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：
8238683
负责人：
L. Jeannine Brady
金额：
$ 36.57万
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 2016-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8238683
关键词：
Acids Address Adhesions Affect Alzheimer&apos s Disease Amyloid Amyloid Fibrils Amyloid fibers Bacteria Bacterial Adhesins Binding Biological Biological Assay Biology Birefringence Cell Wall Cell surface Cells Characteristics Childhood Clinical Communicable Diseases Congo Red Cryoelectron Microscopy Dental Enamel Dental Plaque Dental caries Development Dietary Sugars Dyes Electron Spin Resonance Spectroscopy Enzymes Evaluation Fiber Formic Acids Genes Gram-Positive Bacteria Health Care Costs Healthcare Human Hybrids Hyphae I-antigen Imagery In Situ Individual Insecta Investigation Life Link Literature Mediating Methodology Microbe Microbial Biofilms Modeling NMR Spectroscopy Names Organism Pathogenesis Pattern Peptide Hydrolases Peptides Peptidoglycan Peptidyltransferase Process Property Protein S Proteins Publishing Residual state Resistance Sampling Side Staining method Stains Streptococcus mutans Structure Surface Techniques Testing Therapeutic Therapeutic Intervention Thioflavin T Tooth structure Transmission Electron Microscopy Virulence Visual X ray diffraction analysis X-Ray Diffraction amyloid formation amyloid structure amyloidogenesis base cariogenic bacteria enzyme substrate fluorophore fungus inhibitor/antagonist insight microbial mutant novel novel strategies oral pathogen pathogen polarized light polyproline prevent programs protein aggregate solid state nuclear magnetic resonance sortase stem uptake

项目摘要

DESCRIPTION (provided by applicant): 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. It is recently recognized that micro- organisms can produce functional amyloids that are integral to biofilm development. In this proposal we show that the S. mutans cell surface-localized adhesin called P1 (Antigen I/II, PAc) is an amyloid forming protein. This conclusion is based on defining properties of amyloids including uptake of the amyloidophilic dyes Congo red and Thioflavin T, visualization of amyloid fibers by transmission electron microscopy, and green birefringent properties of Congo red-stained protein aggregates when viewed under cross-polarized light. Amyloid is present in human dental plaque and is produced by both lab strains and clinical isolates. We provide further evidence that amyloid formation is not limited to P1, as bacterial colonies without this adhesin demonstrate residual green birefringence. However, S. mutans lacking sortase, the transpeptidase enzyme that mediates covalent linkage of it substrates to the cell wall peptidoglycan, including P1 and five other proteins, is not birefringent when stained with Congo red and does not form biofilms. We also show that biofilm formation is inhibited when S. mutans is cultured in the presence of known inhibitors of amyloid fibrillization. Amyloid represents an evolutionarily conserved fibrillar, cross 2-sheet quaternary structure in which the 2-sheets laterally self-assemble to form fibers. We recently showed that crystalline P1 demonstrates a unique tertiary structure in which two globular 2-sandwich domains lie on either side of an extended hybrid alpha-polyproline II helix. In this study we will continue biophysical confirmation of P1 amyloidogenesis including its X-ray diffraction pattern, and capitalize on the new tertiary structure information utilizing solid state NMR, electron paramagnetic resonance (EPR), and cryo-electron microscopy methodologies to elucidate P1's ultrastructure particularly as related to its amyloid forming properties (Aim 1). In Aim 2 we will identify and then characterize additional amyloid forming proteins of S. mutans using a similar approach as that followed for P1. Genes encoding amyloid forming proteins will be deleted singly and in combination. In Aim 3 we will evaluate amyloid formation with respect to biofilm development using both static and flow models, and by assessing the effects of known amyloid inhibitors on S. mutans biofilm formation by wild-type and mutant strains. We will then confirm our results using the biophysical assays described in Aim 1. Together these Aims will reveal the contribution of amyloid formation to S. mutans biology, and demonstrate the feasibility of amyloid inhibition as a therapeutic approach to preventing biofilm formation by this pathogen. PUBLIC HEALTH RELEVANCE: The bacteria named Streptococcus mutans cause cavities by living in sticky layers (biofilms) adhered to teeth and then destroying tooth enamel by the acid they make from dietary sugars. Amyloids are aggregates of proteins that form fibers. We show that biofilm formation by Streptococcus mutans involves amyloid fibrils built by the microbe; therefore, we will study how this process occurs and how to inhibit it.

描述（由申请人提供）：龋齿是世界上最常见的传染病，这在很大程度上是由革兰氏阳性细菌链球菌突变引起的。相关的年度医疗保健成本数万美元，美国的儿童龋齿率正在上升。明确的必须满足这种未完成的医疗保健需求，并确定茎龋发病机理的新方法。引起空腔的生物会形成顽固生物膜，从饮食糖中产生酸，并耐酸最终产物。最近认识到，微生物可以产生与生物膜发育不可或缺的功能性淀粉样蛋白。在该提案中，我们表明，链球菌细胞表面钙化的粘附素称为P1（抗原I/II，PAC）是一种淀粉样蛋白。该结论基于定义淀粉样蛋白的特性，包括吸收淀粉样蛋白染料刚果红色和硫非黄酮T，通过透射电子显微镜可视化淀粉样蛋白纤维的可视化以及在交叉极光光下观察时刚果红色染色蛋白质聚集体的绿色双发性性能。淀粉样蛋白存在于人牙斑块中，由实验室菌株和临床分离株产生。我们提供进一步的证据表明淀粉样蛋白形成不限于P1，因为没有这种粘附素的细菌菌落表现出残留的绿色双折射。然而，缺乏分类酶的链球菌，即介导IT底物的共价键上的转肽酶酶与细胞壁肽聚糖（包括P1和其他五种蛋白质），当用刚果红染色并且不形成生物膜时，并不是双重的。我们还表明，在存在已知的淀粉样蛋白纤维化抑制剂的情况下培养链球菌时，生物膜的形成就会抑制。淀粉样蛋白代表一种进化保守的原纤维，交叉2页季结构，其中2个表面横向自组装形成纤维。我们最近表明，晶体P1展示了一个独特的三级结构，其中两个球状2-sandwich域位于扩展的杂化α-聚丙烯II螺旋的两侧。在这项研究中，我们将继续对P1淀粉样生成的生物物理确认，包括其X射线衍射模式，并利用利用固态NMR，电子顺磁共振（EPR）和冷冻电子显微镜方法的新的第三级结构信息，以阐明P1的超级结构，特别是与其Amyle属性相关联（与其Amyloid promaties（Aim tormities）相关联（瞄准）。在AIM 2中，我们将使用类似于P1的类似方法来识别并表征链球菌的其他淀粉样蛋白。编码淀粉样蛋白蛋白的基因将单独删除并组合。在AIM 3中，我们将使用静态和流动模型评估淀粉样蛋白形成，并通过评估已知的淀粉样蛋白抑制剂对野生型和突变菌株的生物膜形成的影响。然后，我们将使用AIM 1中描述的生物物理测定法确认结果。这些目标将揭示淀粉样蛋白形成对链球菌生物学的贡献，并证明淀粉样蛋白抑制作用是一种治疗方法，以防止这种病原体形成生物膜。公共卫生相关性：名为链球菌突变的细菌通过生活在牙齿上的粘性层（生物膜）中，从而引起空腔，然后用饮食中的糖酸破坏牙齿搪瓷。淀粉样蛋白是形成纤维的蛋白质的聚集体。我们表明，突变链球菌的生物膜形成涉及微生物建造的淀粉样蛋白原纤维。因此，我们将研究该过程的发生方式以及如何抑制它。