BIOGENESIS AND INHIBITION OF BIOFILM-ASSOCIATED BACTERIAL AMYLOID

描述（由申请人提供）：许多慢性/复发性人类感染性疾病状态被认为涉及细菌生物膜。这些包括尿路感染（UTI）， 慢性皮肤感染、中耳炎和囊性纤维化肺部感染。在每一种情况下，细菌群落形成，包裹在细胞外基质中，在那里它们免受宿主免疫机制和目前使用的抗生素的作用。这极大地使这些生物体的清除和治愈这些感染的能力复杂化。被称为卷曲的淀粉样纤维在尿路致病性大肠杆菌（UPEC）形成生物膜细胞外基质中至关重要。这种纤维由主要亚基CsgA和次要亚基CsgB组成。利用硫磺素T荧光光谱、圆二色光谱和原子力显微镜研究了CsgB与CsgA体外成核作用的机理。在这些研究中使用的方法将作为一个模型，检查curli系统的其他组件之间的相互作用。在体内，卷曲淀粉样纤维的形成是由特定的伴侣蛋白样蛋白（CsgE和CsgF）在细胞外发生在外膜中的CsgG组装位点。这些蛋白质还用于限制过早的细胞内淀粉样蛋白纤维化，否则淀粉样蛋白纤维化将是有毒的。在这个建议中，我们利用生物物理，生物化学，结构，遗传学和化学遗传学的方法相结合，以阐明的结构，功能和作用机制的伴侣样蛋白CsgE，CsgF和外膜组装蛋白CsgG在促进CsgB-模板化的CsgA。这些研究将阐明卷曲形成和更普遍的淀粉样蛋白形成的基本原理。然后，使用噻唑环稠合的2-吡啶酮支架，我们将研究卷曲组装和卷曲介导的生物膜形成的小分子抑制剂（“curlicides”）的作用机制。Curlicides将被用作分子手术刀， curli生物发生所需的蛋白质间相互作用。Curli促进生物和非生物表面定殖，稳定细胞-细胞接触，允许细胞聚集和生物膜层增厚，并赋予生物膜对环境压力和杀生物剂的抗性。因此，将在体外和导管相关尿路感染的鼠模型中测试curlicides在预防硅胶导管管路上形成UPEC生物膜方面的功效。这项工作将为医学上重要的生物膜形成所必需的过程提供关键见解，并将作为更好地了解神经退行性疾病中重要的淀粉样蛋白斑块的模型。

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.