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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.

摘要