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

 描述(申请人提供)：在美国，肠道沙门氏菌是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其他一些底物，抗生素底物专一性很高。缺乏MacAB的突变体在动物模型中是无毒的，这表明在感染中发挥了重要作用。MacAB是一种ABC类型的MES，已知的是弱外排大环内酯类抗生素。在已发表的工作中，我们已经证明，MacAB通过分泌一种热稳定的化合物来解毒细胞外的活性氧物种(ROS)，从而保护STM免受过氧化氢介导的死亡。ROS是宿主中性粒细胞和巨噬细胞对STM和其他细菌感染做出反应时产生的大量抗微生物化合物。我们未发表的遗传学证据有力地表明，肠杆菌素的一个或多个线性代谢物(DHBS3、DHBS2、DHBs)可能是MacAB分泌的保护沙门氏菌抵抗ROS的天然抗氧化底物。不能产生DHBs三聚体、二聚体或单体的突变体在ROS暴露期间具有macAB突变体的表型，并且在共培养时不能将macAB突变体从过氧化氢介导的杀伤中拯救出来。虽然在大环内酯外排过程中，MacAB功能的外膜伙伴是TolC，但在氧化应激过程中，这种蛋白似乎不能与MacAB一起分泌抗氧化剂化合物。另外两个可能的外膜蛋白STM2172和STM2690在氧化应激过程中也具有macAB突变体的过氧化敏感性，当这些外膜蛋白一起缺失时，双突变体未能将macAB突变体从ROS介导的杀伤中拯救出来。为了从分子和机制上理解MacAB系统在H_2O_2抗性中的功能和组成，我们提出了两个特定目标的假说。首先，(目标I)我们假设肠杆菌素的线性产物(DHBS3，DHBS2，DHBs)是MacAB系统的底物，可以使过氧化氢解毒。其次，(目标II)我们假设STM2172和STM2690是完成MacAB装置的外膜组件，允许底物从细菌中输出。我们处于解决这些假设的独特地位，因为我们拥有完成我们提议的工作所需的关键初步数据、专业知识和工具。我们在MacAB上描述的研究，以及我们了解多种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：它们在这些生物的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，将为新型抗菌药物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 Our unpublished genetic evidence strongly suggest that one or more linear metabolite(s) of enterobactin (DHBS3, DHBS2, DHBS) are likely the natural antioxidant substrates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.