A novel transport pathway for outer membrane lipoproteins in Gram-negative bacteria

项目摘要：预计猖獗的抗生素耐药性将对世界卫生造成毁灭性影响， 下一个三十年。简单感染的治疗正变得越来越复杂的细菌 对已知抗生素有抗药性耐药性对医疗保健的影响因缺乏新的 抗生素开发特别是革兰氏阴性菌越来越受到关注。所有革兰氏阴性 细菌有一个外膜（OM），它的存在使新抗生素的发现变得复杂， 作为治疗剂的障碍并使许多现有的抗生素无效。OM，由 几种分子机器，在革兰氏阴性菌中是必不可少的。每台OM生物发生机需要 至少一种脂蛋白才能正常发挥作用许多脂蛋白都是毒力因子，其中一些是 细胞过程因此，革兰氏阴性菌必须应对转运脂蛋白的挑战 从内膜（IM）穿过水周质到达OM。Lol系统转运脂蛋白 到OM。LolCDE从IM中去除脂蛋白。LolA是一种周质分子伴侣， LolCDE并将它们运输穿过周质，然后通过LolB插入OM中。令人惊讶的是，我 实验室最近发现，在没有LolAB的情况下，脂蛋白仍然可以到达OM。功能性脂蛋白 在没有LolAB的情况下的转运证明必须存在脂蛋白转运的替代途径。这 这一发现挑战了目前脂蛋白转运的范式，因为以前认为Lol系统是 脂蛋白到达OM的唯一机制。因此，我假设一种替代性脂蛋白 转运途径将脂蛋白递送至OM。使用我实验室的大肠杆菌ΔlolAB菌株，我是唯一一个 定位以识别和定义脂蛋白运输的替代途径。我会用生物化学和遗传学 我的假设有两个目的。在目标1中，我将描述LolCDE与替代药物的相互作用。 脂蛋白转运途径。尽管在LolAB缺失的情况下脂蛋白仍被转运至OM， 在脂蛋白转运途径中，我的实验室已经证实LolCDE对脂蛋白转运的所有途径都是绝对必要的。我会 突变LolCDE中的残基并测试突变体在替代脂蛋白转运途径中的功能。我 然后将测试药物敏感性以评估LolC突变体中的OM渗透性。OM生物发生机纯化 膜分离将用于测试突变体转运脂蛋白的能力。在目标2中，我将 识别和表征对替代脂蛋白转运途径重要的基因。我会用一个公正的 转座子突变体的全球筛选，以确定这些基因。我会用OM生物发生机净化 和膜分离以分类对替代途径重要的基因。这两个目标合在一起， 填补了我们对革兰氏阴性菌中脂蛋白转运理解的空白。由于其重要性， 脂蛋白及其运输到OM，这项研究将提供重要的见解，将支持未来 发现了针对OM生物发生的疗法。

Project Summary: Rampant antibiotic resistance is predicted to cause devastating effects on world health over the next thirty years. The treatment of simple infections is becoming increasingly complicated by bacteria resistant to known antibiotics. The effects of resistance on healthcare are exacerbated by a lack of novel antibiotic development. In particular, Gram-negative bacteria are of increasing concern. All Gram-negative bacteria have an outer membrane (OM), the presence of which complicates the discovery of new antibiotics, acting as a barrier to therapeutics and rendering many current antibiotics useless. The OM, which is built by several molecular machines, is essential in Gram-negative bacteria. Each OM biogenesis machine requires at least one lipoprotein to function properly. Many lipoproteins are virulence factors, and several are essential to cellular processes. Gram-negative bacteria must, therefore, navigate the challenge of transporting lipoproteins from the inner membrane (IM) across the aqueous periplasm to the OM. The Lol system transports lipoproteins to the OM. LolCDE removes lipoproteins from the IM. LolA, a periplasmic chaperone, receives lipoproteins from LolCDE and transports them across the periplasm where they are inserted into the OM by LolB. Surprisingly, my lab recently found that lipoproteins can still reach the OM in the absence of LolAB. Functional lipoprotein transport in the absence of LolAB demonstrates that an alternate route of lipoprotein transport must exist. This finding challenges the current paradigm of lipoprotein transport, as the Lol system was previously thought to be the only mechanism by which lipoproteins reach the OM. Therefore, I hypothesize that an alternate lipoprotein transport pathway delivers lipoproteins to the OM. Using my lab’s ΔlolAB strain of Escherichia coli, I am uniquely positioned to identify and define alternate routes of lipoprotein transport. I will use biochemical and genetic assays to test my hypothesis in two aims. In Aim 1, I will characterize the interaction of LolCDE with the alternate route of lipoprotein transport. Although lipoproteins are still transported to the OM in the absence of the LolAB pathway, my lab has confirmed that LolCDE is absolutely essential to all pathways of lipoprotein transport. I will mutate residues in LolCDE and test the function of the mutants in the alternate lipoprotein transport pathway. I will then test drug sensitivity to assess OM permeability in LolC mutants. OM biogenesis machine purification and membrane fractionation will be used to test the ability of mutants to transport lipoproteins. In Aim 2, I will identify and characterize genes important to the alternate lipoprotein transport pathway. I will use an unbiased global screen of transposon mutants to identify these genes. I will then use OM biogenesis machine purification and membrane fractionation to classify genes important to the alternate pathway. Together, these two aims will close a gap in our understanding of lipoprotein transport in Gram-negative bacteria. Due to the essentiality of lipoproteins and their transport to the OM, this research will provide important insight that will support future discovery of therapeutics targeting OM biogenesis.