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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

基本信息

批准号：
10190957
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
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10190957
关键词：
Address Adhesions Adhesives Adopted Amyloid Amyloid Fibrils Antibiotic Resistance Back Bacteria Biological Assay Biophysics Blood Vessels 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 Structure Surface Techniques Temperature Testing Therapeutic Time United States Variant Work Zinc acute care amyloid fibril formation amyloid formation antimicrobial base biophysical analysis chelation chronic infection cohesion cross-species transmission environmental stressor experience genetic manipulation healthcare-associated infections insight medical implant molecular modeling mortality mutant novel strategies polymicrobial biofilm prevent protein B protein folding self assembly single molecule tool

项目摘要

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.

本研究旨在明确表皮葡萄球菌和链球菌感染的机制。金黄色葡萄球菌单物种和混合物种生物膜，重点是细胞壁锚定蛋白Aap和SasG。这该应用程序建立在以前资助的R 01项目的基础上，在该项目中，我们定义了Zn 2 +- 介导的二聚化的粘附B-重复区的Aap和决定因素的稳定性，这种不寻常的蛋白质折叠我们还表明Aap包含两种具有不同组装和稳定性的B重复亚型特征，使我们能够破译葡萄球菌生物膜中细胞间粘附的“组装密码”。重要的是，我们已经证明Aap是一种多功能粘附蛋白。在它的全长，自动- 抑制形式，介导对宿主细胞的粘附，但在蛋白水解加工后，抑制作用被释放，细胞间粘附区未被掩蔽。AAP有两种组装模式：可逆组装模式低聚化（类似于我们的晶体结构中观察到的）和淀粉样原纤维的形成，能抵抗环境压力。此外，最近的报道表明，Aap能够异嗜性与其他生物膜蛋白如小碱性蛋白（SBP）和来自S.金黄色， SasG我们证明了S.表皮葡萄球菌和表皮葡萄球菌。金黄色葡萄球菌可以形成坚固的、协同的混合物种生物膜这对许多疾病状态有重要意义。该应用程序的目标是广泛地表征涉及Aap的可逆自组装模式和异嗜性相互作用; Aap组装成功能性淀粉样蛋白状态;以及控制Aap组装的自抑制机制。在新生生物膜中宿主附着和细胞间粘附之间切换。我们正在与一个在葡萄球菌遗传学领域的领导者，在S.表皮细胞面我们将使用这些表达Aap变体的菌株来明确地测试以下的相对贡献：生物膜生长中可逆的Aap组装和淀粉样纤维形成，以及嗜异性蛋白的作用涉及SBP和SasG的组装事件在单物种和混合物种生物膜的形成。我们将在一种新的流动池装置中在低剪切和高剪切条件下测试每种菌株，以模拟生物膜，在血管或导管中形成。相关性：医疗相关感染（HAI）是患者发病和死亡的主要原因; a 最近的CDC报告估计HAI在美国每年造成75，000人死亡。葡萄球菌是 HAI中最常见的感染因子。葡萄球菌形成生物膜的倾向对抗生素有抗性的表面粘附菌落导致复发性的、难以治疗的感染。的拟议的研究将提供关于葡萄球菌细胞如何在细胞内相互锚定的见解。生物膜和援助的抗菌治疗的靶向方法的发展。