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、产孢细菌，常见于重症或 医院或长期护理机构的老年患者。艰难梭菌感染（CDI），这是最 在发达国家，腹泻相关性腹泻的常见原因主要是由两种 同源外毒素TcdA和TcdB。这些毒素靶向并破坏结肠上皮，导致 腹泻和结肠炎。TcdA（~308 kDa）和TcdB（~270 kDa）含有 四个功能结构域：N-末端葡糖基转移酶结构域（GTD），半胱氨酸蛋白酶结构域（CPD）， 中央跨膜递送和受体结合结构域（DRBD），和C末端组合的重复序列， 寡肽（CROPs）结构域。广泛接受的是，毒素通过细胞膜结合细胞表面受体。 DRBD和CROPs，并通过内吞作用进入细胞。核内体的酸化 毒素的构象变化，促使DRBD形成孔并递送GTD和CPD 穿过内体膜在细胞质中，CPD被真核生物特异性肌醇激活 六磷酸盐，随后经历自蛋白水解以释放GTD。然后GTD 葡糖基化Rho家族的小GTP酶，包括Rho、Rac和Cdc 42。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，细胞变圆，并最终凋亡 细胞死亡因此，GTD是治疗干预的理想分子靶点，其直接靶向 疾病症状和细胞损伤的根本原因。虽然这两种毒素的相对作用， CDI的发病机制还不完全清楚，TcdB被认为比TcdA更具毒性， 更重要的是诱导宿主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，这个建议的目标是了解TcdB的分子机制， 通过葡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） 理解GTD识别Rho GTP酶的结构基础，以及（2）理解GTD与Rho GTP酶的亲和力。 和GTD-Rho识别的特异性要求。我们将使用X射线晶体学和结构- 基于诱变，以检查GTD和Rho蛋白在分子水平上的相互作用，以及 以揭示基板的特异性和脆弱性的GTD的结构决定因素。这些发现 将为GTD的功能和TcdB的致病性提供新的见解，这可以指导 设计通过抑制GTD的活性来治疗CDI的新型治疗剂。

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.