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

细菌感染仍然是卫生保健面临的一个主要挑战，而且由于抗生素耐药性的发展或获得，这一问题更加严重。革兰氏阴性菌组尤其难以管理，因为它们的细胞包膜结构包括一层外层膜，该外层膜可对抗生素和抗菌素提供天然保护。例如，最广泛使用的青霉素类抗生素的分子靶标被保护在细菌外膜内。细菌的内部膜也受到保护，它在维持细胞的生存状态中起着决定性的作用。两种膜具有连续的双层结构，结构和组成明显不同。多粘菌素是一类具有抗革兰氏阴性菌活性的抗菌化合物，已被发现限制临床使用作为最后一线治疗药物。它们显示出相对较高的毒性，尽管有大量的研究和临床使用的历史，但它们的作用机制仍然知之甚少。在它们的作用过程中，多粘菌素利用并破坏细菌表面受体脂多糖，穿过保护性的外膜，进入内膜，在那里它们行使它们的杀伤作用。在本研究中，我们旨在揭示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阶段，并研究耐药细菌适应性变化导致细菌内外膜的详细结构和分子变化。我们从多粘菌素与其靶点脂多糖之间的相互作用中寻找分子模板，这将为多粘菌素抗菌剂的工程设计提供信息，增强其对耐药革兰氏阴性病原体的活性。为了实现这一目标，我们结合了先进的力显微镜，可以在分子分辨率上绘制细菌表面，磁共振波谱提供分子和结构信息在原子水平上的细节，研究多粘菌素与细菌内外膜的相互作用，它们的结构，组织和稳定性的变化。我们假设多粘菌素最初破坏外膜，破坏了两膜之间分子物质的正常交换，并调节了内外膜的组织。为了研究这种失调的致命后果，我们将监测细菌活力，并使用电子显微镜监测细菌包膜中重要结构元件的顺序崩溃。最后，我们将采用这种方法揭示细菌膜结构和分子结构的变化赋予耐药细菌对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.