Functional amyloid formation in streptococcus mutans

摘要 龋病是世界上最常见的传染病，在很大程度上是由革兰氏阳性菌引起的。 变形链球菌相关的年度医疗保健费用数百亿美元， 儿童龋齿在美国S.正在上升。显然，必须解决这一未得到满足的卫生保健需求， 确定干龋发病机制的新方法。导致蛀牙的生物体会形成反柠檬酸生物膜， 从膳食糖中产生酸，并耐受酸性终产物。虽然淀粉样蛋白首次被发现是在 在病理学背景下，它并不总是代表蛋白质错误折叠途径。功能性淀粉样蛋白现在 认可.淀粉样蛋白是一种进化上保守的纤维状、交叉β折叠四级结构， 其中β片层横向自组装形成纤维。淀粉样蛋白聚集体具有共同的生物物理特性， 特性，包括对淀粉样蛋白染料的吸收，通过透射电子显微镜观察时的典型直径 显微镜下观察，以及当用刚果红染色并在交叉偏振光下观察时的双折射性质。 现在已知几种微生物产生功能性淀粉样蛋白，其是生物膜不可或缺的 发展我们的小组是第一个确定变形链球菌是一种淀粉样蛋白形成生物。我们 现在已经将这项工作扩展到在这种细菌中鉴定出总共三种淀粉样蛋白形成蛋白。我们有 提供了粘附素P1的广泛的三级和四级结构表征，并鉴定了其 羧基末端C123截短衍生物作为淀粉样蛋白生成部分。我们还确定了S。变形 WapA和以前未表征的蛋白Smu_63c作为淀粉样蛋白形成蛋白。与P1一样，WapA是一个 表面定位分选酶底物，其截短衍生物阿加是淀粉样蛋白生成的。Smu_63c是一种分泌型 作为遗传能力和生物膜细胞密度的负调节剂的蛋白质。所有这三 蛋白质对S.变形杆菌生物膜的形成，这可以通过小分子的 淀粉样纤维化生物膜开发由S.缺乏编码淀粉样蛋白基因的变异株 形成蛋白质对靶向淀粉样蛋白的化合物的抑制作用明显不敏感， 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 病原体在此更新申请中，我们将利用最先进的方法，包括溶液和固体 状态NMR，以鉴定和表征淀粉样蛋白纤维化的结构转变。 淀粉样蛋白形成蛋白。变异株（Aim 1）。这将使我们能够跟踪它们的结构进展， 单体转化为淀粉样蛋白，并将揭示抑制性化合物的作用机制。我们将 还开发和优化方法，以区分单体和纤维形式的S。变形淀粉样蛋白 在体外和生物膜内形成蛋白质（目的2）。最后，我们将应用这些检测方法来评估 并确定相关的环境触发，调节蛋白单体淀粉样蛋白的转换在S。 变形杆菌生物膜（目标3），这是一个过程，尚未很好地了解任何系统研究的日期。

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.