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：
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
项目类别：
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

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.

该提案旨在明确表皮葡萄球菌和金黄色葡萄球菌形成的机制单物种和混合物种生物膜，重点关注细胞壁锚定蛋白 Aap 和 SasG。该应用程序建立在之前资助的 R01 项目的基础上，在该项目中我们定义了 Zn2+- 的结构基础 Aap 粘合 B 重复区域介导的二聚化以及这种不寻常稳定性的决定因素我们还表明 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“组装代码”。重要的是，我们已经证明 Aap 是一种全长、自动的多功能粘附蛋白。抑制形式，它介导与宿主细胞的粘附，但在蛋白水解加工后，抑制被释放并细胞间粘附区域未被掩盖，Aap 具有两种组装模式：可逆。寡聚化（类似于在我们的晶体结构中观察到的）和淀粉样原纤维的形成此外，最近的报告表明 Aap 具有异嗜性。与其他生物膜蛋白的相互作用，例如小碱性蛋白 (SBP) 和金黄色葡萄球菌的 Aap 直向同源物， SasG。我们已经证明表皮葡萄球菌和金黄色葡萄球菌可以形成强大的、协同的混合物种生物膜。对许多疾病状态具有重要意义。该应用的目标是广泛地。描述 Aap 的可逆自组装模式和异亲相互作用； Aap 组装成功能性淀粉样蛋白状态；以及控制 Aap 的自动抑制机制我们正在与新生生物膜中的宿主附着和细胞间粘附之间的切换。葡萄球菌遗传学领域的领导者，在表皮葡萄球菌细胞上表达全长 Aap 变体我们将使用这些表达 Aap 变体的菌株来明确测试 AAP 的相对贡献。可逆的 Aap 组装和淀粉样原纤维形成在生物膜生长中，以及异嗜性的作用涉及 SBP 和 SasG 在单一物种和混合物种生物膜形成中的组装事件。将在新的流动细胞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：医疗保健相关感染 (HAI) 是患者发病和死亡的主要原因； CDC 最近的报告估计，HAI 每年在美国导致 75,000 人死亡。 HAI 中最常见的感染因子葡萄球菌形成生物膜的倾向——专门化。对抗生素具有抗药性的表面粘附菌落会导致复发性、难以治疗的感染。拟议的研究将深入了解葡萄球菌细胞如何在生物膜并有助于开发有针对性的抗菌治疗方法。