STUDIES OF METAL-DEPENDENT INTERCELLULAR ADHESION IN STAPHYLOCOCCAL BIOFILMS

金黄色葡萄球菌生物膜中金属依赖性细胞间粘附的研究

• 批准号: 9769766
• 负责人: ANDREW B HERR
• 金额: $ 33.91万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 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 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9769766
• 关键词: Acute; Address; Adhesions; Adhesives; Adopted; Amyloid; Amyloid Fibrils; Antibiotic Resistance; Back; Bacteria; Biological Assay; Biophysics; Blood Vessels; Caring; Catheters; Cell Wall; Cell surface; Cell-Matrix Junction; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Characteristics; Code; Communities; Crystallization; Data; Development; Dimerization; Disease; Engineering; Event; Family; Funding; Genetic; Genus staphylococcus; Goals; Growth; Hospitals; Immune response; Incidence; Infection; Interdisciplinary Study; Laboratories; Lectin; Length; Mediating; Medical Device; Membrane Proteins; Metals; Microbial Biofilms; Modeling; Molecular; Morbidity - disease rate; N-terminal; Orthologous Gene; Pathogenesis; Patients; Peptides; Physiological; Positioning Attribute; Progress Reports; Proteins; Proteolytic Processing; Publishing; Recurrence; Reporting; Research; Research Personnel; Research Project Grants; Resistance; Role; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus epidermidis; Stress; Structure; Surface; Techniques; Temperature; Testing; Therapeutic; Time; United States; Variant; Work; Zinc; amyloid fibril formation; amyloid formation; antimicrobial; base; biophysical analysis; chelation; chronic infection; cohesion; experience; healthcare-associated infections; insight; medical implant; molecular modeling; mortality; mutant; novel strategies; polymicrobial biofilm; prevent; protein B; protein folding; self assembly; single molecule; tool

This proposal aims to define the mechanisms by which Staphylococcus epidermidis and S. aureus form both mono-species and mixed-species biofilms, focusing on the cell wall-anchored proteins Aap and SasG. This application builds on the previously funded R01 project in which we defined the structural basis for Zn2+- mediated dimerization of the adhesive B-repeat region of Aap and the determinants for stability of this unusual protein fold. We also showed that Aap contains two B-repeat subtypes with distinct assembly and stability characteristics, allowing us to decipher an `assembly code' for intercellular adhesion in staphylococcal biofilms. Importantly, we have demonstrated that Aap is a multi-functional adhesion protein. In its full-length, auto- inhibited form, it mediates adhesion to host cells, but after proteolytic processing, the inhibition is released and the intercellular adhesion region is unmasked. Aap is capable of two assembly modes: reversible oligomerization (similar to that observed in our crystal structures) and formation of amyloid-like fibrils that are resistant to environmental stresses. Furthermore, recent reports indicate that Aap is capable of heterophilic interactions with other biofilm proteins such as small basic protein (SBP) and the Aap ortholog from S. aureus, SasG. We have shown that S. epidermidis and S. aureus can form robust, synergistic mixed-species biofilms that have important implications for a number of disease states. The goal of this application is to broadly characterize the reversible self-assembly modes and heterophilic interactions involving Aap; the irreversible assembly of Aap into the functional amyloid state; and the mechanism for auto-inhibition that governs the switch between host attachment and intercellular adhesion in the nascent biofilm. We are collaborating with a leader in the field of staphylococcal genetics to express full-length Aap variants on the S. epidermidis cell surface. We will use these strains expressing Aap variants to explicitly test the relative contribution of reversible Aap assembly and amyloid fibril formation in biofilm growth, as well as the role of heterophilic assembly events involving SBP and SasG in the formation of mono-species and mixed-species biofilms. We will test each strain under low- and high-shear conditions in a new flow cell apparatus to mimic biofilms that form in blood vessels or catheters. Relevance: Healthcare-associated infections (HAIs) are a major cause of patient morbidity and mortality; a recent CDC report estimated that HAIs cause 75,000 deaths annually in the United States. Staphylococci are the most common infective agents in HAIs. The propensity of Staphylococci to form biofilms—specialized surface-adherent colonies that are resistant to antibiotics—leads to recurrent, hard-to-treat infections. The proposed research will provide insights into how staphylococcal cells are anchored to one another in the biofilm and aid in the development of targeted approaches for antimicrobial therapy.

该建议旨在定义葡萄球菌表皮和金黄色葡萄球菌形成的机制 单物种和混合物种生物膜，重点是细胞壁锚定的蛋白AAP和SASG。这 申请基于先前资助的R01项目，在该项目中，我们定义了Zn2+ - 的结构基础。 AAP的粘合剂B重复区域的介导二聚化和此不寻常的稳定性的决定剂 蛋白质折叠。我们还表明，AAP包含两个具有不同组装和稳定性的B-重复子类型 特征，使我们能够解读葡萄球菌生物膜中细胞间粘附的“装配代码”。 重要的是，我们已经证明AAP是一种多功能粘合剂蛋白。以其全长的自动 抑制形式，它介导了对宿主细胞的粘附，但是在蛋白水解处理后，释放抑制作用，并 细胞间粘附区域是未掩盖的。 AAP能够采用两种装配模式：可逆 寡聚（类似于在我们的晶体结构中观察到的）和形成的淀粉样蛋白样纤维 抵抗环境应力。此外，最近的报道表明AAP能够异嗜能力 与其他生物膜蛋白的相互作用，例如小碱性蛋白（SBP），以及来自金黄色葡萄球菌的AAP直系同源物， sasg。我们已经表明表皮链球菌和金黄色葡萄球菌可以形成坚固的协同混合物种生物膜 这对许多疾病状态具有重要意义。该应用的目的是广泛 表征可逆的自组装模式和涉及AAP的异质相互作用；不可逆的 AAP组装到功能性淀粉样蛋白状态；以及控制自动抑制的机制 在新生生物膜中的宿主附着和细胞间粘附之间切换。我们正在与 葡萄球菌遗传学领域的领导者在表皮链球菌细胞上表达全长AAP变体 表面。我们将使用表达AAP变体的这些菌株明确测试 可逆的AAP组装和淀粉样纤维生长中的淀粉样纤维形成，以及异性的作用 涉及SBP和SASG形成单种和混合物种生物膜的组装事件。我们 将在新流动池中的低剪切条件下测试每个菌株，以模拟生物膜 在血管或导管中形成。 相关性：与医疗保健相关的感染（HAI）是患者发病率和死亡率的主要原因； CDC最近的报告估计，HAIS每年在美国造成75,000人死亡。葡萄球菌是 Hais中最常见的感染剂。葡萄球菌形成生物膜的承诺 - 特有 对抗生素具有抗性的表面粘附菌落 - 鉴于反复进行的，难以治疗的感染。这 拟议的研究将提供有关如何将葡萄球菌细胞固定在彼此中的见解 生物膜并有助于开发抗菌治疗的靶向方法。