BIOGENESIS AND INHIBITION OF BIOFILM-ASSOCIATED BACTERIAL AMYLOID

生物膜相关细菌淀粉样蛋白的生物发生和抑制

• 批准号: 8815254
• 负责人: CARL FRIEDEN
• 金额: $ 38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8815254
• 关键词: 2-hydroxypyridine; Address; Adhesions; Adhesives; Alternative Therapies; Alzheimer' s Disease; Amyloid; Amyloid fibers; Antibiotic Therapy; Antibiotics; Atomic Force Microscopy; Bacteria; Behavior; Biochemical; Biocide; Biogenesis; Catheters; Cell Aggregation; Cell surface; Cells; Chronic; Circular Dichroism; Communicable Diseases; Communities; Cystic Fibrosis; Data; Development; Disease; Enterobacteriaceae; Escherichia coli; Escherichia coli O157; Extracellular Matrix; Fiber; Fluorescence; Genetic; Genetic study; Goals; Health; Human; Immune; In Vitro; Infection; Infectious Skin Diseases; Knowledge; Lead; Life; Lipoprotein (a); Lung; Maintenance; Measures; Mediating; Membrane; Methodology; Methods; Microbial Biofilms; Minor; Modeling; Molecular; Molecular Chaperones; Molecular Weight; Mus; Neurodegenerative Disorders; Nucleic Acids; Organism; Otitis Media; Parkinson Disease; Process; Proteins; Recurrence; Resistance; Resolution; Seeds; Senile Plaques; Silicones; Site; Skin; Stress; Structure; Surface; System; Testing; Thiazoles; Thioflavin T; Time; Urinary tract infection; Uropathogenic E. coli; Work; amyloid fibril formation; amyloid formation; antimicrobial; base; catheter associated UTI; chemical genetics; combat; cystic fibrosis patients; design; foodborne pathogen; in vivo; inhibitor/antagonist; insight; membrane assembly; monomer; novel; pathogen; pathogenic bacteria; premature; prevent; protein protein interaction; scaffold; scalpel; small molecule; therapeutic development; transmission process; urinary; 2-hydroxypyridine; Address; Adhesions; Adhesives; Alternative Therapies; Alzheimer' s Disease; Amyloid; Amyloid fibers; Antibiotic Therapy; Antibiotics; Atomic Force Microscopy; Bacteria; Behavior; Biochemical; Biocide; Biogenesis; Catheters; Cell Aggregation; Cell surface; Cells; Chronic; Circular Dichroism; Communicable Diseases; Communities; Cystic Fibrosis; Data; Development; Disease; Enterobacteriaceae; Escherichia coli; Escherichia coli O157; Extracellular Matrix; Fiber; Fluorescence; Genetic; Genetic study; Goals; Health; Human; Immune; In Vitro; Infection; Infectious Skin Diseases; Knowledge; Lead; Life; Lipoprotein (a); Lung; Maintenance; Measures; Mediating; Membrane; Methodology; Methods; Microbial Biofilms; Minor; Modeling; Molecular; Molecular Chaperones; Molecular Weight; Mus; Neurodegenerative Disorders; Nucleic Acids; Organism; Otitis Media; Parkinson Disease; Process; Proteins; Recurrence; Resistance; Resolution; Seeds; Senile Plaques; Silicones; Site; Skin; Stress; Structure; Surface; System; Testing; Thiazoles; Thioflavin T; Time; Urinary tract infection; Uropathogenic E. coli; Work; amyloid fibril formation; amyloid formation; antimicrobial; base; catheter associated UTI; chemical genetics; combat; cystic fibrosis patients; design; foodborne pathogen; in vivo; inhibitor/antagonist; insight; membrane assembly; monomer; novel; pathogen; pathogenic bacteria; premature; prevent; protein protein interaction; scaffold; scalpel; small molecule; therapeutic development; transmission process; urinary

