A Small Molecule Approach Toward Understanding Bacterial Toxin Transport

描述(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之一。尽管它们的细胞内靶点不同，但它们毒力的一个共同和必要的步骤是到达胞浆的能力，大多数毒素在胞液中发挥酶作用。细菌产物STX和CTX，以及植物毒素蓖麻毒素，在绕过溶酶体降解的同时，以逆行方式从内体通过高尔基体进入内质网。越来越明显的是，这种逆行运输途径并不是细菌毒素所特有的。相反，细菌毒素被认为劫持了现有的宿主运输途径。这些毒素利用的连续逆行过程已经转化为一种独特的系统，用于探索宿主的内吞机制，并加深了我们对逆行运输的理解。为了努力剖析和抑制STX的逐步运输，我们开发了一种抑制毒素运输的替代方法。以前旨在确定毒素贩运必不可少的成分的遗传方法已被证明是不一致的，并揭示了这些途径背后的复杂性。因此，我们应用了一种高灵敏度、高通量的基于荧光素酶的分析方法来筛选小分子化合物的文库，以了解它们阻止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烈防止志贺中毒的化合物。一种新的化合物B06产生了类似布雷菲尔丁A的高尔基体分散体，初步证据表明，它可以作为一种有用的工具来阐明ARF1介导的通过高尔基体的转运。此外，我们还希望探讨蛋白激酶在毒素转运中的作用。虽然有证据表明，激酶在毒素内吞作用中起作用，但它们在细胞内毒素转运中的作用尚不清楚。基于荧光素酶的分析将筛选一个新的动态组siRNA文库，以确定STX运输所必需的激酶。更重要的是，识别STX转运途径中不同阶段所涉及的激酶将有助于我们进一步了解它们在调节细胞内转运中的功能。在毒素转运的不同步骤中涉及的激酶的识别将同样提供针对毒素介导的疾病的治疗可能性。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小分子方法针对毒素介导的疾病的治疗效用。

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.