Functional Amyloid Formation in Streptococcus mutans

描述（由申请人提供）：龋齿是世界上最常见的传染病，大部分由革兰氏阳性细菌变形链球菌引起。相关的年度医疗保健费用高达数百亿美元，美国儿童龋齿的发病率正在上升。有一个明确的当务之急，以解决这一未满足的医疗保健需求，并确定新的方法，干龋齿发病机制。导致蛀牙的生物体形成柠檬酸生物膜，从膳食糖中产生酸，并耐受酸的最终产物。最近认识到微生物可以产生功能性淀粉样蛋白，其是生物膜发育不可或缺的。在这个建议中，我们表明，S。称为P1（抗原I/II，PAc）的变形细胞表面定位的粘附素是淀粉样蛋白形成蛋白。这一结论是基于定义的淀粉样蛋白的性质，包括吸收的淀粉样蛋白染料刚果红和硫磺素T，可视化的淀粉样蛋白纤维的透射电子显微镜，和绿色双折射特性的刚果红染色的蛋白质聚集体时，在交叉偏振光下观察。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，淀粉样蛋白的形成并不限于P1，因为没有这种粘附素的细菌菌落显示残留的绿色双折射。然而，S.缺乏分选酶的变形杆菌，介导其底物与细胞壁肽聚糖共价连接的转肽酶，包括P1和五种其他蛋白质，当用刚果红染色时不双折射，并且不形成生物膜。我们还表明，生物膜的形成受到抑制时，S。在已知的淀粉样蛋白纤维化抑制剂的存在下培养变形蛋白。淀粉样蛋白代表进化上保守的纤维状、交叉2-片四级结构，其中2-片横向自组装以形成纤维。我们最近表明，结晶P1展示了一个独特的三级结构，其中两个球状的2-夹心结构域位于任何一方的一个扩展的混合α-聚脯氨酸II螺旋。在这项研究中，我们将继续生物物理确认P1淀粉样蛋白的形成，包括其X-射线衍射图案，并利用新的三级结构信息，利用固态NMR，电子顺磁共振（EPR），和冷冻电子显微镜方法来阐明P1的超微结构，特别是有关其淀粉样蛋白形成的特性（目标1）。在目标2中，我们将鉴定并表征S.使用与P1相似的方法对变异株进行了检测。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评估淀粉样蛋白形成与生物膜发展的关系，并通过评估已知的淀粉样蛋白抑制剂对S。通过野生型和突变菌株的变形菌生物膜形成。然后，我们将使用目标1中描述的生物物理测定来确认我们的结果。这些目的将共同揭示淀粉样蛋白形成对S.变形杆菌生物学，并证明淀粉样蛋白抑制作为预防该病原体形成生物膜的治疗方法的可行性。

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.