DESCRIPTION (provided by applicant): Many chronic/recurrent human infectious disease states are recognized to involve bacterial biofilms. These include urinary tract infections (UTIs), chronic skin infections, otitis media and cystic fibrosis lung infections. In each, bacterial communities form, encased in an extracellular matrix where they are shielded from host immune mechanisms and from the action of currently used antibiotics. This greatly complicates the clearance of these organisms and the ability to cure these infections. Amyloid fibers called curli are critical in the formation of a biofilm extracellular matrix by uropathogenic Escherichia coli (UPEC). This fiber is made up of the major subunit CsgA, and a minor subunit CsgB. Using Thioflavin T fluorescence, circular dichroism and atomic force microscopy the in vitro mechanism by which CsgB nucleates CsgA fibrillization has been elucidated. The methods used in these studies will serve as a model for examining interactions between other components of the curli system. In vivo, the formation of curli amyloid fibers is directed by specific chaperone-like proteins (CsgE and CsgF) to occur extracellularly at the CsgG assembly site in the outer membrane. These proteins also serve to restrict premature, intracellular amyloid fibrillization, which otherwise would be toxic. In this proposal we utilize a combination of biophysical, biochemical, structural, genetic and chemical genetic methodologies to elucidate the structure, function and mechanism of action of the chaperone-like proteins CsgE, CsgF and the outer membrane assembly protein CsgG in facilitating the CsgB-templated fibrillization of CsgA. These studies will elucidate basic principles of curli formation and more generally amyloid formation. Then, using a thiazole ring-fused 2-pyridone scaffold we will investigate the mechanism of action of small molecule inhibitors ("curlicides") of curli assembly and curli-mediated biofilm formation. Curlicides will be used as molecular scalpels to dissect the details of the protein-protein interactions required for curli biogenesis. Curli promote biotic and abiotic surface colonization, stabilize cell-cell contacts allowing cell aggregation and thickening of the biofilm layer and confer resistance to the biofilm against environmental stresses and biocides. Thus, curlicides will be tested for their efficacy in preventing the formation of UPEC biofilms on silicone catheter tubing both in vitro and in a murine model of catheter- associated urinary tract infection. This work will provide key insights into processes necessary for the formation of medically important biofilms and will serve as a model to better understand amyloid plaques important in neurodegenerative disorders.

描述（由申请人提供）：许多慢性/复发性人类感染疾病态被认为涉及细菌生物膜。这些包括尿路感染（UTI）， 慢性皮肤感染，中耳炎和囊性纤维化肺部感染。在每种情况下，细菌群落形成，被包裹在细胞外基质中，在这些基质中它们被遮盖了宿主免疫机制，并避免了当前使用的抗生素的作用。这极大地使这些生物的清除和治愈这些感染的能力变得复杂。称为CURLI的淀粉样纤维对于通过尿道病大肠杆菌（UPEC）形成生物膜外基质至关重要。该纤维由主要亚基CSGA和小型亚基CSGB组成。利用硫非类T荧光，圆形二色性和原子力显微镜阐明了CSGB成核CSGA纤维化的体外机制。这些研究中使用的方法将作为检查冰结构系统其他组件之间相互作用的模型。在体内，夹层淀粉样蛋白纤维的形成是由特异性伴侣样蛋白（CSGE和CSGF）指向的，在外膜的CSGG组装位点外发生。这些蛋白质还可以限制过早的细胞内淀粉样蛋白纤维化，否则这将是有毒的。 In this proposal we utilize a combination of biophysical, biochemical, structural, genetic and chemical genetic methodologies to elucidate the structure, function and mechanism of action of the chaperone-like proteins CsgE, CsgF and the outer membrane assembly protein CsgG in facilitating the CsgB-templated fibrillization of CsgA.这些研究将阐明卷质形成和更普遍的淀粉样形成的基本原理。然后，使用噻唑环融合的2-吡啶酮支架，我们将研究小分子抑制剂（“冰柱”）的作用机理，磨牙组装和磨砂介导的生物膜形成的作用机理。策展人将用作分子手术刀，以剖析 Curli生物发生所需的蛋白质蛋白质相互作用。 Curli促进生物和非生物表面定植，稳定细胞 - 细胞接触，从而使生物膜层的细胞聚集和增厚，并赋予生物膜对环境应激和杀菌剂的抗性。因此，将测试策划区在防止在体外和导管相关的尿路感染的鼠模型中预防UPEC生物膜形成的疗效。这项工作将为形成医学上重要的生物膜的形成所需的过程提供关键的见解，并将作为更好地理解在神经退行性疾病中重要的淀粉样蛋白斑块的模型。