Studies of metal-dependent intercellular adhesion in Staphylococcal biofilms

描述（申请人提供）：近三分之二的医院获得性感染（HAIs）是由表皮葡萄球菌和金黄色葡萄球菌引起的。这些葡萄球菌特别麻烦，因为它们倾向于形成生物膜，生物膜是附着在基质上并形成微菌落的细菌群落，通常被多糖基质包围。由生物膜引起的复发性细菌感染对抗生素治疗和宿主免疫反应是难治性的，因此通常必须通过手术切除。最近的研究表明，在葡萄球菌生物膜的形成过程中存在一个单一的蛋白家族。这些蛋白在表皮葡萄球菌中被称为Aap，在金黄色葡萄球菌中被称为SasG，它们在b重复区含有多达17个串联G5结构域重复序列，这是葡萄球菌生物膜形成所必需的和充分的。我们已经证明G5结构域作为锌依赖的粘附模块，在生物膜中触发葡萄球菌细胞之间形成“锌拉链”粘附结构。培养基中锌的螯合抑制表皮葡萄球菌和金黄色葡萄球菌（包括几种MRSA菌株）的生物膜形成。我们已经解决了短共识b重复结构的晶体结构，揭示了一个高度拉长的“褶皱带”折叠，在锌存在下形成反平行二聚体。我们现在正在研究更长的b重复结构，以匹配体内生物膜形成所需的长度。在锌或铜的存在下，这些结构形成具有淀粉样特征的纤维聚集体。我们将定义葡萄球菌生物膜中aap介导的细胞间粘附和淀粉样蛋白形成的分子机制。除了短共识重复与锌和铜络合的晶体结构，我们将确定较长的b重复结构通过技术的组合。最后，我们将分析Aap在培养葡萄球菌生物膜形成中的作用，并将其与负责生物膜多糖生物合成的icaADBC操纵子的功能进行比较。这些研究的可行性被我们强有力的已发表和初步数据，我们开发的试剂，我们在解决短共识重复晶体结构方面的成功，以及我们在生物物理和晶体学方法方面的专业知识所增强。我们还将受益于与细菌遗传学，生物化学和生物膜专家Dan Hassett博士的密切合作，以及与低温电子显微镜，淀粉样蛋白形成和分析性超离心方面的关键领导者的互动。这项资助的跨学科性质将使我们能够通过实验生物物理方法以及临床相关的生物膜实验来测试我们的假设。相关性：医院获得性感染（HAIs）是美国第四大死亡原因，每年导致超过100,000人死亡。葡萄球菌引起了近三分之二的艾滋病。葡萄球菌形成生物膜的倾向导致复发性、顽固性感染。这些研究将有助于更好地理解生物膜形成的主要机制，并将有助于开发廉价和广泛有效的方法来防止生物膜的形成。

DESCRIPTION (provided by applicant): Nearly two-thirds of all hospital-acquired infections (HAIs) are caused by Staphylococcus epidermidis and S. aureus. These Staphylococci are particularly troublesome due to their propensity to form biofilms, which are communities of bacteria that adhere to a substrate and form microcolonies, typically surrounded by a polysaccharide matrix. Recurrent bacterial infections caused by biofilms are refractory to antibiotic treatment and host immune responses and thus must often be surgically removed. Recent work has implicated a single protein family in the formation of staphylococcal biofilms. These proteins, called Aap in S. epidermidis and SasG in S. aureus, contain up to 17 tandem G5 domain repeats in the B-repeat region that are necessary and sufficient for the formation of staphylococcal biofilms. We have shown that G5 domains act as zinc-dependent adhesion modules that trigger formation of a "zinc zipper" adhesive structure between staphylococcal cells in biofilms. Chelation of zinc from the media inhibits biofilm formation by both S. epidermidis and S. aureus, including several MRSA strains. We have solved the crystal structure of a short consensus B-repeat construct, revealing a highly elongated "pleated ribbon" fold that forms an anti-parallel dimer in the presence of zinc. We are now studying longer B-repeat constructs that match the length required for biofilm formation in vivo. In the presence of zinc or copper, these constructs form fibrous aggregates with amyloid-like characteristics. We will define the molecular mechanism for Aap-mediated intercellular adhesion and amyloid formation in staphylococcal biofilms. In addition to crystal structures of the short consensus repeat complexed with zinc and copper, we will determine the structure of longer B-repeat constructs by a combination of techniques. Finally, we will analyze the role of Aap in the formation of cultured staphylococcal biofilms, and compare its function with that of the icaADBC operon responsible for biosynthesis of the biofilm polysaccharide. The feasibility of these studies is enhanced by our strong published and preliminary data, the reagents we have developed, our success in solving the crystal structure of the short consensus repeat, and our expertise in biophysical and crystallographic approaches. We will also benefit from a close collaboration with Dr. Dan Hassett, an expert in bacterial genetics, biochemistry, and biofilms, as well as interactions with key leaders in cryo-electron microscopy, amyloid formation, and analytical ultracentrifugation. The interdisciplinary nature of this grant will allow us to test our hypotheses by experimental biophysical approaches as well as clinically-relevant biofilm experiments. Relevance: Hospital-acquired infections (HAIs) are the fourth-leading cause of death in the United States, leading to over 100,000 deaths per year. Staphylococci cause nearly two-thirds of these HAIs. The propensity of Staphylococci to form biofilms leads to recurrent, recalcitrant infections. These studies will lead to a better understanding of a major mechanism for biofilm formation and will help aid the development of inexpensive and broadly effective approaches for preventing biofilm formation.