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: 8496082
• 负责人: ANDREW B HERR
• 金额: $ 28.21万
• 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/8496082
• 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; 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; screening; 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.

描述(申请人提供)：近三分之二的医院获得性感染(HAI)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，因为它们有形成生物膜的倾向，生物膜是附着在底物上并形成微菌落的细菌群落，通常被多糖基质包围。由生物被膜引起的反复细菌感染对抗生素治疗和宿主免疫反应是难治的，因此必须经常手术切除。最近的研究表明，葡萄球菌生物膜的形成与单一蛋白家族有关。这些蛋白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-重复区域包含多达17个串联的G5结构域重复序列，这是形成葡萄球菌生物膜所必需的，也是足够的。我们已经证明，G5结构域作为锌依赖的黏附模块，触发生物膜中葡萄球菌细胞之间形成“锌拉链”黏附结构。培养液中锌的螯合作用抑制了表皮葡萄球菌和金黄色葡萄球菌的生物膜形成，包括几种MRSA菌株。我们已经解决了一个短的共识B-重复结构的晶体结构，揭示了一个高度拉长的“褶皱带状”折叠，在锌存在下形成一个反平行的二聚体。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。在锌或铜存在的情况下，这些结构形成具有淀粉样特征的纤维聚集体。我们将确定AAP介导的细胞间黏附和葡萄球菌生物膜中淀粉样蛋白形成的分子机制。除了与锌和铜络合的短共识重复结构的晶体结构外，我们还将通过组合技术来确定较长B-重复结构的结构。最后，我们将分析AAP在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。这些研究的可行性由于我们强大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的晶体结构方面的成功以及我们在生物物理和结晶学方法方面的专业知识而得到加强。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，以及与冷冻电子显微镜、淀粉样蛋白形成和分析超速离心法方面的主要领导者的互动。这笔赠款的跨学科性质将使我们能够通过实验生物物理方法以及与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)是美国第四大死亡原因，每年导致超过10万人死亡。其中近三分之二是由葡萄球菌引起的。葡萄球菌形成生物膜的倾向导致反复、顽固性感染。这些研究将有助于更好地了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预防生物膜形成的方法。