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

20世纪早期抗生素的发现对人类产生了重大影响，多年来拯救了数百万人的生命。然而，这些抗生素正变得越来越无效，而且对多药耐药，即所谓的“超级细菌”正在增加。最近的研究表明，仅在2019年，抗药性感染就在西欧夺走了5.1万人的生命，全球估计有127万人死亡，而且这些数字预计还会上升。在英国，抗药性感染预计将使患者的治疗成本增加一倍，并将成为NHS的一大负担。抗生素在医疗保健、兽医和农业环境中的广泛使用促进了对这类药物的耐药性的发展，这种情况需要紧急关注。耐药性增加的原因是细菌可以通过进化和/或通过获得这些耐药性来发展耐药性，这些耐药性导致其他细菌的基因水平。接合是一个介导水平基因转移的过程，在所有细菌中普遍存在。细菌的这种高度复杂的接合过程是由一套分子机器推动的。这其中包括一个被称为‘4型分泌系统’(T4SS)的大分子复合体，一个被称为‘菌毛’的细胞外管道，以及一个被称为‘松弛小体’的DNA处理机制。在理解T4SS机制方面已经取得了合理的进展，但人们对含有抗生素耐药基因的DNA是如何处理和运输的知之甚少。为了发生接合作用，DNA的双链状态必须被破坏并打开以使其能够运输。这种DNA开放是由松弛小体介导的，松弛小体是一种多蛋白复合体，它促进了一种名为松弛酶的中央酶的访问。这种松弛酶执行启动和DNA解离步骤，并可能引导DNA转移。目前还不清楚这些步骤是如何发生的，以实现DNA的接合转移。我们将详细研究松弛小体复合体和松弛酶蛋白，以获得对结合DNA处理机制的基本理解。为了解决DNA结合过程背后的分子机制，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络合物的结构细节。这项技术使我们能够看到这个复合体的三维排列到一个分辨率，其中单个成分，如氨基酸和碱基可以定位。此外，我们将进行生化和细胞研究，以全面了解这一过程。通过我们的工作，我们将能够对使细菌结合的DNA处理机制提供见解。这项工作的结果可能被用于药物发现，最终将有助于解决细菌中抗生素耐药性的传播问题。

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.