Studies of metal-dependent intercellular adhesion in Staphylococcal biofilms

葡萄球菌生物膜中金属依赖性细胞间粘附的研究

• 批准号: 8185263
• 负责人: ANDREW B HERR
• 金额: $ 29.27万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8185263
• 关键词: Acquired Immunodeficiency Syndrome; Address; Adhesions; Adhesives; Affect; American Medical Association; Amyloid; Amyloid fibers; Anabolism; Anti-Infective Agents; Antibiotic Resistance; Antibiotic Therapy; Bacteria; Bacterial Infections; Bacteriology; Biochemistry; Cause of Death; Cells; Cessation of life; Characteristics; Chemicals; Collaborations; Communities; Complex; Confocal Microscopy; Consensus; Copper; Cryoelectron Microscopy; Data; Development; Feasibility Studies; Future; Genetic; Genus staphylococcus; Goals; Grant; Growth; Hospitals; Immune response; Implant; Individual; Infection; Investigation; Ions; Journals; Laser Scanning Confocal Microscopy; Lead; Length; Measures; Mediating; Medical Device; Metals; Microbial Biofilms; Modeling; Molecular; Morbidity - disease rate; Morphology; Mutagenesis; Nature; Nosocomial Infections; Operon; Patients; Physiology; Polysaccharides; Protein Family; Proteins; Publishing; Reagent; Recurrence; Refractory; Relative (related person); Reporting; Research; Resistance; Roentgen Rays; Role; Screening procedure; Sepsis; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus epidermidis; Structure; Surface; Sushi Domain; Techniques; Testing; Therapeutic; United States; Work; X-Ray Crystallography; Zinc; amyloid formation; analytical ultracentrifugation; automobile accident; bacterial genetics; base; biophysical chemistry; cardiovascular infection; chelation; chemical stability; clinically relevant; cost; dimer; experience; in vivo; malignant breast neoplasm; methicillin resistant Staphylococcus aureus; mortality; multidisciplinary; novel strategies; prevent; protein oligomer; research study; small molecule; success

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. PUBLIC HEALTH RELEVANCE: Biofilms are communities of bacteria that attach to a surface and become highly resistant to antibiotics or immune responses; biofilms can cause recurrent, hard-to-treat infections, particularly with implanted medical devices. In this proposal we will investigate the details of how Staphylococcus bacterial cells stick to one another in a biofilm, and how they become resistant to physical or chemical challenges. The results from this research will provide new approaches for preventing biofilm formation or potentially reversing pre-formed biofilms.

描述（由申请人提供）：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，生物膜是粘附在基质上并形成微菌落的细菌群落，通常被多糖基质包围。由生物膜引起的反复细菌感染对抗生素治疗和宿主免疫反应均无效，因此通常必须通过手术去除。最近的工作表明单个蛋白质家族参与了葡萄球菌生物膜的形成。这些蛋白质在表皮葡萄球菌中称为 Aap，在金黄色葡萄球菌中称为 SasG，在 B 重复区域中含有多达 17 个串联 G5 结构域重复，这些重复对于葡萄球菌生物膜的形成是必要且充分的。我们已经证明，G5 结构域充当锌依赖性粘附模块，触发生物膜中葡萄球菌细胞之间形成“锌拉链”粘附结构。培养基中的锌螯合可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。我们已经解析了一个短共有 B 重复构建体的晶体结构，揭示了高度拉长的“褶带”折叠，该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 重复构建体，以匹配体内生物膜形成所需的长度。在锌或铜存在的情况下，这些构建体形成具有淀粉样蛋白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。除了与锌和铜复合的短共有重复序列的晶体结构外，我们还将通过技术组合确定较长 B 重复序列构建体的结构。最后，我们将分析 Aap 在培养的葡萄球菌生物膜形成中的作用，并将其功能与负责生物膜多糖生物合成的 icaADBC 操纵子的功能进行比较。我们强大的已发表的初步数据、我们开发的试剂、我们在解决短一致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增强了这些研究的可行性。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，以及与冷冻电子显微镜、淀粉样蛋白形成和分析超速离心领域主要领导者的互动。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检验我们的假设。相关性：医院获得性感染 (HAI) 是美国第四大死因，每年导致超过 100,000 人死亡。近三分之二的 HAI 是由葡萄球菌引起的。葡萄球菌形成生物膜的倾向会导致复发性、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且广泛有效的方法来防止生物膜形成。 公共卫生相关性：生物膜是附着在表面上并对抗生素或免疫反应具有高度抵抗力的细菌群落；生物膜会导致复发性、难以治疗的感染，尤其是植入的医疗设备。在这项提案中，我们将研究葡萄球菌细菌细胞如何在生物膜中相互粘附，以及它们如何对物理或化学挑战产生抵抗力。这项研究的结果将为防止生物膜形成或潜在逆转预先形成的生物膜提供新方法。