Illuminating the essential role of the outer membrane component and drug target, lipopolysaccharide

革兰氏阴性外膜（OM）是不对称的，内叶上有磷脂， 脂多糖（LPS）在外小叶上。LPS是革兰氏阴性细胞包膜的一个决定性部分， 是动物感染的主要信号，是最后使用抗生素（如粘菌素）的靶点。通常是必不可少的， 临床使用的粘菌素已选择了不产生LPS的鲍氏不动杆菌菌株。虽然LPS 虽然它与多种功能有关，但其在革兰氏阴性生物学中的确切作用尚不清楚。最近在E. 大肠杆菌的研究表明，LPS能够为细胞膜提供与细胞壁一样多的刚性，这种功能 需要LPS上的负部分和二价阳离子之间的多离子相互作用。条件分析 e. LPS合成和转运缺陷的大肠杆菌突变体揭示了细丝和细胞链的形成， 提示LPS在细胞分裂和分离中的额外作用。LPS在任一过程中的重要性是 不清楚 为了评估LPS在革兰氏阴性菌生物学中的重要作用，我将利用一组E。大肠杆菌突变体， 有条件地敲低LPS合成和转运，操纵LPS电荷（从而操纵其相互作用 具有二价阳离子以提供刚性），并产生最小的LPS结构。在目标1中使用此集合， 将系统地表征LPS缺陷对细胞生长和形态的影响，以了解 LPS对这两种现象的贡献。在目标2中，我将测试我的假设，即提供细胞包膜刚性 是LPS的主要基本功能。目的2.1将评价高渗条件下（ 减少由膨压施加在细胞包膜上的力）以补偿LPS缺陷。因为 膨压力在细胞壁和OM之间传播，目标2.2将测试是否增加刚度 通过细胞壁交联的细胞被膜可以补偿LPS缺陷。在目标3中，我将确定步骤 在细胞分裂和分离中分别受到LPS合成和转运缺陷的影响。这一努力将 阐明了LPS突变体中双链化和链化的机制基础。A的检验。鲍曼不 LPS缺失突变体确定了分裂和细胞存活之间的相关性。因此我特别感兴趣的是 在测试使用互补遗传策略增强分裂是否促进LPS的生长时， 缺陷型E.杆菌如果增强细胞膜的刚性是LPS的重要作用，那么额外的隔膜可能会抵消 通过对细胞整体结构完整性的积极影响，与LPS缺陷相关的致死性。 免疫系统的主要信号分子和抗生素的最后目标，更好地了解 LPS在革兰氏阴性生物学中的重要作用将为减轻和治疗革兰氏阴性菌提供见解。 阴性病原体。通过这个F31奖学金，我将发展在显微镜，蛋白质生物化学， 和单细胞分析。此外，它将给予我必要的时间和支持，以磨练我的技能， 科学导师和沟通者，为我作为教授和独立调查员的职业生涯做好准备。

The gram-negative outer membrane (OM) is asymmetric, with phospholipids on the inner leaflet and lipopolysaccharide (LPS) on the outer leaflet. A defining part of the gram-negative cell envelope, LPS is a major signal for infection in animals and is a target of last resort antibiotics, such as colistin. Normally essential, clinical use of colistin has selected for Acinetobacter baumannii strains that do not produce LPS. Although LPS has been implicated in diverse functions, its precise role in gram-negative biology is unclear. Recent work in E. coli suggests LPS is capable of contributing as much rigidity to the cell envelope as the cell wall, a function that requires polyionic interactions between negative moieties on LPS and divalent cations. Analysis of conditional E. coli mutants defective in LPS synthesis and transport revealed formation of filaments and cell chains, suggesting an additional role for LPS in cell division and separation. The essentiality of LPS in either process is unclear. To evaluate the essential role(s) of LPS in gram-negative biology, I will leverage a set of E. coli mutants to conditionally knockdown LPS synthesis and transport, manipulate LPS charge (and thereby its interactions with divalent cations to provide rigidity), and produce a minimal LPS structure. Using this collection in Aim 1, I will systematically characterize the effect(s) of LPS defects on cell growth and morphology to understand the contribution of LPS to both phenomena. In Aim 2, I will test my hypothesis that providing cell envelope rigidity is a primary, essential function of LPS. Aim 2.1 will evaluate the ability of hyperosmotic conditions (which reduce the force exerted by turgor pressure on the cell envelope) to compensate for LPS defects. Because the force of turgor pressure is spread between the cell wall and the OM, Aim 2.2 will test whether increasing rigidity of the cell envelope via cell wall crosslinking can compensate for LPS defects. In Aim 3, I will identify the steps in cell division and separation impacted by defects in LPS synthesis and transport, respectively. This effort will illuminate the mechanistic basis of filamentation and chaining in LPS mutants. Examination of A. baumannii LPS deletion mutants identified a correlation between division and cell survival. I am thus particularly interested in testing whether enhancing division using complementary genetic strategies promotes growth of LPS deficient E. coli. If enhancing cell envelope rigidity is an essential role of LPS, additional septa may offset lethality associated with LPS defects through a positive impact on the structural integrity of the cell as a whole. A major signaling molecule for the immune system and target for last resort antibiotics, a better understanding of the essential role of LPS in gram-negative biology will provide insights into mitigation and treatment of gram- negative pathogens. Through this F31 fellowship, I will develop expertise in microscopy, protein biochemistry, and single cell analysis. Further, it will grant me the time and support necessary to hone my skills as a scientific mentor and communicator to prepare me for a career as a professor and independent investigator.