Targeting cell separation systems of gram-negative bacteria.

描述(申请人提供)：革兰氏阴性细菌是多种重要人类疾病的病原体。这些微生物感染的成功治疗受到它们周围复杂的细胞膜的阻碍。这种包膜包括第二层(外)膜层，药物很难穿透。因此，革兰氏阴性细菌对抗生素有很高的内在抗药性，对许多对缺乏外膜的细菌有效的药物完全不敏感。在这个项目中，我们打算验证抗生素的新靶点，这些靶点在灭活时会破坏外膜的渗透性屏障。 膜可以杀死革兰氏阴性细菌，或消除它们对已批准的治疗药物的内在耐药性。该项目是基于我们对大肠杆菌细胞分裂的研究。在这个革兰氏阴性细菌模型中，我们最近确定了分裂过程中细胞包膜重塑所需的因素，并开发了研究这些因素的工具。在分裂过程中，细胞动能机制产生的新的细胞壁材料必须经过水解酶处理，以便子细胞分离。我们已经发现，这个过程是由被称为酰胺酶的酶进行的，这些酶由带有LytM结构域的因子激活。一旦细胞壁被加工，第二组被称为Tol-Pal系统的因子催化外膜的收缩，完成分裂过程。在大肠杆菌和其他革兰氏阴性细菌中，酰胺酶或Tol-Pal系统的失活会导致形成无法分离的长链细胞。这些细胞链已被证明具有受损的外膜通透性屏障，并对许多药物过敏。尽管它们在维持外膜通透性屏障方面发挥了作用，但这些细胞分离系统对耐药的重要性尚未在有问题的革兰氏阴性病原体中进行调查。因此，在这个项目中，我们将验证细胞分离系统作为TE病原体铜绿假单胞菌潜在药物靶点的有效性。分子遗传学将被用来研究灭活这些系统对这种有机体的生长和抗药性的影响，并将实施化学筛选来确定这一过程的抑制剂。虽然我们关注的是铜绿假单胞菌，但选择用于靶向的系统在革兰氏阴性细菌中高度保守。因此，我们的结果将与革兰氏阴性感染的新治疗方法的开发广泛相关。

DESCRIPTION (provided by applicant): Gram-negative bacteria are the causative agents of a variety of important human diseases. Successful treatment of infections with these organisms is hampered by the complex cell envelope that surrounds them. This envelope includes a second (outer) membrane layer that is difficult for drugs to penetrate. Gram-negative bacteria thus have a high intrinsic resistance to antibiotics and are completely insensitive to many drugs that are effective against bacteria lacking an outer membrane. In this project, we intend to validate new targets for antibiotics that when inactivated will compromise the permeability barrier of the outer membrane to either kill gram-negative bacteria or eliminate their intrinsic resistance to approved therapeutics. The project is based on our studies of cell division in Escherichia coli. In this model gram-negative bacterium, we recently identified factors required for cell envelope remodeling during division and have developed tools to study them. During division, new cell wall material produced by the cytokinetic machinery must be processed by hydrolytic enzymes for daughter cells to separate. We have discovered that this processing is carried out by enzymes called amidases that are activated by factors with LytM domains. Once the cell wall is processed, a second set of factors called the Tol-Pal system catalyzes the constriction of the outer membrane to complete the division process. Inactivation of either the amidases or the Tol-Pal system in E. coli and other gram-negative bacteria leads to the formation of long chains of cells that cannot separate. These cell chains have been shown to have a compromised outer membrane permeability barrier and are hypersensitive to many drugs. Despite their role in maintaining the outer membrane permeability barrier, the importance of these cell separation systems for drug resistance has not been investigated in problematic gram-negative pathogens. Therefore, in this project, we will validate cell separation systems as potential drug targets in te pathogen Pseudomonas aeruginosa. Molecular genetics will be used to investigate the effect of inactivating these systems on the growth and drug resistance of this organism, and chemical screens will be implemented to identify inhibitors of the process. Although we focus on P. aeruginosa, the systems chosen for targeting are highly conserved in gram-negative bacteria. Thus, our results will be broadly relevant to the development of novel treatments for gram-negative infections.