STUDIES OF METAL-DEPENDENT INTERCELLULAR ADHESION IN STAPHYLOCOCCAL BIOFILMS

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。这个 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 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“组装代码”。 重要的是，我们已经证明 Aap 是一种多功能粘附蛋白。在其完整的、自动的 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 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 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，例如小碱性蛋白 (SBP) 和金黄色葡萄球菌的 Aap 直向同源物， 萨斯G。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 这对许多疾病状态具有重要影响。该应用程序的目标是广泛 表征涉及 Aap 的可逆自组装模式和异亲性相互作用；不可逆转的 将 Aap 组装成功能性淀粉样蛋白状态；以及控制的自动抑制机制 新生生物膜中宿主附着和细胞间粘附之间的切换。我们正在与一个合作 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 表面。我们将使用这些表达 Aap 变体的菌株来明确测试 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 在单物种和混合物种生物膜形成中的组装事件。我们 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；一个 CDC 最近的报告估计，医院感染每年在美国导致 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——专门 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。的 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。

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.