Structural basis of Rho glucosylation by Clostridium difficile toxins

摘要 艰难梭菌是一种革兰氏阳性、厌氧、产孢子菌，常见于重症患者或 医院或长期护理机构中的老年患者。艰难梭菌感染(CDI)是最常见的 在发达国家，抗生素相关性腹泻的常见原因主要由两个原因引起 同源外毒素，TcdA和TcdB。这些毒素针对并破坏结肠上皮，导致 通过受体介导的内吞作用引起腹泻和结肠炎。TcdA(~308 kDa)和Tcdb(~270 kDa)包含 四个功能结构域：N-末端葡萄糖转移酶结构域(GTD)、半胱氨酸蛋白酶结构域(CPD)、 一个中央跨膜递送和受体结合域(DRBD)，和一个C末端结合的重复序列 寡肽(农作物)领域。人们普遍认为，这些毒素与细胞表面受体结合是通过 DRBD和作物，并通过内吞进入细胞。内吞体内的酸化会触发 促使DRBD形成毛孔并传递GTD和CPD的毒素的构象变化 穿过内质体膜。在胞浆中，CPD被真核特有的肌醇激活。 六聚磷酸，然后进行自身蛋白分解以释放GTD。然后是GTD 糖基化Rho家族的小GTP酶，包括Rho、Rac和CDC42。Rho蛋白的糖基化 抑制它们的功能，导致肌动蛋白细胞骨架的改变，细胞圆化，最终导致细胞凋亡 细胞死亡。因此，GTD是治疗干预的理想分子靶点，它直接针对 CDI中疾病症状和细胞损伤的根本原因。而这两种毒素在体内的相对作用 CDI的发病机制尚不完全清楚，Tcdb被认为比TcdA和TcdA更具毒性 更重要的是诱导宿主的炎症和先天免疫反应。因此，我们将重点关注 Tcdb在这个项目中，这项建议的目标是了解Tcdb通过的分子机制 通过糖基化共价修饰其底物，Rho家族GTP酶。我们提出两个具体目标：(1) 了解GTD识别Rho GTP酶的结构基础，以及(2)了解亲和力 以及GTD-RHO识别的特异性要求。我们将使用X射线结晶学和结构- 基于突变在分子水平上检测GTD和Rho蛋白之间的相互作用 以揭示GTD底物专一性和脆弱性的结构决定因素。这些发现 将对GTD的功能和TcdB的致病性提供新的见解，从而指导 设计通过抑制GTD活性来治疗CDI的新型治疗试剂。

Abstract Clostridium difficile, a Gram-positive, anaerobic, sporogenic bacterium, is often seen in severely ill or elderly patients in hospitals or in long-term care facilities. Clostridium difficile infection (CDI), which is the most common cause of antibiotic-associated diarrhea in developed countries, is primarily caused by two homologous exotoxins, TcdA and TcdB. These toxins target and disrupt the colonic epithelium, leading to diarrhea and colitis through receptor mediated endocytosis. TcdA (~308 kDa) and TcdB (~270 kDa) contain four functional domains: an N-terminal glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a central transmembrane delivery and receptor-binding domain (DRBD), and a C-terminal combined repetitive oligopeptides (CROPs) domain. It is widely accepted that the toxins bind to cell surface receptors via the DRBD and the CROPs, and enter the cells through endocytosis. Acidification in the endosome triggers conformational changes in the toxins that prompt the DRBD to form a pore and deliver the GTD and the CPD across the endosomal membrane. In the cytosol, the CPD is activated by eukaryotic-specific inositol hexakisphosphate and subsequently undergoes autoproteolysis to release the GTD. The GTD then glucosylates small GTPases of the Rho family, including Rho, Rac, and Cdc42. Glucosylation of Rho proteins inhibits their functions, leading to alterations in the actin cytoskeleton, cell-rounding, and ultimately apoptotic cell death. Therefore the GTD is an ideal molecular target for therapeutic interventions, which directly targets the root cause of disease symptoms and cellular damage in CDI. While the relative roles of these two toxins in the pathogenesis of CDI are not completely understood, TcdB is considered to be more virulent than TcdA and more important for inducing the host inflammatory and innate immune responses. Therefore, we will focus on TcdB in this project, and the goal of this proposal is to understand the molecular mechanism by which TcdB covalently modifies its substrates, Rho family GTPases, by glucosylation. We propose two specific aims: (1) to understand the structural basis for recognition of Rho GTPases by the GTD, and (2) to understand the affinity and specificity requirements for the GTD–Rho recognition. We will use X-ray crystallography and structure- based mutagenesis to examine interactions between the GTD and Rho proteins at the molecular level, as well as to reveal the structural determinants of substrate specificity and vulnerabilities of the GTD. These findings will provide new insights into the function of the GTD and the pathogenicity of TcdB, which could guide the design of novel therapeutic reagents to treat CDI by inhibiting the activity of the GTD.