Mechanisms for sensing and responding to interbacterial antagonism

项目摘要 越来越明显的是，在细菌界，物种间的拮抗是生命所固有的。和 然而，尽管我们对细菌使用的抗菌机制的理解与它们的同胞发生了冲突 最近呈指数级增长，我们对细菌感知和反应的方式的了解 对抗性威胁仍然有限。在这个提议中，我们检验了细菌对这种存在做出反应的假设。 通过激活多方面的防御程序来对抗细菌竞争者。这 假说源于我们的发现铜绿假单胞菌激活了广泛的转录后转录 针对细菌间拮抗反应的调控计划。这条路径，我们称之为Para (铜绿假单胞菌对拮抗的反应)，当铜绿假单胞菌的一个亚群细胞时触发 由于对抗攻击而屈服于裂解。细胞内未知分子(S)的检测 裂解产物激活小RNA介导的Gac/RSM全球转录后调控 程序。这种反应的激活对铜绿假单胞菌在攻击过程中的生存至关重要，因为突变株无法 在与拮抗生物体的竞争中，登山反应遭受了严重的适应缺陷。最后，我们 证明防御性反应需要多种同时作用的机制，包括 未知功能的通路。在本提案的目标1中，我们将描述一条这样的途径，我们 名称：ARC1(拮抗反应复合体1)。我们的初步数据表明，Arc1是一个很大的膜- 铜绿假单胞菌对毒素介导的拮抗作用的相关蛋白复合体 由竞争物种的VI型分泌系统提供。我们对Arc1的研究将阐明 它为其提供保护的威胁，并提供对其防御功能的机械性洞察。在……里面 目标2，我们将寻求互补的遗传和生化方法，以表征信号 存在于铜绿假单胞菌细胞裂解物中，负责触发PARA。最后，在目标3中，我们超越了P。 并询问副Gac/RSM途径背后的调控成分在多大程度上- 在其他假单胞菌中起到抵御细菌间拮抗作用的作用。我们也 检验Gac/RSM调节子的变异性反映了对特定细菌的适应的假设 不同物种所面临的威胁。通过这项工作，我们将回答长期以来在 领域，包括定义一个重要的全球监管方案在进化上的相关职能，以及 提供了一个长而难以捉摸的信号分子的分子特征。此外，通过 Arc1的表征，我们的工作将定义参与保守的膜的机制基础 在细菌间防御中功能未知的复合体。总的来说，拟议的工作将扩大我们的 了解细菌间的拮抗作用如何影响细菌进化的过程。

Project Summary It is increasingly evident that interspecies antagonism is intrinsic to life in the bacterial kingdom. And yet, while our understanding of the antibacterial mechanisms bacteria employ in conflicts with their brethren has recently grown exponentially, our knowledge of the means by which bacteria sense and respond to antagonistic threats remains limited. In this proposal, we test the hypothesis that bacteria react to the presence of an antagonistic bacterial competitor through the activation of a multifaceted defensive program. This hypothesis grew from our discovery that Pseudomonas aeruginosa activates an extensive posttranscriptional regulatory program in response to interbacterial antagonism. The pathway, which we term PARA (Pseudomonas aeruginosa response to antagonism), is triggered when a subpopulation of P. aeruginosa cells succumb to lysis as a result of an antagonistic attack. Detection of as yet unidentified molecule(s) in cellular lysate leads to the activation of the small RNA-mediated Gac/Rsm global posttranscriptional regulatory program. Activation of this response is crucial for P. aeruginosa survival during attack, as a mutant unable to mount the response suffers a severe fitness defect during competition with antagonistic organisms. Finally, we demonstrate that the defensive response requires multiple, simultaneously acting mechanisms, including pathways of unknown function. In Aim 1 of this proposal, we will characterize one such pathway, which we name ARC1 (antagonism response complex 1). Our preliminary data indicate that ARC1 is a large membrane- associated protein complex that provides P. aeruginosa protection against antagonism mediated by toxins delivered by the type VI secretion system of a competitor species. Our studies of ARC1 will elucidate the range of threats towards which it provides protection and provide mechanistic insight into its defensive functions. In Aim 2, we will pursue complementary genetic and biochemical approaches directed at characterizing the signal present in P. aeruginosa cellular lysate responsible for triggering PARA. Finally, in Aim 3, we move beyond P. aeruginosa and ask to what extent the regulatory components behind PARA – the Gac/Rsm pathway – function generally to defend against interbacterial antagonism in other Pseudomonas species. We also examine the hypothesis that variability in the Gac/Rsm regulon reflects adaptation to the specific bacterial threats encountered by different species. Through this work, we stand to answer longstanding questions in the field, including defining the evolutionarily relevant function of an important global regulatory program and providing a molecular characterization of a long elusive signaling molecule. Additionally, through the characterization of ARC1, our work will define the mechanistic basis for participation of a conserved membrane complex of unknown function in interbacterial defense. Overall, the proposed work stands to broaden our understanding of the ways in which interbacterial antagonism shapes the course of bacterial evolution.