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

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，很大程度上由革兰氏阳性菌引起变形链球菌每年的相关医疗费用高达数百亿美元，并且费用高达美国的儿童职业正在上升，解决这一未满足的医疗保健需求显然势在必行。确定导致龋齿形成顽固生物膜的生物体的新方法，从膳食糖中产生酸，并耐受酸性最终产物，而淀粉样蛋白首先是在淀粉样蛋白中发现的。从病理学的角度来看，它并不总是代表功能性淀粉样蛋白的蛋白质错误折叠途径。淀粉样蛋白代表了进化上保守的原纤维、交叉β片层四级结构。 β-折叠横向自组装形成纤维具有共同的生物物理特性。特性，包括淀粉样蛋白染料的吸收、通过透射电子观察时的典型直径显微镜观察，以及用刚果红染色并在交叉偏振光下观察时的双折射特性。现在已知几种微生物可以产生生物膜不可或缺的功能性淀粉样蛋白我们的小组是第一个将变形链球菌鉴定为淀粉样蛋白形成生物的人。现在我们已经扩展了这项工作，以确定这种细菌中总共三种淀粉样蛋白形成蛋白。提供了粘附素 P1 的广泛三级和四级结构表征，并鉴定了其我们还鉴定了变形链球菌的羧基末端 C123 截短衍生物。 WapA 和以前未表征的淀粉样蛋白形成蛋白 Smu_63c 与 P1 一样，WapA 是一种淀粉样蛋白形成蛋白。表面定位的分选酶底物，其截短衍生物 AgA 是一种分泌型淀粉样蛋白。作为遗传能力和生物膜细胞密度的负调节剂的蛋白质。蛋白质有助于变形链球菌生物膜的形成，这可以被小分子抑制剂抑制缺乏编码淀粉样蛋白的基因的变形链球菌突变体形成淀粉样蛋白纤维化。形成蛋白明显不易受到针对淀粉样蛋白的化合物的抑制，表明淀粉样蛋白抑制作为预防生物膜形成的治疗方法的可行性在此更新应用中，我们将利用最先进的方法，包括溶液和固体。状态 NMR，通过以下方式识别和表征淀粉样蛋白纤维化背后的结构转变变形链球菌的淀粉样蛋白形成蛋白（目标 1）这将使我们能够跟踪其结构进展。我们将揭示生物膜内淀粉样蛋白的单体，并揭示抑制化合物的作用机制。还开发和优化方法来区分变形链球菌淀粉样蛋白的单体和纤维形式最后，我们将应用这些检测方法来评估体外和生物膜内形成的蛋白质。并确定调节链球菌内蛋白质单体向淀粉样蛋白转化的相关环境触发因素。变形生物膜（目标 3），迄今为止在任何研究的系统中尚未充分理解这一过程。