Molecular Mechanism Of DNA Processing For Antibiotic Resistance Gene Transfer In Bacteria

细菌抗生素抗性基因转移 DNA 加工的分子机制

• 批准号: BB/X016900/1
• 负责人: Aravindan Ilangovan
• 金额: $ 65.88万
• 依托单位: Queen Mary University of London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX016900%2F1
• 关键词: Molecular; Mechanism; DNA; Processing; Antibiotic

The discovery of antibiotics in the earlier part of the 20th century has had a significant impact on humans and has saved millions of lives over the years. These antibiotics however are increasingly becoming ineffective and multi drug resistant so called "superbugs" are on the rise. Recent studies have suggested, in the year 2019 alone, drug resistance infections have claimed 51,000 lives in western Europe and an estimated 1.27 million lives globally, and these numbers are expected to rise. In the UK, drug resistant infections are expected to double the treatment cost of patients and will become a big burden on the NHS. The extensive use of antibiotics in the healthcare, veterinary and agricultural settings has contributed to the development of resistance against such drugs and this situation requires urgent attention. The reason for this increase in resistance is because bacteria can develop resistance through evolution and/or by obtaining these resistance causing genes horizontally from other bacteria. Conjugation is a process that mediate horizontal gene transfer and is ubiquitous in all bacteria. This highly sophisticated process of conjugation in bacteria is facilitated by a set of molecular machines. These includes a large macromolecular complex called the 'Type 4 secretion system' (T4SS), an extra cellular conduit called the 'pilus' and a DNA processing machinery called the 'relaxosome'. Reasonable progress has been made towards understanding the T4SS machinery, however little is known about how the DNA, harbouring antibiotic resistance genes, is processed and transported.For conjugation to occur, the double stranded state of the DNA has to be disrupted and opened to enable its transport. This DNA opening is mediated by the relaxosome, a multi-protein complex that facilitates access to a central enzyme called relaxase. This relaxase performs the initiation and DNA unwinding steps and possibly pilots the DNA transfer. It is unclear how these steps occur to enable transfer of DNA by conjugation. We will be studying the relaxosome complex and relaxase protein in detail to derive a fundamental understanding into the conjugative DNA processing mechanism. To address the molecular mechanism behind conjugative DNA processing, we will exploit the recent advances in high-resolution cryo electron microscopy technique to obtain structural details of these complexes. This technique allows us to see the three dimensional arrangement of this complex to a resolution where individual components such as the amino acids and the nucleobases could be located. Further to this we will carry out biochemical and cellular studies to obtain a holistic understanding into this process. Through our work we will be able to provide insights into the mechanism of DNA processing that enables bacterial conjugation. The outcome from this work could be exploited for drug discovery that will ultimately help tackle the spread of antibiotic resistance in bacteria.

20世纪早期抗生素的发现对人类产生了重大影响，多年来挽救了数百万人的生命。然而，这些抗生素越来越无效，耐多药的所谓“超级细菌”正在增加。最近的研究表明，仅在2019年，耐药性感染就在西欧夺走了5.1万人的生命，在全球夺走了约127万人的生命，预计这些数字还会上升。在英国，耐药感染预计将使患者的治疗费用增加一倍，并将成为NHS的一大负担。在卫生保健、兽医和农业环境中广泛使用抗生素导致对这类药物产生耐药性，这一情况需要紧急关注。耐药性增加的原因是细菌可以通过进化和/或从其他细菌水平获得这些耐药性基因而产生耐药性。偶联是介导水平基因转移的过程，在所有细菌中普遍存在。细菌中这种高度复杂的结合过程是由一套分子机器促成的。其中包括一个叫做“4型分泌系统”（T4SS）的大分子复合物，一个叫做“菌毛”的额外的细胞导管和一个叫做“松弛体”的DNA加工机器。在了解T4SS机制方面已经取得了合理的进展，然而，对于含有抗生素抗性基因的DNA是如何加工和运输的，我们知之甚少。为了偶联发生，DNA的双链状态必须被破坏并打开以使其能够运输。这种DNA打开是由松弛体介导的，松弛体是一种多蛋白复合物，有助于进入一种称为松弛酶的中心酶。这种松弛执行起始和DNA解绕步骤，并可能引导DNA转移。目前尚不清楚这些步骤是如何发生的，从而使DNA通过偶联转移。我们将详细研究松弛体复合体和松弛蛋白，以获得对结合DNA加工机制的基本理解。为了解决共轭DNA加工背后的分子机制，我们将利用高分辨率低温电子显微镜技术的最新进展来获得这些复合物的结构细节。这种技术使我们能够看到这种复合体的三维排列到一个分辨率，其中的单个成分，如氨基酸和核碱基可以定位。此外，我们将进行生化和细胞研究，以获得对这一过程的整体理解。通过我们的工作，我们将能够深入了解DNA加工的机制，使细菌结合。这项工作的结果可以用于药物发现，最终有助于解决细菌中抗生素耐药性的传播。