Cell envelope stress responses and the mechanism of antibiotic tolerance in Gram-negative pathogens

项目摘要 细菌通常抵抗通常杀菌抗生素的杀灭，导致临床治疗失败， 抗生素耐药性的发展。在暴露于抗生素引起的损伤中存活的能力被称为 宽容耐受性可能是导致停用抗生素后感染复发的原因 治疗，并提供了一个水库的细菌种群，可以发展全面的阻力。一个极端 耐受性的一个例子是形成持留细胞，这些细胞不会经历由以下原因引起的过敏性损伤： 休眠然而，我们和其他人已经发现许多革兰氏阴性病原体（霍乱弧菌， 铜绿假单胞菌、阴沟肠杆菌、流感嗜血杆菌和鲍曼不动杆菌）是 对细胞壁作用抗生素（β-内酰胺类）诱导的损伤完全敏感，但在极高的温度下仍能存活 程度.通过形成缺乏可检测的细胞壁的活球， 在撤回抗生素后恢复到正常形状。在我们的模式生物中， 病原菌霍乱弧菌，耐受性是由细胞膜应激反应，特别是双组分 系统WigKR。WigKR是由细胞壁作用抗生素诱导的，并产生一种复杂的反应， 能够从球形状态恢复。这种反应包括细胞壁合成功能的上调， 外膜合成、磷脂合成以及运动和铁获得基因的下调。 人们对这种反应如何促进耐受性知之甚少，其他免疫反应中的耐受机制也是如此。 革兰氏阴性菌。在这里，我们的目的是询问霍乱弧菌的细胞包膜应激反应和它们的免疫反应。 与β内酰胺耐受性和抗生素后恢复的关系。利用基因和生化技术 方法，我们将发现由组氨酸激酶WigK感知的难以捉摸的诱导信号。利用 广泛的数据集全面描述WigKR调节子，我们将测量每个单独的调节子 成员对β内酰胺耐受性的贡献。最后，我们将应用我们在V. cholesterol中所学到的知识， 对其他表现出高β内酰胺耐受性的革兰氏阴性病原体，特别是E. cloxacin和P. 铜绿。我们的实验将对抗生素耐受性的机制产生新的见解，并导致 β-内酰胺类抗耐受佐剂候选药物靶点的鉴定。

Project Summary Bacteria often resist killing by normally bactericidal antibiotics, resulting in clinical treatment failure and the development of antibiotic resistance. The ability to survive damage elicited by exposure to antibiotics is termed tolerance. Tolerance is likely responsible for the recurrence of infections after discontinuation of antimicrobial therapy, and provides a reservoir of a bacterial population that can develop full scale resistance. An extreme case of tolerance is the formation of persister cells, which do not experience antibiotic-induced damage due to dormancy. However, we and others have found that many Gram-negative pathogens (Vibrio cholerae, Pseudomonas aeruginosa, Enterobacter cloacae, Haemophilus influenzae and Acinetobacter baumannii) are fully susceptible to damage induced by cell wall acting antibiotics (beta lactams), but yet survive at very high levels. Survival is enabled through the formation of viable spheres that are devoid of detectable cell wall material and that recover to normal shape upon withdrawal of the antibiotic. In our model organism, the cholera pathogen V. cholerae, tolerance is promoted by cell envelope stress responses, especially the two-component system WigKR. WigKR is induced by cell wall acting antibiotics and mounts a complex response that ultimately enables recovery from the spherical state. This response includes upregulation of cell wall synthesis functions, outer membrane synthesis, phospholipid synthesis and downregulation of motility and iron acquisition genes. How this response promotes tolerance is poorly understood, and so are the mechanisms of tolerance in other Gram-negative bacteria. Here, we aim to interrogate V. cholerae's cell envelope stress responses and their relationship with beta lactam tolerance and post-antibiotic recovery. Using genetic and biochemical approaches, we will find the elusive induction signal sensed by the histidine kinase WigK. Leveraging extensive datasets comprehensively describing the WigKR regulon, we will measure each individual regulon member's contribution to beta lactam tolerance. Lastly, we will apply what we have learned in the V. cholerae model to other Gram-negative pathogens exhibiting high beta lactam tolerance, specifically E. cloacae and P. aeruginosa. Our experiments will yield novel insight into the mechanisms of antibiotic tolerance and result in the identification of candidate drug targets for anti-tolerance adjuvants of beta lactams.