Super-persistent cells and the paradox of untreatable infections

描述（由申请人提供）：传染病通常是无法治愈的，即使是由对抗生素不耐药的病原体引起的。这是一个悖论，也是我们要解决的问题。微生物种群产生持久的、休眠的细胞，它们不是突变体，而是对抗生素耐受的野生型的表型变异。持久性细菌的存在似乎可以解释治疗悖论：一种抗生素消灭了大部分细菌，一旦浓度下降，幸存的持久性细菌开始分裂并重新感染。然而，在体外用抗生素长期治疗，应该模仿体内治疗，消除这些休眠细胞。假设。我们假设导致无法治疗的感染的病原体是一种携带高持久性突变并诱导应激反应的超级持久性细胞。在体外反复应用高水平的抗生素选择大肠杆菌（高持久性）突变体，其持久性细胞水平增加。根据我们的数据，与野生型持久性细胞相比，臀部细胞也更耐药。我们推断，对慢性感染患者定期使用致死剂量的抗生素将同样选择髋关节突变体。对一囊性纤维化患者感染铜绿假单胞菌纵向分离株的分析表明，晚期而非早期分离株确实是髋关节突变体。重复剂量的抗生素治疗似乎有可能在许多（如果不是所有）病原体中选择髋关节突变体，而正是这些目前被忽视的耐药（而不是耐药）突变体最终导致了疾病的发病率和患者的死亡。除了髋部突变外，似乎还有另一个被忽视的，但潜在的关键因素有助于耐受性-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，关闭功能并产生持久性细胞；并诱导应激反应（热休克、DNA修复、氧化应激等），积极保护细胞免受有害条件的侵害。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蛋白，那么它将关闭抗生素目标，同时保留有助于其生存的保护性蛋白。在体内，病原体暴露于氧化剂、DNA损伤剂、膜作用剂中，似乎几种应激反应的表达是一种常态。最终的幸存者是一个携带髋部突变的人，这种突变是在表达应激反应的人群中形成的。正是这种超级顽固分子可能导致了许多无法治愈的疾病，也将成为我们研究的重点。实验计划将解决以下相关问题：髋关节突变是慢性感染的重要组成部分吗？是否存在将髋部突变与应激反应表达结合起来的超级坚持者？和抗性一样，耐受性也是一种可传播的特性吗？

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.