Elucidating the mechanisms of transient polymyxin resistance in pathogenic E. coli.

确定内毒素修饰在不同的大肠杆菌致病类型中的调控差异 项目摘要 细胞遇到持续不断的细胞外提示，它们只使用有限数量的 信号转导通路编码在它们的基因组中。当我们理解个体如何发出信号时 转导系统运行，对不同的信号系统如何相互作用进行整合知之甚少 信息和/或扩展它们的信号响应。本项目的总体目标是了解信令如何 灵活性有利于不同的大肠杆菌菌株对阳离子多肽的反应。尽管很多人都在关注 越来越多的证据表明，肠杆菌科细菌获得了抗生素耐药性标记物 这种细菌还可以通过上调染色体编码而对抗生素产生暂时性耐药性。 记号笔。例如，沙门氏菌和大肠杆菌编码的pmrc基因是 对粘菌素类抗生素产生耐药性的MCR-1基因。我们最近证明了这种暂时性的 在用铁刺激后，对多粘菌素B产生耐药性的尿路致病大肠杆菌菌株。我们 随后发现，瞬时多粘菌素B抗性是通过激活pmrB而产生的 传感器激酶，是PmrAB双组分系统(TCS)的成员。细菌TCS包括一层膜- 嵌入的组氨酸激酶是信号受体，以及一种反应调节蛋白，它引导 相应的细胞变化。尽管有一些基于序列的决定因素决定了 同源TCS合作伙伴，我们发现PmrAB和QseBC TCS之间有很强的相互作用，其中 PmrB组氨酸激酶容易激活其同源伙伴PmrA和非同源反应调节器 QSEB对铁的反应，导致MIC增加了16倍。我假设协调监管 PmrA和QseB的缺失导致脂质A修饰的关键基因上调，从而保护细菌 从多粘菌素B和其他阳离子多肽的侮辱。我将在三个目标中检验这一假设，其中 我将：(1)定义响应多粘菌素B的PmrA和QseB调节子，并定义其作用机制 PmrA和QseB激活导致多粘菌素B耐药。(2)确定QseBC和PmrAB如何 相互作用已经进化到有利于细菌在不同的利基环境中的适应性，以及；(3)确定 不同系统发育分支的大肠杆菌QseBC-PmrAB信号级联中的保守性 并且有不同的致病策略。为了实现这些目标，将采取跨学科的方法， 包括分子生物学、全基因组转录分析和强大的小鼠感染模型。 结合这些研究，将提供一种机制的细节，使细菌能够存活在 最后的抗生素，并将提供洞察力的QseBC-PmrAB电路的保守不同 大肠埃希菌的致病型。

Defining Differences in how LPS modification is Regulated in Different E. coli pathotypes Project Summary Cells encounter a constant barrage of extracellular cues to which they respond using only the finite number of signal transduction pathways encoded within their genome. While we understand how individual signal transduction systems operate, little is known about how distinct signaling systems interact to integrate information and/or expand their signal responses. The overall goal of this project is to understand how signaling flexibility benefits the responses of different E. coli strains to cationic polypeptides. Although much attention is placed on the acquisition of antibiotic resistance markers by the Enterobacteriaceae, there is increasing evidence that bacteria can also mount transient resistance to antibiotics via the upregulation of chromosomally encoded markers. For example, the pmrC gene encoded by Salmonella spp and E. coli species is an orthologue of the mcr-1 gene that imparts resistance to colistin antibiotics. We have recently demonstrated that transient resistance to polymyxin B arises in strains of uropathogenic E. coli, following stimulation with ferric iron. We subsequently found that the transient polymyxin B resistance is brought about via the activation of the PmrB sensor kinase, a member of the PmrAB two-component system (TCS). Bacterial TCSs comprise a membrane- embedded histidine kinase that is the signal receptor, and a response regulator protein that directs the corresponding cellular changes. Although there are sequence-based determinants that dictate specificity among cognate TCS partners, we discovered strong interactions between the PmrAB and QseBC TCSs, in which the PmrB histidine kinase readily activates both its cognate partner PmrA and the non-cognate response regulator QseB in response to ferric iron, leading to a 16-fold increase in the MIC. I hypothesize that coordinated regulation of PmrA and QseB leads to upregulation of genes critical for lipid A modification that in turn protects bacteria from the insults of polymyxin B and other cationic polypeptides. I will test this hypothesis in three aims, in which I will: (1) Define the PmrA and QseB regulons in response to polymyxin B and define the mechanism by which PmrA and QseB activation leads to polymyxin B resistance. (2) Determine how the QseBC and PmrAB interactions have evolved to benefit bacterial fitness in different niches, and; (3) Ascertain how the amount of conservation present in the QseBC-PmrAB signaling cascade in E. coli strains from different phylogenetic clades and with different pathogenic strategies. Towards these goals, an inter-disciplinary approach will be followed, encompassing molecular biology, genome-wide analyses of transcription and robust murine models of infection. Combined these studies will provide mechanistic details into a mechanism that allows bacteria to survive one of the last resort antibiotics and will provide insights into the conservation of the QseBC- PmrAB circuitry in different E. coli pathotypes.