A Genomics Approach to Drug Tolerance

描述(由申请人提供)：当前拨款的总体目标是识别导致形成休眠的、耐多药的持久基因的基因。这是一个具有挑战性的问题，也是在理解持之以恒的本质方面取得进展的主要障碍。这仍然是一项重要目标，并将在项目资助期结束时实现。有了持续体基因，我们计划在项目的后续阶段研究持续体形成的详细分子机制。然而，我们最近发现，持续体可以形成对DNA损伤的反应，并已确定毒素/抗毒素模块TisAB，显然会导致休眠，并是拟议的2年恢复法案竞争修订基金申请通知编号NOT-OD-09-058的主题。 这一发现为我们提供了一个有吸引力的机会来加快项目的速度，并在未来两年内描述持久细胞形成的第一个分子机制，而不是等到项目的下一个阶段。我们也没有计划研究持续性复苏，假设一旦我们知道了它们形成的机制，这将在未来的某个时候完成。TISB也为我们在今后两年解决这一问题提供了一个极好的机会。 在补编中，我们建议大大提前建立第一个详细的持久形成机制。拟议的研究将包括通过细胞分选分离依赖TISB的持久者并获得其转录组；详细研究TISB作用的分子机制；以及依赖TISB的持久者复苏的机制。就TISB而言，复苏机制耐人寻味。TISB没有可以对抗其作用的蛋白质抗毒素--抗毒素是一种只影响表达的RNA。TISB是一种膜作用肽，可破坏PMF并导致ATP下降。这将导致细胞内系统的全面关闭和多药耐受。在没有抗毒素的情况下，尚不清楚细胞是如何从如此深度休眠的状态中复苏的。筛选能够在异位表达TISB的菌株中生长的突变体将导致通过全基因组测序鉴定复苏基因。 除了发现持久的形成和复苏机制外，这项工作将首次将两种看似相反的细胞生存策略联系在一起--通过应激反应进行主动保护；以及休眠。从我们的研究中出现的新范式是，压力导致休眠，这一点将在拟议的补充研究中详细说明。这项研究将指出一个耐人寻味的可能性，即其他应激反应与持久形成之间存在联系。除了DNA损伤剂外，病原菌还暴露在宿主环境中的许多应激因素中-氧化剂、高温、低pH、膜作用剂。有可能所有的应激反应都会诱导形成一个小而有弹性的存活的持久者亚群。本研究将为研究应激反应在体内引起多药耐受中的作用奠定基础。一旦我们正确理解多药耐受的本质，这将指导我们努力寻找有效的应对措施。 与公共卫生相关：该项目还将通过雇用两名新的博士后研究员、向一名研究生提供研究助学金、购买资本设备以及将该项目的一些工作外包出去，来实现刺激经济的目标。

DESCRIPTION (provided by applicant): The overall goal of the current grant is to identify the genes leading to the formation of dormant, multidrug tolerant persister genes. This is a challenging problem and the main impediment to progress in understanding the nature of persistence. This remains an important goal and will be achieved by the end of the funded period of the project. With the persister genes in hand, we then planned to study the detailed molecular mechanism of persister formation in a subsequent phase of the project. However, we recently discovered that persisters can be formed in response to DNA damage, and have identified a toxin/antitoxin module TisAB that apparently leads to dormancy, and is the subject of the proposed 2-year Recovery Act Funds for Competitive Revision Application Notice Number NOT-OD-09-058. This finding provides us with an attractive opportunity to accelerate the speed of the project and describe the first molecular mechanism of persister cell formation within the next two years, rather than wait till the next phase of the project. We also did not plan to study persister resuscitation, assuming that this will be done some time in the future once we know the mechanism by which they are formed. TisB provides us with a great opportunity to address this question in the next two years as well. In the Supplement, we propose to establish the first detailed mechanism of persister formation considerably ahead of schedule. Proposed studies will include isolating TisB-dependent persisters by cell sorting and obtaining their transcriptome; a detailed study of the molecular mechanism of TisB action; and the mechanism by which TisB-dependent persisters resuscitate. In case of TisB, the mechanism of resuscitation is intriguing. TisB does not have a protein antitoxin that could antagonize its action - the antitoxin is an RNA that only affects expression. TisB is a membrane-acting peptide that disrupts the pmf and causes a decrease in ATP. This will produce an overall systems shutdown in the cell and multidrug tolerance. In the absence of an antitoxin, it is not clear how the cell manages to resuscitate from such a state of deep dormancy. A screen for mutants able to grow in a strain ectopically expressing TisB will lead to identification of the resuscitation genes by whole genome sequencing. Apart from finding a persister formation and resuscitation mechanism, this work will for the first time link together two seeming opposite strategies of cell survival - active protection through stress responses; and dormancy. The new paradigm emerging from our studies is that stress causes dormancy, and this will be detailed in the proposed Supplement studies. This study will point to an intriguing possibility of a link between other stress responses and persister formation. Pathogens are exposed to many stress factors in the host environment apart from DNA damaging agents - oxidants, high temperature, low pH, membrane-acting agents. It is possible that all stress responses induce the formation of a small but resilient subpopulation of surviving persisters. The present study will lay the foundation for examining the role of stress responses in causing multidrug tolerance in vivo. Once we properly understand the nature of multidrug tolerance, this will instruct our efforts on finding effective counter-measures. PUBLIC HEALTH RELEVANCE: This project will also address the goals of stimulating the economy by hiring two new Postdoctoral Fellows; providing a Research Assistantship to a graduate student; purchasing capital equipment; and contracting out some of the work of this project.