Defining the molecular mechanisms of MacAB in protection of Salmonella from oxidative stress

 描述（由申请方提供）：肠道沙门氏菌是美国细菌性食源性肠炎的主要原因，也是食源性疾病导致死亡的主要原因。鼠伤寒沙门氏菌（STm）编码至少11个多药外排系统（MES），已知从细胞中输出抗生素和一些其他底物，对抗生素具有高度冗余的底物特异性。缺乏MacAB的突变体，已知弱外排大环内酯类抗生素的ABC型MES，在动物模型中是无毒的，表明在感染中的重要作用。在已发表的工作中，我们已经表明，MacAB通过分泌一种热稳定化合物来解毒细胞外活性氧（ROS），从而保护STm免受过氧化物介导的杀伤。ROS是由宿主嗜中性粒细胞和巨噬细胞响应于STm和其他细菌感染而大量产生的抗微生物化合物。我们未发表的遗传证据强烈表明，肠杆菌素的一种或多种线性代谢产物（DHBS 3，DHBS 2，DHBS）可能是MacAB分泌的天然抗氧化剂底物，可保护沙门氏菌免受ROS的侵害。不能产生DHBS三聚体、二聚体或单体的突变体在ROS暴露期间共享macAB突变体的表型，并且当共培养时不能从过氧化物介导的杀伤中拯救macAB突变体。虽然MacAB功能的外膜伴侣在大环内酯外排是TolC，这种蛋白质似乎没有功能与MacAB分泌的抗氧化剂化合物在氧化应激。另外两个推定的外膜蛋白，STM2172和STM2690，共享过氧化氢敏感性的macAB突变体在氧化应激过程中，当这些外膜蛋白一起删除，和双突变体未能拯救macAB突变体从ROS介导的杀伤。为了发展的功能和组成部分的MacAB系统在H2O2抗性的分子和机制的理解，我们已经开发了两个假设，在两个特定的目标。首先，（目的I）我们假设肠杆菌素的线性产物（DHBS 3，DHBS 2，DHBS）是MacAB系统的底物，可以解毒过氧化物。第二，（目的II）我们假设STM 2172和STM 2690是完成MacAB装置的外膜组分，允许将底物输出到细菌外。我们在解决这些假设方面具有独特的优势，因为我们拥有完成我们提出的工作所需的关键初步数据、专业知识和工具。我们在这里描述的关于MacAB的研究，以及我们了解多药物外排系统的自然功能的长期目标，解决了我们理解细菌外排泵的一个关键空白：它们在这些生物体生物学中的自然作用。最后，对MacAB功能的详细了解，以及更广泛的MES功能，将为新型抗菌治疗的开发提供新的靶点。

 DESCRIPTION (provided by applicant): Salmonella enterica is the leading cause of bacterial food borne enteritis and the leading cause of death from food borne disease in the United States. Salmonella Typhimurium (STm) encodes at least 11 multidrug efflux systems (MES), known to export antibiotics and a few other substrates from the cell, with high redundancy in substrate specificity for antibiotics. Mutants lacking MacAB, an ABC-type MES known to weakly efflux macrolide antibiotics, are avirulent in animal models suggesting an important role in infection. In published work we have shown that MacAB protects STm from peroxide mediated killing by secreting a heat stable compound that detoxifies extracellular reactive oxygen species (ROS). ROS are antimicrobial compounds produced in abundance by host neutrophils and macrophages in response to STm and other bacterial infections. Our unpublished genetic evidence strongly suggest that one or more linear metabolite(s) of enterobactin (DHBS3, DHBS2, DHBS) are likely the natural antioxidant substrates secreted by MacAB that protect Salmonella against ROS. Mutants that cannot produce DHBS trimers, dimers or monomers share the phenotypes of a macAB mutant during ROS exposure, and cannot rescue the macAB mutant from peroxide mediated killing when co-cultured. Although the outer membrane partner for MacAB function during macrolide efflux is TolC, this protein does not appear to function with MacAB for secretion of antioxidant compounds during oxidative stress. Two other putative outer membrane proteins, STM2172 and STM2690, share peroxide sensitivity of macAB mutants during oxidative stress when these outer membrane proteins are deleted together, and the double mutant fails to rescue a macAB mutant from ROS mediated killing. In order to develop a molecular and mechanistic understanding of the function and components of the MacAB system in H2O2 resistance, we have developed two hypotheses addressed in two specific aims. First, (Aim I) we hypothesize that linear products of enterobactin (DHBS3, DHBS2, DHBS) are the substrates of the MacAB system that detoxify peroxide. Second, (Aim II) we hypothesize that STM2172 and STM2690 are the outer membrane components that complete the MacAB apparatus allowing export of the substrate out of the bacterium. We are uniquely positioned to address these hypotheses because we possess critical preliminary data, expertise and the tools necessary to complete the work we propose. Our studies described here on MacAB, and our long-term goal to understand the natural functions of multi-drug efflux systems, address a key gap in our understanding bacterial efflux pumps: their natural role in the biology of these organisms. Finally, a detailed understanding of MacAB function, and MES function more broadly, will provide new targets for the development of novel antimicrobial therapy.