effects. The bacterial products Stx and Ctx, as well as the plant toxin ricin, traffick in a retrograde fashion from endosomes to the endoplasmic reticulum via the Golgi while bypassing lysosomal degradation. It has become increasingly apparent that this retrograde trafficking pathway is not unique to bacterial toxins. Rather, bacterial toxins are believed to hijack existing host transport pathways. The sequential retrograde progression utilized by these toxins has translated into a unique system for probing host endocytic mechanisms and have enhanced our understanding of retrograde transport. In an effort to dissect and inhibit the stepwise trafficking of Stx, we have developed an alternative approach toward inhibiting toxin transport. Previous genetic approaches aimed at identifying components essential to toxin trafficking have proven inconsistent and revealed the complexity underlying these pathways. As a result, we have applied a highly sensitive, high-throughput luciferase-based assay to screen a library of small molecule compounds for their ability to block inhibition of protein synthesis. From an initial screen, we have identified various compounds that strongly protect against Shiga intoxication. One novel compound, B06, produces a brefeldin A-like dispersal of the Golgi, and preliminary evidence suggests that it could serve as a useful tool in elucidating ARF1-mediated transport through the Golgi apparatus. In addition, we hope to explore the role of kinases in toxin trafficking. Though there is evidence for the role of kinases in toxin endocytosis, their functions in intracellular toxin trafficking remain unclear. A novel kinome siRNA library will be screened by the luciferase-based assay to identify kinases essential to Stx transport. More importantly, the identification of kinases involved at various stages along the Stx trafficking pathway will further our understanding of their functions in mediating intracellular transport. Identification of kinases involved at distinct steps in toxin trafficking will equally provide therapeutic possibilities against toxin-mediated diseases. Bacterial and plant toxins are significant agents of human disease and potential vehicles for bioterrorism. Our high-throughput assay underscores the therapeutic utility of a small molecule approach for targeting toxin-mediated diseases.