Disruption in bacterial cell envelope following polymyxin challenge and adaptations in tolerant strains

细菌感染仍然是医疗保健的主要挑战，而对抗生素产生或获得抗药性加剧了这一问题。这类革兰氏阴性细菌特别容易受到管理的影响，因为它们细胞包膜的结构包括一层外膜，可以提供自然保护，免受抗生素和抗菌剂的伤害。例如，青霉素组中使用最广泛的抗生素的分子靶标在细菌外膜内受到保护。同样受到保护的还有细菌内膜，它在维持细胞的生存状态方面起着决定性的作用。这两种膜都具有连续的双层结构，具有明显不同的结构和组成。多粘菌素是一类具有抗革兰氏阴性菌活性的抗菌化合物，其临床应用受到限制。它们表现出较高的毒性，尽管有丰富的研究和临床使用历史，但它们的作用机制仍然知之甚少。在它们的作用过程中，多粘菌素利用并颠覆细菌表面的受体--脂多糖，穿过保护的外膜，进入内膜，在那里它们行使它们的杀伤作用。在这项提案中，我们的目标是破译多粘菌素杀死细菌的分子细节，描述多粘菌素诱导细菌死亡的各个阶段，并研究由于耐药细菌的适应性变化而导致的细菌外膜和内膜的详细结构和分子变化。我们从耐药细菌中多粘菌素与其靶标脂多糖之间的相互作用中寻找分子模板，这将为多粘菌素抗微生物工程提供信息，以增强对耐药革兰氏阴性病原体的活性。为了实现这一目标，我们将先进的力显微镜与核磁共振光谱相结合，以研究多粘菌素与细菌外膜和内膜的相互作用，以及它们的结构、组织和稳定性的变化。我们假设最初的外膜被多粘菌素破坏，扰乱了两个膜之间正常的分子物质交换，并扰乱了外膜和内膜的组织。为了研究这种失调的致命后果，我们将监测细菌的生存能力，并将使用电子显微镜来监测细菌被膜中重要结构元件的顺序崩溃。最后，我们将使用这种方法来揭示细菌膜结构和分子结构的变化对耐药细菌中多粘菌素的保护作用。

Bacterial infections remain a major challenge to healthcare and the problem is exacerbated by the development or acquisition of resistance to antibiotics. The group of Gram-negative bacteria are particularly refractive to management, as the structure of their cell envelope includes an outer membrane that confers natural protection from antibiotics and antimicrobials. For example, the molecular targets for the most widely used antibiotics from the penicillin group, are protected within the bacterial outer membrane. Also protected is the inner bacterial membrane, which plays a defining role in maintaining the living state of cells. Both membranes have a continuous bilayer structure with distinctly different architecture and composition.Polymyxins are one class of antimicrobial compounds with activity against Gram-negative bacteria that have found restricted clinical use as last line therapeutics. They show comparatively high toxicity and despite a wealth of research and history of clinical use, their mechanism of action remains poorly understood. During their action, polymyxins utilise and subvert a bacterial surface receptor, lipopolysaccharide, to cross the protective outer membrane and access the inner membrane, where they exercise their killing action. In this proposal, we aim to decipher the molecular details of bacterial killing by polymyxins, describe the stages of polymyxin-induced bacterial death, and investigate the detailed structural and molecular changes in bacterial outer and inner membranes that result from adaptive changes in resistant bacteria. We seek a molecular template from the interaction between polymyxin and its target, lipopolysaccharide, from resistant bacteria, which will inform engineering of polymyxin antimicrobials with enhanced activity against resistant Gram-negative pathogens.To achieve this, we combine advanced force microscopy that can map bacterial surfaces at molecular resolution, with magnetic resonance spectroscopy providing molecular and structural information at atomic level of detail, to investigate the interaction of polymyxins with bacterial outer and inner membranes, the changes in their structure, organisation and stability. we hypothesise initial outer membrane breach by polymyxin disrupts the normal exchange of molecular material between the two membranes and dysregulates organisation of both outer and inner membranes. To investigate the lethal consequences of this dysregulation, we will monitor bacterial viability and will use electron microscopy to monitor the sequential collapse of important structural elements in the bacterial envelope. Finally, we will employ this methodology to reveal what changes in bacterial membrane structure and molecular architecture confer protection against polymyxins in resistant bacteria.