STUDIES OF METAL-DEPENDENT INTERCELLULAR ADHESION IN STAPHYLOCOCCAL BIOFILMS

这项建议旨在定义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，重点是细胞壁锚定蛋白AAP和SasG。这 应用程序建立在之前资助的R01项目的基础上，在该项目中，我们定义了锌的结构基础- AAP粘附性B-重复区域的介导性二聚化及其稳定性的决定因素 蛋白质折叠。我们还发现AAP包含两种不同的B-重复亚型，它们具有明显的组装和稳定性 特性，使我们能够破译葡萄球菌生物膜细胞间黏附的“汇编代码”。 重要的是，我们已经证明了AAP是一种多功能的黏附蛋白。它的全长，自动- 抑制形式，它介导与宿主细胞的黏附，但在蛋白分解处理后，抑制被释放并 细胞间粘附区未被遮盖。AAP支持两种组装模式：可逆 齐聚(类似于我们在晶体结构中观察到的)和淀粉样纤维的形成 能抵抗环境压力。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，如小碱性蛋白(SBP)和金黄色葡萄球菌的AAP同源基因， 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。 这对许多疾病状态都有重要影响。此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亲相互作用；不可逆 将AAP组装成功能淀粉样蛋白状态；以及控制AAP的自身抑制机制 在新生生物膜中，在宿主附着和细胞间黏附之间切换。我们正在与一家 葡萄球菌遗传学领域的领先者在表皮葡萄球菌细胞上表达全长AAP变体 浮出水面。我们将使用这些表达AAP变体的菌株来明确测试 AAP的可逆组装和淀粉样原纤维在生物膜生长中的形成以及异嗜性的作用 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。我们 将在一种新的流动细胞装置中测试每种菌株在低剪切和高剪切条件下的情况，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)是患者发病率和死亡率的主要原因；a 美国疾病控制与预防中心最近的一份报告估计，禽流感每年在美国造成7.5万人死亡。葡萄球菌是 HAI中最常见的感染性因素。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附着菌落会导致复发的、难以治疗的感染。这个 拟议的研究将为葡萄球菌细胞如何在体内相互锚定提供见解 生物被膜和协助开发有针对性的抗菌治疗方法。

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.