Functional Amyloid Formation in Streptococcus mutans

描述(申请人提供)：龋齿是世界上最常见的传染病，很大程度上是由革兰氏阳性细菌变形链球菌引起的。相关的每年医疗保健费用高达数百亿美元，而美国儿童龋齿的发病率正在上升。显然，迫切需要解决这一未得到满足的卫生保健需求，并确定新的方法来阻止龋病的发生。导致龋齿的微生物形成顽固的生物膜，从饮食中的糖中产酸，并耐受酸的最终产物。最近人们认识到，微生物可以产生对生物膜发育不可或缺的功能性淀粉样蛋白。在这个方案中，我们证明了变形链球菌细胞表面定位的粘附素P1(抗原I/II，PAC)是一种淀粉样蛋白形成蛋白。这一结论是基于对淀粉样蛋白的性质的定义，包括对淀粉样染料刚果红和硫代黄素T的吸收，用透射电子显微镜观察淀粉样纤维，以及在交叉偏振光下观察刚果红色染色的蛋白质聚集体的绿色双折射特性。淀粉样蛋白存在于人类牙菌斑中，由实验室菌株和临床分离株产生。我们提供了进一步的证据表明淀粉样蛋白的形成并不局限于P1，因为没有这种粘附素的细菌菌落显示出残留的绿色双折射。然而，缺乏索糖酶的变形链球菌，转肽酶是介导其底物与细胞壁肽聚糖(包括P1和其他5种蛋白质)共价连接的酶，当被刚果红染色时不双折射，也不形成生物膜。我们还表明，当变形链球菌在已知的淀粉样纤化抑制剂存在的情况下培养时，生物膜的形成被抑制。淀粉样蛋白代表一种进化上保守的交叉2页四级结构，其中2页横向自组装形成纤维。我们最近发现晶体P1显示了一种独特的三级结构，其中两个球形的2-夹心结构域位于扩展的杂交α-多聚脯氨酸II螺旋的两侧。在这项研究中，我们将继续对P1淀粉样蛋白的发生进行生物物理确认，包括其X射线衍射谱，并利用新的三级结构信息，利用固体核磁共振、电子顺磁共振和冷冻电子显微镜方法来阐明P1‘S的超微结构，特别是与其淀粉样蛋白形成特性相关的超微结构(目标1)。在目标2中，我们将使用与P1类似的方法来鉴定和表征其他变形链球菌的淀粉样蛋白形成蛋白。编码淀粉样蛋白形成蛋白的基因将被单独和组合删除。在目标3中，我们将使用静态和流动模型来评估淀粉样蛋白的形成与生物膜的发育，并通过评估已知的淀粉样抑制剂对野生型和突变株变形链球菌生物膜形成的影响来评估淀粉样蛋白的形成。然后，我们将使用目标1中描述的生物物理分析来验证我们的结果。这些目标将揭示淀粉样蛋白形成对变形链球菌生物学的贡献，并论证淀粉样蛋白抑制作为防止这种病原体生物膜形成的一种治疗方法的可行性。

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.