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

项目摘要 细菌经常抵抗通常的杀菌抗生素的杀灭，导致临床治疗失败和 抗生素耐药性的发展。因接触抗生素而造成的损伤的存活能力被称为 宽容。耐受性可能是停用抗菌药物后感染复发的原因 治疗，并提供了一个细菌种群的储藏库，可以产生全面的耐药性。一种极端的 耐受的情况是形成持久细胞，这些细胞不会经历抗生素诱导的损害，这是由于 休眠。然而，我们和其他人发现，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 对细胞壁作用的抗生素(β-内酰胺类)造成的损害完全敏感，但在非常高的条件下仍能存活 级别。生存是通过形成没有可检测到的细胞壁的有活力的球体来实现的。 停用抗生素后可恢复正常形状的材料。在我们的模型生物体中，霍乱 霍乱弧菌的耐受性是由细胞被膜应激反应促进的，尤其是两组分 系统WigKRWigKR由细胞壁作用的抗生素诱导，并产生复杂的反应，最终 允许从球形状态恢复。该响应包括细胞壁合成功能的上调， 外膜合成、磷脂合成以及运动性和铁获取基因的下调。 这种反应是如何促进耐受性的，人们对此知之甚少，其他国家的耐受性机制也是如此 革兰氏阴性细菌。在这里，我们的目标是询问霍乱弧菌的细胞膜应激反应和它们的 β-内酰胺耐药与抗生素后恢复的关系。利用遗传和生化 方法，我们将发现难以捉摸的诱导信号由组氨酸蛋白激酶WigK感应。利用 全面描述WigKR规则的大量数据集，我们将测量每个单独的规则 成员对β-内酰胺耐受性的贡献。最后，我们将把我们所学到的知识应用于霍乱弧菌 其他对β-内酰胺类耐药的革兰氏阴性病原菌的模型，特别是阴沟肠杆菌和P. 铜绿假单胞菌。我们的实验将对抗生素耐受的机制产生新的见解，并导致 β-内酰胺类抗耐药佐剂候选药物靶点的确定。

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.