MOLECULAR AND FUNCTIONAL ANALYSES OF THE PMRA/PMRB REGULATORY SYSTEM

描述(申请人提供)：革兰氏阴性菌的最外层是脂多糖(即脂多糖)。这种分子构成了一种通透性屏障，保护细菌免受各种有害物质的伤害，它被不同动物的宿主固有免疫系统识别，并被几种天然抗菌肽靶向。许多革兰氏阴性病原菌已经进化出一种机制来修改它们的内毒素，以减少宿主的识别并增加对抗菌肽和某些有毒金属的抵抗力。在伤寒和胃肠炎的病原体肠沙门氏菌中，许多内毒素的修饰只有在激活PmrA/pmrB调节系统的条件下才能观察到。本提案描述了旨在了解如何动态控制PmrA/PmrB系统的活动以响应内部和外部输入的实验。我们的研究将集中在一种同时编码小肽和小RNA的新基因，以及一组在巨噬细胞内生存所需的沙门氏菌特异性基因。此外，我们还将分析PmrD蛋白在沙门氏菌中发挥的不同特性，在沙门氏菌中，它的功能是激活PmrA蛋白，而在相关物种中，它缺乏这一能力。我们将检查当大肠杆菌在促进沙门氏菌中这种激活的诱导条件下进行PmrA控制的脂多糖修饰时，共生大肠杆菌在小鼠肠道的定植能力是否受到损害。这些目标的实现将揭示PmrA/pmrB系统-肠道细菌内毒素修饰的主要调节因子-如何控制其细胞表面的重塑，以保护其免受抗菌素的侮辱。

DESCRIPTION (provided by applicant): The outermost part of Gram-negative bacteria is the lipopolysaccharide (i.e. LPS). This molecule constitutes a permeability barrier that protects bacteria from a variety of noxious agents, and it is recognized by the host innate immune system of different animals and targeted by several natural antimicrobial peptides. Many Gram-negative pathogens have evolved mechanisms to modify their LPS in ways that decrease recognition by the host and increase resistance to antimicrobial peptides and certain toxic metals. In Salmonella enterica, which is the etiologic agent of typhoid fever and gastroenteritis, many of these LPS modifications are observed only under conditions that activate the PmrA/PmrB regulatory system. This proposal describes experiments aimed at understanding how the activity of the PmrA/PmrB system is dynamically controlled in response to internal and external inputs. Our studies will focus on a novel gene that encodes both a small peptide and small RNA, and on a set of Salmonella-specific genes that are required for survival inside macrophages. In addition, we will analyze the distinct properties that the PmrD protein plays in Salmonella, where it functions to activate the PmrA protein, and in the related species Escherichia coli, where it lacks this ability. We will examine whether the ability of commensal E. coli to colonize the mouse intestine is compromised when E. coli carries out the PmrA-controlled LPS modifications under inducing conditions that promote such activation in Salmonella. An accomplishment of these goals will uncover how the PmrA/PmrB system - a major regulator of LPS modifications in enteric bacteria - controls the remodeling of its cell surface in ways that protect it from antimicrobial insult.