Functional amyloid formation in streptococcus mutans

变形链球菌中功能性淀粉样蛋白的形成

基本信息

批准号：
9892876
负责人：
L. Jeannine Brady
金额：
$ 45万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-05 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9892876
关键词：
Acids Address Adhesions Adhesives Alzheimer&apos s Disease Amyloid Amyloid Fibrils Atherosclerosis Bacteria Bacterial Adhesins Bacterial Endocarditis Binding Caliber Cell Density Cell Wall Cell surface Chemicals Childhood Clinical Communicable Diseases Competence Complex Confocal Microscopy Congo Red Dental Plaque Dental caries Detection Development Dietary Sugars Dyes Electron Microscopy Environmental Risk Factor Evaluation Extracellular Matrix Fiber Foundations Funding Future Gene Expression Genes Genetic Goals Gram-Positive Bacteria Grant Growth Health Care Costs Healthcare Human Hyphae Immunologics In Vitro Infectious Agent Investigation Isotopes Laboratories Link Liquid substance Literature Methodology Methods Microbial Biofilms Microfluidic Microchips Molecular Molecular Sieve Chromatography Morphology Oral cavity Organism Pathogenesis Pathogenicity Pathology Pathway interactions Peptides Process Production Property Proteins Publishing Reagent Reporting Research Resolution Screening procedure Signaling Molecule Stains Streptococcus mutans Structural Protein Structure Surface System Techniques Therapeutic Therapeutic Intervention Transmission Electron Microscopy Virulence Work amyloid formation amyloid structure beta pleated sheet biophysical properties enzyme substrate in vivo microbial microorganism monomer mutant novel strategies oral bacteria pathogen polarized light polymicrobial biofilm prevent protein aggregation protein misfolding small molecule inhibitor solid state nuclear magnetic resonance sortase stem structural biology therapeutic target uptake

Acids Address Adhesions Adhesives Alzheimer&apos s Disease Amyloid Amyloid Fibrils Atherosclerosis Bacteria Bacterial Adhesins Bacterial Endocarditis Binding Caliber Cell Density Cell Wall Cell surface Chemicals Childhood Clinical Communicable Diseases Competence Complex Confocal Microscopy Congo Red Dental Plaque Dental caries Detection Development Dietary Sugars Dyes Electron Microscopy Environmental Risk Factor Evaluation Extracellular Matrix Fiber Foundations Funding Future Gene Expression Genes Genetic Goals Gram-Positive Bacteria Grant Growth Health Care Costs Healthcare Human Hyphae Immunologics In Vitro Infectious Agent Investigation Isotopes Laboratories Link Liquid substance Literature Methodology Methods Microbial Biofilms Microfluidic Microchips Molecular Molecular Sieve Chromatography Morphology Oral cavity Organism Pathogenesis Pathogenicity Pathology Pathway interactions Peptides Process Production Property Proteins Publishing Reagent Reporting Research Resolution Screening procedure Signaling Molecule Stains Streptococcus mutans Structural Protein Structure Surface System Techniques Therapeutic Therapeutic Intervention Transmission Electron Microscopy Virulence Work amyloid formation amyloid structure beta pleated sheet biophysical properties enzyme substrate in vivo microbial microorganism monomer mutant novel strategies oral bacteria pathogen polarized light polymicrobial biofilm prevent protein aggregation protein misfolding small molecule inhibitor solid state nuclear magnetic resonance sortase stem structural biology therapeutic target uptake

