Molecular and Structural Bases of Polymyxin Resistance

描述(由申请人提供)：对多种抗生素具有耐药性的细菌病原体的不断分离要求开发新的治疗策略。这不仅需要识别抗性决定因素，还需要了解调控这些决定因素表达的环境线索。沙门氏菌PmrA/PmrB双组分系统对多肽抗生素多粘菌素B和来自人多形核白细胞的几种抗菌蛋白具有耐药性。调节蛋白PmrA控制内毒素(LPS)与4-氨基阿拉伯糖(导致多粘菌素抗性)和磷乙醇胺(其意义尚不清楚)修饰的蛋白质的表达。我们已经证实：(1)pmrB蛋白是一种感受器，通过激活PmrA蛋白来响应细胞外Fe3+的水平；(2)低浓度的镁离子也可以在PmrD蛋白介导的过程中激活PmrA蛋白；(3)pmrA或pmrB基因缺陷的突变体对Fe3+的杀伤高度敏感。这项建议描述了旨在了解多个环境线索激活PmrA/PmrB双组分系统以促进对不同化合物的抗药性的一连串事件的实验。我们将研究低pH激活PmrA蛋白的分子机制；研究PmrD蛋白如何在转录后水平激活PmrA蛋白；识别PmrA调节的决定因素，介导对多粘菌素和Fe3+的抗性；并确定PmrA控制的磷乙醇胺修饰内毒素的生理作用。这些目标的实现将揭示细菌信号转导和抗菌肽耐药性的分子基础。此外，由于目前正在开发各种多肽和蛋白质作为新型抗菌剂，拟议的实验可能有助于我们了解这些化合物如何发挥其杀菌性能。

DESCRIPTION (provided by applicant): The unremitting isolation of bacterial pathogens exhibiting resistance to multiple antibiotics demands the development of novel therapeutic strategies. This entails not only identifying resistance determinants but also understanding the environmental cues that regulate expression of such determinants. The Salmonella PmrA/PmrB two-component system is required for resistance to the peptide antibiotic polymyxin B and to several antimicrobial proteins from human polymorphonuclear leukocytes. The regulatory protein PmrA governs expression of proteins mediating modifications of the lipopolysaccharide (LPS) with 4-aminoarabinose, which confers polymyxin resistance, and with phosphoethanolamine, the significance of which has remained unknown. We have established that: (1) the PmrB protein is a sensor that responds to extracellular levels of Fe 3+ by activating the PmrA protein, (2) low Mg2+ can also activate the PmrA protein in a process mediated by the PmrD protein, and (3) mutants defective in the pmrA or pmrB genes are hypersensitive to killing by Fe3+. This proposal describes experiments aimed at understanding the cascade of events by which multiple environmental cues activate the PmrA/PmrB two-component system to promote resistance to different compounds. We will examine the molecular mechanism by which low pH activates the PmrA protein; investigate how the PmrD protein can activate the PmrA protein at a posttranscriptional level; identify the PmrA-regulated determinants mediating resistance to polymyxin and to Fe3+; and define the physiological role of PmrA-controlled phosphoethanolamine modification of the LPS. An accomplishment of these goals will uncover the molecular bases for bacterial signal transduction and antimicrobial peptide resistance. Moreover, as a variety of peptides and proteins are currently being developed as novel antimicrobial agents, the proposed experiments may help in our understanding of how these compounds exert their microbicidal properties.