A Genomics Approach to Drug Tolerance

描述（由申请人提供）：目前赠款的总体目标是确定导致形成休眠，多药耐受持续基因的基因。这是一个具有挑战性的问题，也是在理解持久性的本质方面取得进展的主要障碍。这仍然是一个重要目标，将在项目供资期结束时实现。有了持续存在的基因，我们计划在项目的后续阶段研究持续存在形成的详细分子机制。然而，我们最近发现，持久性可以形成响应DNA损伤，并确定了一个毒素/抗毒素模块TisAB，显然会导致休眠，是拟议的2年恢复法基金竞争性修订申请通知号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，可以加快项目的速度，并在未来两年内描述持续细胞形成的第一个分子机制，而不是等到项目的下一阶段。我们也没有计划研究持续复苏，假设一旦我们知道它们的形成机制，将来某个时候就会进行这项研究。TisB也为我们在未来两年解决这个问题提供了一个很好的机会。 在补编中，我们建议大大提前建立第一个详细的持久性形成机制。拟议的研究将包括通过细胞分选分离TisB依赖的坚持者并获得其转录组; TisB作用的分子机制的详细研究;以及TisB依赖的坚持者复苏的机制。在TisB的情况下，复苏的机制是有趣的。TisB没有一种蛋白质抗毒素可以拮抗它的作用-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减少。这将在细胞中产生整体系统关闭和多药耐受性。在缺乏抗毒素的情况下，尚不清楚细胞如何设法从这种深度休眠状态中复苏。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。 除了找到一个持久的形成和复苏机制，这项工作将首次将两个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;和休眠。从我们的研究中出现的新范式是压力导致休眠，这将在拟议的补充研究中详细说明。这项研究将指出一个有趣的可能性之间的联系，其他压力反应和persister形成。病原体在宿主环境中暴露于除DNA损伤剂之外的许多应激因素-氧化剂、高温、低pH、膜作用剂。所有的应激反应都可能诱导形成一个小而有弹性的存活坚持者亚群。本研究为进一步研究应激反应在体内引起多药耐受中的作用奠定了基础。一旦我们正确理解多药耐药性的性质，这将指导我们努力寻找有效的应对措施。 公共卫生相关性：该项目还将通过雇用两名新的博士后研究员来实现刺激经济的目标;为一名研究生提供研究助理;购买资本设备;并将该项目的一些工作外包。

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.