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项目摘要

ABSTRACT Dental caries is the most common infectious disease in the world caused in large part by the Gram-positive bacterium Streptococcus mutans. Associated annual health care costs tens of billions of dollars, and rates of childhood caries in the U. S. are rising. There is a clear imperative to address this unmet health care need and identify new approaches to stem caries pathogenesis. Organisms that cause cavities form recalcitrant biofilms, generate acids from dietary sugars, and tolerate acid end products. While amyloid was first identified in the context of pathology, it does not always represent a protein mis-folding pathway. Functional amyloid is now recognized. Amyloid represents an evolutionarily conserved fibrillar, cross β-sheet quaternary structure in which the β-sheets laterally self-assemble to form fibers. Amyloid aggregates have common biophysical properties including uptake of amyloidophilic dyes, typical diameters when viewed by transmission electron microscopy, and birefringent properties when stained with Congo red and viewed under cross-polarized light. Several microorganisms are now known to produce functional amyloids that are integral to biofilm development. Our group was the first to identify Streptococcus mutans as an amyloid-forming organism. We have now extended that work to identify a total of three amyloid forming proteins in this bacterium. We have provided extensive tertiary and quaternary structural characterization of adhesin P1 and have identified its carboxy-terminal C123 truncation derivative as the amyloidogenic moiety. We have also identified S. mutans WapA and a previously uncharacterized protein Smu_63c as amyloid forming proteins. Like P1, WapA is a surface-localized sortase substrate whose truncation derivative, AgA, is amyloidogenic. Smu_63c is a secreted protein that serves as a negative regulator of genetic competence and biofilm cell density. All three of these proteins contribute to S. mutans biofilm development, which can be inhibited by small molecule inhibitors of amyloid fibrillization. Biofilm development by S. mutans mutants lacking genes encoding the amyloid forming proteins is significantly less susceptible to inhibition by compounds that target amyloids indicating the feasibility of amyloid inhibition as a therapeutic approach to preventing biofilm formation by this pathogen. In this renewal application we will utilize state of the art methods, including solution and solid state NMR, to identify and characterize the structural transitions underlying amyloid fibrillization by the amyloid forming proteins of S. mutans (Aim 1). This will enable us to follow their structural progression from monomer to amyloid within biofilms and will reveal mechanisms of action of inhibitory compounds. We will also develop and optimize methods to differentiate monomeric and fibrillar forms of the S. mutans amyloid forming proteins in vitro and within biofilms (Aim 2). Lastly we will apply these detection methods to assess and identify relevant environmental triggers that regulate protein monomer to amyloid conversion within S. mutans biofilms (Aim 3), a process that is not yet well understood in any system studied to date.

抽象的龋齿是世界上最常见的传染病细菌链球菌突变。相关的年度医疗保健成本数百亿美元，费率为美国的儿童汽车正在上升。显然必须满足这种未满足的医疗保健需求和确定用于茎汽车发病机理的新方法。引起空腔的生物形成顽固生物膜，从饮食糖中产生酸，并耐酸最终产物。淀粉样蛋白首先在病理的背景，它并不总是代表蛋白质误折叠途径。功能性淀粉样蛋白现在认可。淀粉样蛋白代表一种进化构建的原纤维，交叉β-折季季结构 β-折叠横向自组装形成纤维。淀粉样蛋白聚集体具有常见的生物物理特性，包括肌醇染料的吸收当用刚果红染色并在交叉偏振光下染色时，显微镜和双重特性。现在已知几种微生物会产生与生物膜不可或缺的功能性淀粉样蛋白发展。我们的小组是第一个将突变链球菌变成淀粉样蛋白酶形成生物的人。我们现在已经扩展了这项工作，以确定该细菌中总共三种淀粉样蛋白形成的蛋白质。我们现在有提供了粘附素P1的大量第三和第四纪结构表征，并确定了其羧基末端C123截断衍生物作为淀粉样蛋白生成部分。我们还确定了S. mutans WAPA和先前未表征的蛋白SMU_63C作为淀粉样蛋白。像P1一样，WAPA是一个截短衍生物AGA的表面钙化分子酶底物具有淀粉样蛋白生成。 SMU_63C是一个分泌的作为遗传能力和生物膜细胞密度的负调节剂的蛋白质。这三个蛋白质有助于突变链球菌生物膜发育，可以被小分子抑制剂抑制淀粉样蛋白纤维化。 S.突变体突变体缺乏编码淀粉样蛋白的基因的生物膜开发靶向淀粉样蛋白的化合物明显不太容易受到抑制的影响。淀粉样蛋白抑制作用作为预防生物膜形成的治疗方法的可行性病原。在此续签应用中，我们将利用最新方法，包括解决方案和固体状态NMR，以识别和表征通过淀粉样蛋白纤维化的结构过渡淀粉样蛋白链球菌的蛋白质（AIM 1）。这将使我们能够遵循它们的结构发展单体至生物膜内的淀粉样蛋白，并将揭示抑制性化合物作用机制。我们将还开发并优化了区分链霉菌的单体和原纤维形式的方法体外和生物膜内形成蛋白质（AIM 2）。最后，我们将应用这些检测方法评估并确定相关的环境触发器，这些触发器调节蛋白质单体向S.内的淀粉样蛋白转化率 Mutans Biofilm（AIM 3），迄今为止，在任何研究系统中都尚未良好理解的过程。