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

项目摘要：猖獗的抗生素耐药性预计将对世界健康造成毁灭性影响 接下来的三十年。单纯性感染的治疗因细菌而变得越来越复杂 对已知的抗生素具有抗药性。抗药性对医疗保健的影响因缺乏新颖性而加剧 抗生素的发展。特别是，革兰氏阴性细菌越来越引起人们的关注。全部革兰氏阴性 细菌有一个外膜(OM)，它的存在使新抗生素的发现变得复杂， 成为治疗的障碍，使许多现有的抗生素失效。OM，它是由 在革兰氏阴性细菌中，有几个分子机器是必不可少的。每台OM生物发生机器都需要 至少有一种脂蛋白能够正常发挥作用。许多脂蛋白是毒力因子，其中几种是必不可少的 细胞过程。因此，革兰氏阴性细菌必须克服运输脂蛋白的挑战。 从穿过房水周质的内膜(IM)到OM。LOL系统转运脂蛋白 向OM致敬。LolCDE从IM中去除脂蛋白。Lola是一种周质伴侣，它从 LolCDE并通过周质运输它们，在那里它们被LOLB插入OM。令人惊讶的是，我的 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的情况下运输表明，必须存在另一种脂蛋白运输途径。这 这一发现挑战了目前脂蛋白运输的范式，就像之前认为的Lol系统一样 脂蛋白到达OM的唯一机制。因此，我假设一种替代的脂蛋白 运输途径将脂蛋白输送到OM。使用我实验室的ΔLolab菌株的大肠杆菌，我是独一无二的 定位于识别和定义脂蛋白运输的替代路径。我会用生化和基因 测试我的假设有两个目的。在目标1中，我将描述LolCDE与替换物的相互作用 脂蛋白的运输途径。尽管在没有Lolab的情况下，脂蛋白仍然被运输到OM 我的实验室已经证实，LolCDE对于脂蛋白运输的所有途径都是绝对必要的。这就做 突变LolCDE中的残基，并测试突变体在交替脂蛋白运输途径中的功能。我 然后将测试药物敏感性，以评估LolC突变体的OM通透性。OM生化机提纯 膜分离将被用来测试突变体运输脂蛋白的能力。在《目标2》中，我会 识别和表征对交替脂蛋白运输途径重要的基因。我会用一个不带偏见的 全球筛选转座子突变体以鉴定这些基因。然后我会用OM生化机提纯 以及膜分离，以分类对替代途径重要的基因。这两个目标加在一起将 填补了我们对革兰氏阴性细菌中脂蛋白运输的理解上的一个空白。由于……的重要性 脂蛋白及其向OM的转运，这项研究将提供重要的见解，将支持未来 针对OM生物发生的治疗药物的发现。

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.