Structural basis of Rho glucosylation by Clostridium difficile toxins

艰难梭菌毒素 Rho 糖基化的结构基础

基本信息

批准号：
10308686
负责人：
Rongsheng Jin
金额：
$ 23.55万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10308686
关键词：
Actins Affinity Amino Acids Anaerobic Bacteria Animal Model Antibiotics Apoptotic Bacteria Binding Biological Assay C-terminal Caspase Cell Death Cell Surface Receptors Cells Centers for Disease Control and Prevention (U.S.)Cessation of life Chimeric Proteins Clinical Clostridium difficile Clostridium difficile tcdA protein Colitis Complex Crystallization Cytoskeleton Cytosol Developed Countries Development Diarrhea Disease Drug Targeting Endocytosis Endosomes Engineering Epithelial Exotoxins FDA approved Family Gastroenteritis Glucosyltransferase Goals Guanosine Triphosphate Phosphohydrolases Health care facility Hospitals Hot Spot Infection Infection prevention Inflammatory Innate Immune Response Lead Length Long-Term Care Mediating Membrane Molecular Molecular Conformation Molecular Weight Monomeric GTP-Binding Proteins Mutagenesis N Domain N-terminal Oligopeptides Pathogenesis Pathogenicity Pathology Patient-Focused Outcomes Peptides Phytic Acid Plant Roots Positioning Attribute Protease Domain Protein Isoforms Protein Sequence Analysis Proteins Reagent Recurrence Resolution Role Specificity Structure Substrate Specificity Symptoms TNFRSF6B gene Targeted Toxins Tertiary Protein Structure Therapeutic Intervention Time Toxin Validation Virulence Virulent X-Ray Crystallography alpha Toxin antibiotic-associated diarrhea antitoxin base cell injury design drug development electron density enzyme substrate gut bacteria gut microbiome gut microbiota human model improved in vivo inhibitor insight member molecular targeted therapies mouse model mutant neutralizing antibody novel novel therapeutics older patient protein complex public health relevance receptor binding receptor mediated endocytosis resistant strain rho rho GTP-Binding Proteins standard of care success targeted treatment three dimensional structure

项目摘要

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.

抽象的艰难梭菌，一种革兰氏阳性，厌氧，孢子菌细菌，通常在严重病中或医院或长期护理设施中的老年患者。艰难梭菌感染（CDI），最多发达国家中与抗生素相关的腹泻的常见原因主要由两个同源外毒素，TCDA和TCDB。这些毒素靶向并破坏结肠上皮，导致腹泻和结肠炎通过受体介导的内吞作用。 TCDA（〜308 kDa）和TCDB（〜270 kDa）包含四个功能结构域：N末端葡萄糖基转移酶结构域（GTD），半胱氨酸蛋白酶结构域（CPD），中央跨膜输送和受体结合结构域（DRBD）和C末端组合重复性寡肽（农作物）结构域。人们普遍认为，毒素通过 DRBD和农作物，并通过内吞作用进入细胞。内体触发器中的酸化毒素的构象变化，促使DRBD形成孔并传递GTD和CPD 跨越内体膜。在细胞质中，CPD被真核特异性肌感激活六磷酸盐和随后进行自动解体以释放GTD。然后GTD Rho家族的小GTP酶，包括Rho，RAC和CDC42。 Rho蛋白的葡萄糖基化抑制它们的功能，导致肌动蛋白细胞骨架，细胞创新和最终凋亡的改变细胞死亡。因此，GTD是热干预措施的理想分子靶标，该靶标直接靶向疾病症状的根本原因和CDI的细胞损伤。而这两个毒素在 CDI的发病机理尚未完全了解，TCDB被认为比TCDA更具毒性，并且对于诱导宿主的炎症和先天免疫反应而言，更重要。因此，我们将专注于 TCDB在该项目中，该提案的目的是了解TCDB的分子机制通过葡萄糖基化，共价修饰其底物Rho家族GTPases。我们提出了两个具体目标：（1）了解GTD识别Rho GTPases的结构基础，（2）了解亲和力 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，以检查分子水平的GTD和RHO蛋白之间的相互作用为了揭示GTD底物特异性和脆弱性的结构决定者。这些发现将为GTD的功能和TCDB的致病性提供新的见解，这可以指导通过抑制GTD的活性来治疗CDI的新型治疗剂的设计。