Synthesis and properties of a bacterial bioadhesive

描述（由申请人提供）：细菌通常利用多糖作为粘附结构附着于表面，形成生物膜，并感染宿主细胞。此外，多糖在人类活动的许多领域中作为生物粘合剂，包括作为牙科和外科粘合剂，具有很强的前景。新月柄杆菌能合成一种叫做固着剂的多糖，这种多糖具有令人印象深刻的粘附力。与大多数商业粘合剂相反，固着剂在淡水和海洋环境中紧密地粘附到各种表面。这种性质对于人体的医学应用至关重要。本研究的总体目标是使用从遗传学到生物物理学的多学科方法来研究holdfast属性的化学和生物物理基础，并了解holdfast属性是如何通过脱乙酰化和细胞外DNA（eDNA）的抑制来调节的。该项目有三个具体目标。第一个目的是确定holdfast的生物物理基础。原子力显微镜（AFM）将被用来系统地研究表面粗糙度，剪切应力，表面成分，离子强度，和pH值对固着附着力的影响，以提供固着附着力和附着力控制的机制更好地理解。第二个目的是确定脱乙酰化在固着锚定和粘合性能中的作用。固着多糖脱乙酰基酶突变体引起溶液中非粘附固着物的释放。将从正常和脱乙酰酶突变细胞中确定固着多糖的组成和结构。AFM力谱和高分辨率荧光显微镜将被用来确定脱乙酰化的作用上的固着力和凝聚力。生物化学实验将用于研究脱乙酰化在将固着物锚定到细胞中的作用。最后，对海洋物种的固着性的类似研究将为人体盐水环境中的潜在应用提供更好的生物材料。第三个目的是确定最近发现的eDNA抑制固着粘附机制的生物学基础。将研究毒素-抗毒素系统在程序性细胞死亡产生eDNA中的作用，并确定固着抑制的序列特异性的基础。AFM压痕研究和同步AFM成像和拉曼散射光谱的固着结合或不结合到eDNA将被用来确定如何特定的DNA改变固着的结构和结构特性。从拟议的研究结果将提供深入了解的基本机制，令人印象深刻的粘合性能的固着剂和调制这些属性，铺平了道路的固着剂作为一种生物粘合剂的未来发展。此外，这些研究的结果将提供以下见解： 多糖粘附的一般机制，以及抑制多糖粘附的策略，例如在病原体感染期间。 公共卫生相关性：细菌通常利用多糖作为粘附结构来附着于表面，形成生物膜，并感染宿主细胞，并且这些多糖在人类活动的许多领域中作为生物粘合剂具有很强的前景，包括作为牙科和外科粘合剂。我们将研究已知的最强的生物粘合剂之一，通过调查的化学和生物物理基础的固着特性和固着性的抑制剂的作用机制的固着性多糖的新月柄杆菌。这些研究将提供必要的数据，在各种应用中使用多糖的策略的设计，以及在细菌病原体感染过程中抑制多糖粘附的策略。

DESCRIPTION (provided by applicant): Bacteria often utilize polysaccharides as adhesive structures to attach to surfaces, to form biofilms, and to infect host cells. In addition, polysaccharides hold strong promise as biological adhesives in many areas of human activity, including as dental and surgical adhesives. The bacterium Caulobacter crescentus synthesizes a polysaccharide called the holdfast that exhibits and impressive adhesive force. Contrary to most commercial adhesives, holdfasts adhere tightly to a variety of surfaces in both freshwater and marine environments. Such a property is critical for medical applications in the human body. The general goal of this research is to use a multidisciplinary approach ranging from genetics to biophysics to study the chemical and biophysical basis for holdfast properties and to understand how holdfast properties are modulated by deacetylation and inhibition by extracellular DNA (eDNA). The project has three specific aims. The first aim is to determine the biophysical basis for holdfast adhesiveness. Atomic force microscopy (AFM) will be used to systematically study the influence of surface roughness, shear stress, surface composition, ionic strength, and pH on holdfast adhesion in order to provide a better understanding of the mechanism of holdfast adhesion and adhesion control. The second aim is to determine the role of deacetylation in holdfast anchoring and adhesive properties. A holdfast polysaccharide deacetylase mutant causes the release of non-adherent holdfast in solution. The composition and structure of the holdfast polysaccharide will be determined from normal and deacetylase mutant cells. AFM force spectroscopy and high-resolution fluorescence microscopy will be used to determine the role of deacetylation on holdfast adhesiveness and cohesiveness. Biochemical experiments will be used to study the role of deacetylation in anchoring the holdfast to the cell. Finally, similar studies of the holdfast of marine species will provide better biomaterials for potential applications in the saline environment of the human body. The third aim is to determine the biological basis for the recently discovered mechanism of eDNA inhibition of holdfast adherence. The role of a toxin-antitoxin system in the production of eDNA by programmed cell death will be studied and the basis for the sequence specificity of holdfast inhibition will be determined. AFM indentation studies and simultaneous AFM imaging and Raman scattering spectroscopy of holdfasts bound or not to eDNA will be used to determine how specific DNA alters the structure and structural properties of the holdfast. Results from the proposed studies will provide insight into the basic mechanisms for the impressive adhesive properties of the holdfast and modulation of these properties, paving the way for the future development of the holdfast as a biological adhesive. In addition, results of these studies will provide insights into the mechanism of polysaccharide adhesiveness in general, as well as for strategies to inhibit polysaccharide adhesion, for example during infection by pathogens. PUBLIC HEALTH RELEVANCE: Bacteria often utilize polysaccharides as adhesive structures to attach to surfaces, to form biofilms, and to infect host cells, and these polysaccharides hold strong promise as biological adhesives in many areas of human activity, including as dental and surgical adhesives. We will study one of the strongest known biological adhesives, the holdfast polysaccharide of Caulobacter crescentus by investigating the chemical and the biophysical basis for holdfast properties and the mechanism of action of a specific inhibitor of holdfast adhesiveness. These studies will provide data essential to the design of strategies to use polysaccharides in various applications, as well as strategies to inhibit polysaccharide adhesion during infection by bacterial pathogens.