Identification and characterisation of barriers to antimicrobial resistance gene transfer

抗菌素耐药性（AMR）是一个日益严重的全球公共卫生问题，每年造成数十万人死亡，而且还在不断增加。AMR基因通常携带在可转移的DNA片段上，称为移动遗传元件（MGE），可以通过水平基因转移（HGT）在细菌之间共享。因此，抗生素在医院的广泛使用提供了一种选择性压力，有助于AMR基因的快速传播，导致耐甲氧西林金黄色葡萄球菌（MRSA）等耐药菌株的建立。然而，金黄色葡萄球菌的HGT尚未完全了解，特别是对于医院和社区相关的MRSA（分别为HA-MRSA和CA-MRSA）。在金黄色葡萄球菌中，HGT的主要方法是通过一个称为转导的过程。感染细菌的病毒（噬菌体）将非噬菌体DNA错误地包装成病毒颗粒，然后将DNA转移到新的宿主。细菌已经进化出一系列系统来阻止MGE和噬菌体的入侵，重要的例子是限制性修饰（R-M）和CRISPR-Cas系统，它们靶向并降解外源DNA。因此，这些系统也会成为HGT的障碍。我们对金黄色葡萄球菌的分子转导过程的了解是不完整的，很少有新的研究出现。因此，本项目的目的是提高我们对MRSA中HGT的机制和屏障的理解。为了研究金黄色葡萄球菌中HGT的转导障碍，伦敦大学圣乔治分校的J. Lindsay团队开发了一种检测方法，可以识别潜在的HGT屏障基因。该试验使用CA-MRSA菌株中1952个非必需基因的突变文库。然后，它允许AMR基因的HGT率的量化。HGT的显著增加意味着被敲除的基因起到了阻断转导的作用，将其确定为候选基因，其中几个已经被确定。因此，该项目将继续这项研究并优化分析。另一个项目目标是利用生物信息学和分子技术表征这些候选基因。对基因序列以及基因操纵子的计算分析，将允许识别具有相似DNA序列的保守结构域和同源物，从而使我们对基因功能有一个概念。这可以通过分子实验进一步探索，或通过集中在分析的模块组件。已鉴定基因在金黄色葡萄球菌测序群体中的分布也将提供对其在进化中的作用的深入了解。噬菌体在感染时将其基因组插入宿主染色体。基因组可以作为前噬菌体在染色体中保持不活跃状态，直到环境发生变化，导致它切除、复制、产生病毒颗粒、裂解宿主细胞并传播。大多数金黄色葡萄球菌的基因组中有1-4个噬菌体。然而，临床和HA-MRSA噬菌体知之甚少。R-M系统阻断来自不同宿主菌株的MGEs的HGT。因此，这个项目的目标是在我们的突变体库中淘汰这个系统。这将使我们能够对CA-MRSA以外的菌株（包括直接从患者身上分离的MRSA）重复HGT率测定，使我们能够比较不同菌株之间的HGT率。这使我们有机会对临床MRSA噬菌体进行测序和表征。本项目旨在通过噬菌体提高我们对金黄色葡萄球菌HGT分子过程的理解，使我们能够更好地了解MRSA群体在全球和临床环境中的进化动态。正如R-M和CRISPR-Cas工具所证明的那样，对MRSA HGT屏障进行更深入的研究也可能导致新的生物技术的发现。该项目涉及定量技能、跨学科技能和整个生物体生理学的使用。

Antimicrobial resistance (AMR) is a mounting global public health issue, causing hundred of thousands of deaths annually, which is ever increasing. AMR genes are often carried on transferable sections of DNA called mobile genetic elements (MGE) which can be shared between bacteria through horizontal gene transfer (HGT). The widespread use of antibiotics in hospitals has therefore provided a selective pressure that has aided the rapid spread of AMR genes, leading to the establishment of resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) However, HGT in S. aureus is not completely understood, particularly for hospital and community associated MRSA (HA-MRSA and CA-MRSA respectively).In S. aureus, the primary method of HGT is through a process called transduction. Bacteria-infecting viruses (bacteriophage) mis-package non-phage DNA into viral particles, which then transfer the DNA to a new host. Bacteria have evolved a range of systems to block MGE and bacteriophage invasion and - important examples are restriction modification (R-M) and CRISPR-Cas systems, which target and degrade foreign DNA. These systems therefore also act as barriers to HGT. Our knowledge of the molecular process of transduction in S. aureus is incomplete, with little new research emerging.The aim of this project is therefore to improve our understanding of the mechanism and barriers of HGT in MRSA. In order to investigate the barriers of transduction HGT in S. aureus, the J. Lindsay team at St. George's, University of London have developed an assay to allow the identification of potential HGT barrier genes. The assay uses a mutant library of 1952 non-essential genes in a strain of CA-MRSA. It then allows for the quantification of the rate of HGT of AMR genes. A significant increase in HGT implies that the knocked-out gene was acting to block transduction, identifying it as a gene candidate, of which several have already been identified. This project will therefore continue this research and optimise the assay.Another project goal is the characterisation of these candidate genes using bioinformatic and molecular techniques. Computational analysis of the gene sequence, as well as the gene operon, would allow for the identification of conserved domains and orthologues with similar DNA sequences, giving us an idea of the gene function. This can then be further explored through molecular experimentation, or by focussing on modular components of the assay. The distribution of identified genes within sequenced S. aureus populations will also provide insight into their role in evolution.Bacteriophage insert their genome into the host chromosome upon infection. The genome can remain inactive in the chromosome as a prophage until a change conditions causes it to excise, replicate, create viral particles, lyse the host cell and spread. Most S. aureus have 1-4 prophage in their genome. However, clinical and HA-MRSA prophage are poorly understood. The R-M system blocks HGT of MGEs from strains different from the host. A goal of this project is therefore to knock this system out in our mutant library. This will allow us to repeat the HGT rate assay with strains other than our CA-MRSA, including MRSA isolated directly from patients, allowing us to compare HGT rates between isolates. This give us the opportunity to sequence and characterise clinical MRSA prophage.This project aims to improve our understanding of the molecular process of HGT in S. aureus through bacteriophage, allowing us to better understand MRSA population evolution dynamics globally and in clinical settings. As the tools R-M and CRISPR-Cas demonstrate, greater research into MRSA HGT barriers could also lead to the discovery of new biotechnology.This project involves the use of quantitative skills, interdisciplinary skills and whole organism physiology.