Identification and characterisation of barriers to antimicrobial resistance gene transfer

抗菌素耐药性基因转移障碍的鉴定和表征

• 批准号: 2434040
• 金额: --
• 依托单位: St George's, University of London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2434040
• 关键词: Identification; characterisation; barriers; antimicrobial; resistance

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.

抗菌药物耐药性（AMR）是一个日益严重的全球公共卫生问题，每年导致数十万人死亡，而且这一数字还在不断增加。AMR基因通常携带在称为移动的遗传元件（MGE）的DNA的可转移部分上，其可以通过水平基因转移（HGT）在细菌之间共享。因此，医院中抗生素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。金黄色葡萄球菌的耐药性尚不完全清楚，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断MGE和噬菌体入侵，重要的例子是限制性修饰（R-M）和CRISPR-Cas系统，其靶向并降解外源DNA。因此，这些系统也成为HGT的障碍。我们对S.金黄色葡萄球菌HGT的研究还不完整，几乎没有新的研究出现。因此，本项目的目的是提高我们对MRSA中HGT的机制和障碍的理解。为了研究HGT在S.金黄色葡萄球菌中，伦敦大学圣乔治的J.林赛团队已经开发了一种检测方法来鉴定潜在的HGT屏障基因。该检测使用CA-MRSA菌株中1952个非必需基因的突变体文库。然后，它允许量化AMR基因的HGT率。HGT的显著增加意味着敲除的基因起到阻断转导的作用，将其鉴定为候选基因，其中几个已经被鉴定。因此，本项目将继续这项研究，并优化检测方法。另一个项目目标是利用生物信息学和分子技术对这些候选基因进行表征。对基因序列以及基因操纵子的计算分析将允许鉴定具有相似DNA序列的保守结构域和直系同源物，从而使我们对基因功能有一个概念。然后可以通过分子实验或通过关注测定的模块化组件来进一步探索。已鉴定的基因在测序的S.金黄色葡萄球菌种群也将提供深入了解它们在进化中的作用。2噬菌体在感染时将它们的基因组插入宿主染色体。基因组可以作为原噬菌体在染色体中保持无活性，直到改变条件使其切除、复制、产生病毒颗粒、裂解宿主细胞并扩散。大多数S。金黄色葡萄球菌基因组中有1-4个前噬菌体。然而，临床和HA-MRSA前噬菌体知之甚少。R-M系统阻断来自与宿主不同的菌株的MGE的HGT。因此，这个项目的目标是在我们的突变体库中敲除这个系统。这将使我们能够用CA-MRSA以外的菌株重复HGT率测定，包括直接从患者中分离的MRSA，使我们能够比较分离株之间的HGT率。本项目的目的是进一步了解HGT在S.金黄色葡萄球菌通过噬菌体，使我们能够更好地了解MRSA的人口演变动态全球和临床环境。正如R-M和CRISPR-Cas工具所证明的那样，对MRSA HGT障碍的更多研究也可能导致新生物技术的发现。该项目涉及使用定量技能，跨学科技能和整个生物体生理学。