Super-persistent cells and the paradox of untreatable infections

描述(申请人提供)：传染病通常是无法治疗的，即使是由对抗生素没有抗药性的病原体引起的。这就是悖论，也是我们致力于解决的问题。微生物群体产生持久的、休眠的细胞，这些细胞不是突变体，而是对抗生素耐受的野生型的表型变体。似乎有可能，坚持者的存在可以解释治疗悖论：抗生素会消除大部分种群，一旦其浓度下降，幸存的坚持者开始分裂并重新建立感染。然而，在体外长期使用抗生素治疗，应该模仿体内治疗，可以消除这些休眠细胞。假设。我们假设，导致无法治愈的感染的病原体是一种携带高持久性突变并已诱导应激反应的超持久细胞。在体外重复应用高水平的抗生素，选择具有更高水平的持久细胞的大肠杆菌HIP(高持久细胞)突变体。根据我们的数据，与野生型持久者相比，HIP细胞对药物的耐受性也更强。我们推断，定期对慢性感染患者应用致命剂量的抗生素将同样选择髋关节突变。对感染铜绿假单胞菌的囊性纤维化患者的纵向分离株的分析表明，晚期但不是早期的分离株确实是HIP突变。似乎有可能用反复剂量的抗生素治疗在许多病原体中选择HIP突变，而正是这些目前被忽视的耐受(而不是耐药)突变最终导致了疾病的发病率和患者的死亡。除了髋关节突变，似乎还有另一个被忽视但潜在的关键因素导致了耐受性-应激反应。到目前为止，我们已经知道了两种看似相反的细胞生存策略--休眠，它关闭功能并产生持久细胞；以及诱导应激反应(热休克、DNA修复、氧化应激等)。积极地保护细胞免受有害环境的伤害。我们认为，这两种策略实际上是相辅相成的。如果在表达应激蛋白的种群中形成持久性，那么它将关闭抗生素靶标，同时保留帮助其生存的保护性蛋白质。在人体内，病原体暴露在氧化剂、DNA损伤剂、膜作用剂中，似乎表达几种应激反应是一种常态。最终的幸存者是携带髋关节突变的持久者，这种突变是在表达应激反应的人群中形成的。正是这种超级顽固可能导致了许多无法治愈的疾病，并将成为我们调查的重点。该实验计划将解决以下相互关联的问题：HIP突变是否是慢性感染的重要组成部分？有没有将髋关节突变与应激反应表达相结合的超级持久者？容忍，就像抵抗一样，是一种可传播的特征吗？ 公共卫生相关性：在这个项目中，我们将寻找能够进入对现有抗生素高度耐受的休眠状态的病原体突变体。我们的发现可能会改变我们看待传染病的方式，并将为发现完全根除感染的药物提供合理的方法。

DESCRIPTION (provided by applicant): Infectious disease is often untreatable, even when caused by a pathogen that is not resistant to antibiotics. This is the paradox, and the problem that we aim to solve. Microbial populations produce persisters, dormant cells that are not mutants, but phenotypic variants of the wild type that are tolerant to antibiotics. It seemed possible that the presence of persisters could explain the treatment paradox: an antibiotic eliminates most of the population, and once its concentration drops, the surviving persisters start dividing and reestablish the infection. However, prolonged treatment of persisters in vitro with antibiotics which should emulate the in vivo therapy eliminates these dormant cells. The hypothesis. We hypothesize that the agent responsible for untreatable infections is a super-persister cell which carries a high-persister mutation and has induced stress responses. Repeated application of high levels of antibiotics in vitro selects for E. coli hip (high-persister) mutants that have an increased level of persister cells. According to our data, the hip cells are also more drug-tolerant as compared to wild type persisters. We reasoned that periodic application of lethal doses of antibiotics to patients with chronic infections will similarly select for hip mutants. Analysis of longitudinal isolates from a cystic fibrosis patient infected with P. aeruginosa showed that late, but not early isolates are indeed hip mutants. It seems possible that therapy with repeated doses of antibiotic selects hip mutants in many if not all pathogens, and it is these presently overlooked tolerant (rather than resistant) mutants that are ultimately responsible for morbidity of the disease and for the death of a patient. Apart from hip mutations, there seems to be another overlooked, but potentially critical component contributing to tolerance - stress responses. So far, we have known of two seemingly opposite strategies of cell survival - dormancy, which shuts down functions and creates persister cells; and induction of stress responses (heat shock, DNA repair, oxidation stress, etc.) that actively protect the cell from noxious conditions. We propose that these two strategies actually complement each other. If a persister is formed in a population that had expressed stress proteins, then it will shut down antibiotic targets, while retaining protective proteins which will help it survive. In the body, a pathogen is exposes to oxidants, DNA damaging agents, membrane acting agents, and it seems that expression of several stress responses is a norm. The ultimate survivor is then a persister carrying a hip mutation which is formed in a population expressing stress responses. It is this super-persister that is probably responsible for much of untreatable disease and will be the focus of our investigation. The experimental plan will address the following interrelated questions: are hip mutants an important part of chronic infection? Are there super-persisters that combine hip mutations with expression of stress responses? Is tolerance, similarly to resistance, a transmissible trait? PUBLIC HEALTH RELEVANCE: In this project, we will search for mutants of pathogens that are able to enter into a state of dormancy highly tolerant to existing antibiotics. Our findings are likely to change the way we view infectious diseases and will provide rational approaches for discovering drugs that completely eradicate the infection.