DNA damage and evolution of Salmonella antibiotic persisters during infections

感染期间沙门氏菌抗生素持续存在的 DNA 损伤和进化

• 批准号: 1978467
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1978467
• 关键词: DNA; damage; evolution; Salmonella; antibiotic

Introduction Antibiotic failure represents a major health concern. The recalcitrant nature of many bacterial infections is thought to be due in part to the presence of persister cells. Persisters have been reported in all major pathogens and are key players in the relapse of bacterial infections. For example, in patients with invasive non-typhoidal Salmonella Typhimurium disease, approximately 78% of recurrent infection was caused by the same strain responsible for the initial disease (Okoro et al, 2012). Moreover, high levels of persisters have been observed in late isolates of Pseudomonas aeruginosa from lung infections in patients with chronic cystic fibrosis (Mulcahy et al, 2010). Unlike inherited resistance, persistence refers to a transient phenotypic state in which a slow-growing subpopulation of bacteria becomes tolerant to antibiotic treatment (Fisher, Gollan & Helaine). Formation and survival of persisters Formation of persisters has been shown to be triggered by several stress conditions such as nutrient starvation (Germain, et al, 2015); diauxic carbon-source shifts (Amato & Brynildsen, 2013), pH shift (Helaine et al, 2014) or DNA damage (Dorr et al, 2009). There is also correlation between the activity of toxin-antitoxin (TA) modules and antibiotic persistence (Moyed et al, 1986). Although, persister formation has been extensively studied in laboratory conditions, the ability of these cells to sustain a non-growing state during infection is overlooked. However, in physiologically relevant conditions, infection with intracellular pathogens such as Salmonella Typhimurium and Mycobacterium Tuberculosis results in high numbers of persisters able to survive the macrophage environment (Mouton et al, 2016; Helaine et al; 2014). Persisters can also survive in lung biofilms of patients with cystic fibrosis (Spoering & Lewis, 2001; Moker, Dean & Tao, 2010). Therefore, the mechanism of survival of the persister subpopulation in these environments is important to understand from a clinical point of view. Evolution of persistence In vitro studies in E. coli have shown that intermittent antibiotic stress can lead to evolution of antibiotic tolerance (Van den Bergh, et al, 2016). Unlike persistence, which refers to a phenotypic switch in a subpopulation of cells, tolerance is described as a decrease in antibiotic susceptibility at a whole population level, which can be genetically acquired or not (Balaban, 2016). The evolved strains had mutations that rendered them tolerant, rather than resistant to cyclic stress. This suggests that bacteria can adapt to antibiotic treatment through mechanisms other than resistance. Moreover, it was also suggested that evolution of tolerance can aid the subsequent development of resistant mutations (Fridman et al, 2014; Levin-Reisman et al ,2017). Future objectives: 1. The importance of DNA damage response during persistence Upon internalization, Salmonella are exposed to the antimicrobial environment of the macrophages. These host immune cells attack engulfed bacteria by generating high levels of oxidative stress, potentially inducing bacterial DNA damage (Burton, et al, 2014). Bacterial cells respond to DNA damage by activating the SOS response, which has been correlated with increased level of persistence. Studies in E. coli have shown that induction of the SOS response following treatment with fluoroquinolones is required for the formation of persisters (Dorr, et al, 2009; Volzing & Brynildsen, 2015). We will use a library of Salmonella mutants lacking 21 proteins involved in different DNA repair pathways to investigate their ability to survive in a non-growing antibiotic tolerant state while in the stressful macrophage environment. Our aim is to identify DNA repair pathways involved in persistence and test their role in survival of persisters both in vitro following antibiotic treatment or ex vivo during infection of primary bone marrow derive

介绍抗生素失败是一个主要的健康问题。许多细菌感染的不稳定性被认为部分是由于持续存在的细胞。在所有主要病原体中都有报道，并且是细菌感染复发的关键因素。例如，在侵袭性非伤寒性鼠伤寒沙门氏菌病患者中，约78%的复发性感染是由导致初始疾病的相同菌株引起的（Okoro et al，2012）。此外，在慢性囊性纤维化患者肺部感染的铜绿假单胞菌晚期分离株中观察到了高水平的持续存在（Mulcahy等人，2010）。与遗传抗性不同，持久性是指一种短暂的表型状态，其中缓慢生长的细菌亚群对抗生素治疗变得耐受（Fisher，Gollan和Helaine）。持留菌的形成和存活已显示出持留菌的形成由几种胁迫条件触发，例如营养饥饿（Germain等人，2015）;二次碳源变化（Amato & Brynildsen，2013），pH变化（Helaine等人，2014）或DNA损伤（Dorr等人，2009）。毒素-抗毒素（TA）模块的活性与抗生素持久性之间也存在相关性（Moyed et al，1986）。尽管在实验室条件下已经广泛研究了持续存在的形成，但这些细胞在感染期间维持非生长状态的能力被忽视了。然而，在生理学相关条件下，细胞内病原体（如鼠伤寒沙门氏菌和结核分枝杆菌）的感染导致大量持久菌能够在巨噬细胞环境中存活（Mouton et al，2016; Helaine et al; 2014）。Persisters也可以在囊性纤维化患者的肺生物膜中存活（Spoering &刘易斯，2001; Moker，Dean & Tao，2010）。因此，从临床的角度来看，了解这些环境中持续存在的亚群的生存机制是很重要的。在E.大肠杆菌的研究表明，间歇性抗生素应激可导致抗生素耐受性的进化（货车den Bergh等人，2016）。与持久性不同，持久性是指细胞亚群中的表型转换，耐受性被描述为在整个群体水平上抗生素敏感性的降低，这可能是遗传获得的，也可能不是遗传获得的（Balaban，2016）。进化出的菌株具有突变，使它们能够耐受，而不是抵抗周期性压力。这表明细菌可以通过耐药性以外的机制适应抗生素治疗。此外，还表明耐受性的演变可以帮助随后产生耐药突变（Fridman et al，2014; Levin-Reisman et al，2017）。今后的目标：沙门氏菌在持续存在过程中DNA损伤反应的重要性在内化过程中，沙门氏菌暴露于巨噬细胞的抗菌环境中。这些宿主免疫细胞通过产生高水平的氧化应激来攻击被吞噬的细菌，可能诱导细菌DNA损伤（Burton等人，2014）。细菌细胞通过激活SOS反应来响应DNA损伤，这与持久性水平的增加有关。在E.大肠杆菌已经表明，在用氟喹诺酮类处理后诱导SOS应答是形成持久性所需的（Dorr等人，2009; Volzing & Brynildsen，2015）。我们将使用缺乏21种参与不同DNA修复途径的蛋白质的沙门氏菌突变体库来研究它们在压力巨噬细胞环境中在非生长抗生素耐受状态下生存的能力。我们的目的是确定与持久性有关的DNA修复途径，并在抗生素治疗后的体外或原发性骨髓源性白血病感染期间的离体实验中检测它们在持久性白血病生存中的作用。