N-terminal acylation of Lipoproteins in Firmicutes

项目摘要 该应用程序的长期目标是了解细菌脂蛋白在医学上为什么以及如何 重要厚壁菌门经历结构修饰。脂蛋白是膜相关蛋白 通过酰化的N-末端半胱氨酸锚栓系到细菌表面。它们是无处不在的细胞 革兰氏阳性菌和革兰氏阴性菌的包膜结构，在几乎所有方面都起着关键作用 细菌细胞被膜生理学由于其功能的重要性，丰富，普遍分布， 结构独特的酰化N-末端半胱氨酸，先天免疫系统通过结合 使用Toll样受体2（TLR 2）家族的脂蛋白的N-末端。TLR 2激活触发亲- 炎症细胞因子/趋化因子对清除细菌应答以及协调体液免疫。 然而，有新的证据表明，来自厚壁菌门的脂蛋白的酰化模式不是 典型的TLR 2配体，它们的结构也不是静态的。厚壁菌合成脂蛋白化学型， 酰基链的数量，长度和连接位置，使用辅助脂蛋白生物合成基因的阵列。 基因分布在属和种水平上都不同，甚至在菌株水平上也不同， 循环质粒/转座子编码的脂蛋白重塑基因。脂蛋白成分也是 受生长环境的动态调节，包括已显示诱导表达的铜 某些N端修饰基因的基因该项目旨在揭示不同的生理需求 脂蛋白N-末端修饰系统，并表征所涉及的酶。同时选择脂蛋白 结构修饰赋予宿主相关细菌、脂蛋白N-末端 在环境谱系中也发现了酰化/乙酰化，这表明了广泛的选择压力。给定 铜共诱导脂蛋白N-末端修饰和铜抗性基因， 铜直接与游离的N-末端结合，除非N-修饰，否则会抑制细胞生长。该项目将 通过测量生长，研究N-末端脂蛋白修饰对铜结合的影响。 需要特定脂蛋白功能的铜挑战条件，使用Tn-seq比较化学型特异性 铜致敏基因网络，并测定氧化损伤的模型脂肽分离， 活跃呼吸细胞和体外反应条件下。在第二个目标中，最近发现的 将重构来自金黄色葡萄球菌的脂蛋白N-酰化酶LnsAB。定向突变， 酰基链供体和可能的蛋白质-蛋白质复合物的形成将使用遗传和生物化学方法进行检查。 接近。该项目的最后一部分将研究如何利用遗传学方法制造N-乙酰化脂蛋白。 转座子筛选和生化分析，以及脂蛋白乙酰化对关键表型的影响。 通过了解脂蛋白的合成，该项目将揭示脂蛋白之间复杂的相互作用， 化学型，铜暴露，TLR 2识别，进而是控制宿主-厚壁菌门相互作用的关键轴。

Project Summary The long term goal of the application is to understand why and how bacterial lipoproteins in the medically important Firmicutes phylum undergo structural modifications. Lipoproteins are membrane associated proteins tethered to the bacterial surface through an acylated N-terminal cysteine anchor. They are ubiquitous cell envelope structures in both gram-positive and gram-negative bacteria, playing key roles in nearly every aspect of bacterial cell envelope physiology. Due to their functional importance, abundance, universal distribution, and the structurally unique acylated N-terminal cysteine, the innate immune system detects bacteria by binding the N-terminus of lipoproteins using the Toll-like receptor 2 (TLR2) family. TLR2 activation triggers a pro- inflammatory cytokine/chemokine response to clear bacteria as well as to orchestrate humoral immunity. However, there is emerging evidence that acylation patterns in lipoproteins from the Firmicutes phylum are not canonical TLR2 ligands nor are they static in structure. Firmicutes synthesize lipoprotein chemotypes varying in acyl chain number, length, and attachment position using an array of accessory lipoprotein biosynthetic genes. Gene distribution varies at both the genera and species level, and can even differ at the strain level due to circulating plasmid/transposon encoded lipoprotein remodeling genes. Lipoprotein composition is also dynamically regulated by the growth environment, including copper which has been shown to induce expression of certain N-terminal modifying genes. This project aims to uncover the physiological need(s) for distinct lipoprotein N-terminal modification systems and to characterize the enzymes involved. While select lipoprotein structural modifications confer TLR2 evading capabilities in host associated bacteria, lipoprotein N-terminal acylation/acetylation is also found in environmental lineages which suggests a broad selective pressure. Given copper co-induces both lipoprotein N-terminal modification and copper resistance genes, it has been proposed that copper directly binds to the free N-terminus and inhibits cell growth unless N-modified. This project will investigate the impact of N-terminal lipoprotein modifications on copper binding by measuring growth under copper challenge conditions requiring specific lipoprotein functions, using Tn-seq to compare chemotype-specific copper sensitization gene networks, and assaying oxidative damage to model lipopeptides isolated from both actively respiring cells and under in vitro reaction conditions. In the second aim, the recently discovered lipoprotein N-acylating enzymes LnsAB from Staphylococcus aureus will be reconstituted. Targeted mutations, acyl chain donor, and possible protein-protein complex formation will be examined using genetic and biochemical approaches. The final part of the project will examine how N-acetylated lipoproteins are made using genetic transposon screens and biochemical assays, and what impact lipoprotein acetylation has on key phenotypes. By understanding lipoprotein synthesis, the project will shed light on the complex interplay between lipoprotein chemotype, copper exposure, TLR2 recognition, and in turn a critical axis governing host-Firmicutes interactions.