Functional amyloid formation in streptococcus mutans

摘要 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菌引起的 变形链球菌。相关的年度医疗保健成本高达数百亿美元， 美国的儿童期龋齿正在上升。解决这一未得到满足的医疗保健需求显然是当务之急 找出阻止龋病发生的新方法。导致龋齿的生物体形成顽固的生物膜， 从饮食中的糖中产生酸，并耐受酸的最终产物。而淀粉样蛋白最早是在 在病理学背景下，它并不总是代表蛋白质的错误折叠途径。功能性淀粉样蛋白现在 被认可了。淀粉样蛋白是一种进化上保守的纤维状交叉β-Sheet四级结构 β-Sheet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征 性能，包括淀粉样染料的上染率，用透射电子观察时的典型直径 显微镜，以及刚果红染色并在交叉偏振光下观察时的双折射特性。 目前已知有几种微生物可以产生生物膜所必需的功能性淀粉样蛋白。 发展。我们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。我们 现在已经扩展了这项工作，在这种细菌中总共鉴定了三种淀粉样蛋白形成蛋白。我们有 提供了广泛的粘附素P1的三级和四级结构表征，并确定了其 羧基末端C123截断衍生物作为淀粉样变性部分。我们还鉴定了变形链球菌 WAPA和以前未被鉴定的蛋白SMU_63c作为淀粉样蛋白形成蛋白。与P1一样，WAPA也是一个 表面定位的索酸酶底物，其截断导数AGA是淀粉样变性的产物。SMU_63c是一种分泌型 对遗传能力和生物膜细胞密度起负调节作用的蛋白质。这三个都是 蛋白质有助于变形链球菌生物膜的形成，而变形链球菌的生物被膜可以被小分子抑制剂抑制。 淀粉样纤维化。缺乏编码淀粉样蛋白基因的变形链球菌的生物膜发育 形成蛋白质明显不太容易受到以淀粉样蛋白为靶点的化合物的抑制 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。在此续订申请中，我们将使用最先进的方法，包括溶液和固体 状态核磁共振，以鉴定和表征潜在的淀粉样蛋白纤化的结构转变 变形链球菌淀粉样蛋白形成蛋白(目标1)。这将使我们能够跟踪它们的结构进程 从单体到生物膜内的淀粉样蛋白，并将揭示抑制化合物的作用机制。我们会 还开发和优化了区分变形链球菌淀粉样蛋白单体和纤维形式的方法 在体外和生物膜内形成蛋白质(目标2)。最后，我们将应用这些检测方法来评估 并确定了调节蛋白单体向淀粉样蛋白转化的相关环境因素。 变种生物膜(目标3)，这一过程到目前为止还没有在任何研究系统中得到很好的理解。

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.