BIOGENESIS AND INHIBITION OF BIOFILM-ASSOCIATED BACTERIAL AMYLOID

描述(由申请人提供)：许多慢性/复发性人类传染病状态被认为涉及细菌生物膜。这些疾病包括尿路感染(UTI)、 慢性皮肤感染、中耳炎和囊性纤维化肺部感染。在每个细菌群落中，细菌群落被包裹在细胞外基质中，在那里它们不受宿主免疫机制和当前使用的抗生素的作用的影响。这极大地增加了清除这些微生物和治愈这些感染的能力。被称为卷曲的淀粉样纤维在致尿性大肠杆菌(UPEC)形成生物膜细胞外基质的过程中起着至关重要的作用。该纤维由主亚基CsgA和次亚基CsgB组成。用硫代黄素T荧光、圆二色谱和原子力显微镜研究了CsgB在体外成核CsgA的机理。这些研究中使用的方法将作为检验Curli系统其他组件之间相互作用的模型。在体内，卷曲淀粉样纤维的形成是由特定的伴侣样蛋白(CsgE和CsgF)指导的，发生在外膜的CsgG组装部位。这些蛋白质还用来限制过早的细胞内淀粉样纤维化，否则这将是有毒的。在这项建议中，我们结合生物物理、生化、结构、遗传和化学遗传学方法来阐明伴侣蛋白CsgE、CsgF和外膜组装蛋白CsgG在促进CsgA的CsgB模板化过程中的结构、功能和作用机制。这些研究将阐明卷曲形成的基本原理，更广泛地讲，淀粉样蛋白的形成。然后，我们将使用噻唑环融合的2-吡啶酮支架来研究小分子抑制物(“花环化合物”)对卷曲组装和卷曲介导的生物被膜形成的作用机制。花环菌将被用作分子手术刀来解剖 卷曲生物发生所需的蛋白质-蛋白质相互作用。Curli促进生物和非生物表面的定植，稳定细胞与细胞的接触，允许细胞聚集和生物膜层增厚，并使生物膜对环境压力和杀菌剂具有抵抗力。因此，将在体外和导管相关尿路感染的小鼠模型中测试花环素在防止硅胶导尿管上UPEC生物膜形成方面的有效性。这项工作将为形成具有重要医学意义的生物膜所需的过程提供关键的见解，并将成为更好地了解在神经退行性疾病中重要的淀粉样斑块的模型。

DESCRIPTION (provided by applicant): Many chronic/recurrent human infectious disease states are recognized to involve bacterial biofilms. These include urinary tract infections (UTIs), chronic skin infections, otitis media and cystic fibrosis lung infections. In each, bacterial communities form, encased in an extracellular matrix where they are shielded from host immune mechanisms and from the action of currently used antibiotics. This greatly complicates the clearance of these organisms and the ability to cure these infections. Amyloid fibers called curli are critical in the formation of a biofilm extracellular matrix by uropathogenic Escherichia coli (UPEC). This fiber is made up of the major subunit CsgA, and a minor subunit CsgB. Using Thioflavin T fluorescence, circular dichroism and atomic force microscopy the in vitro mechanism by which CsgB nucleates CsgA fibrillization has been elucidated. The methods used in these studies will serve as a model for examining interactions between other components of the curli system. In vivo, the formation of curli amyloid fibers is directed by specific chaperone-like proteins (CsgE and CsgF) to occur extracellularly at the CsgG assembly site in the outer membrane. These proteins also serve to restrict premature, intracellular amyloid fibrillization, which otherwise would be toxic. In this proposal we utilize a combination of biophysical, biochemical, structural, genetic and chemical genetic methodologies to elucidate the structure, function and mechanism of action of the chaperone-like proteins CsgE, CsgF and the outer membrane assembly protein CsgG in facilitating the CsgB-templated fibrillization of CsgA. These studies will elucidate basic principles of curli formation and more generally amyloid formation. Then, using a thiazole ring-fused 2-pyridone scaffold we will investigate the mechanism of action of small molecule inhibitors ("curlicides") of curli assembly and curli-mediated biofilm formation. Curlicides will be used as molecular scalpels to dissect the details of the protein-protein interactions required for curli biogenesis. Curli promote biotic and abiotic surface colonization, stabilize cell-cell contacts allowing cell aggregation and thickening of the biofilm layer and confer resistance to the biofilm against environmental stresses and biocides. Thus, curlicides will be tested for their efficacy in preventing the formation of UPEC biofilms on silicone catheter tubing both in vitro and in a murine model of catheter- associated urinary tract infection. This work will provide key insights into processes necessary for the formation of medically important biofilms and will serve as a model to better understand amyloid plaques important in neurodegenerative disorders.