Plasmid biology underpinning development of a novel plasmid displacement technology to eliminate antibiotic resistance genes

细菌的抗生素耐药性正在成为一个日益紧迫的问题，被认为是全球公共卫生面临的主要挑战之一。耐药性的上升是由于使用抗生素和其他抗菌剂来控制感染所施加的选择压力，以及细菌本身的遗传可塑性。这使得耐药机制得以进化并在细菌之间迅速传播。这种抵抗机制一旦存在，就很难摆脱。细菌拥有的这个基因库的一个关键部分是质粒，相对于染色体来说质粒的特征是小的，通常是圆形的，能够在细菌细胞内独立于宿主染色体复制的DNA元素。许多质粒具有在细菌之间转移的能力，通过专门的纳米分子机制，在细菌之间创造融合和隧道，以及一个对接过程，使质粒的副本可以从一个细菌运送到另一个细菌。这个过程被称为结合或细菌性，为细菌从细菌群落的其他地方获得有利基因提供了一个强大的机制。事实上，许多质粒能够在许多不同的细菌类型之间转移和繁殖，从而允许抗性基因在不同种类的细菌和不同的生态位之间迅速传播。在选择性环境中，优势质粒往往携带抗生素耐药性。主要是在使用新的抗微生物药物治疗感染的临床环境中，质粒积累了对宿主接触过的多种抗生素的耐药决定因素，因此当它们移动新宿主时，它们会同时对多种抗生素产生耐药性。因此，在由于抗性基因在自传播质粒上的积累而使菌株变得无法治疗的情况下，扭转这种情况的一种可能方法可能是替换抗性质粒本身。因此，如果携带多种耐药基因的质粒可以被转移，那么所有的耐药基因将会丢失，从而允许重复使用本来无效的抗生素。我们已经开发出一种方法，使用广泛的宿主质粒携带一盒遗传功能，阻止目标质粒增殖并阻断其生存机制。我们对用来携带这种抗质粒盒的质粒了解很多，但我们发现它促进质粒位移的能力取决于一个特定的基因，这个基因属于一个广泛存在于不同质粒上的基因家族。了解这种增强如何起作用可能有助于我们设计更好的方法来取代质粒，这形成了第一个工作包。我们将首先在基因中制造突变，看看哪些会影响这种增强。接下来将进行生化分析，以了解这些突变对质粒的哪些特性产生了影响。该工作包的产出将进一步支持固化质粒的开发。第二个工作包侧重于质粒在肠道中从一个细菌传播到另一个细菌的速度。这种传播依赖于细菌表面一种叫做“菌毛”的蛋白质“毛发”。不同种类的质粒有不同种类的菌毛，但长而灵活的菌毛被认为更能稳定质粒转移所需的配对。我们选择的质粒有一个短的刚性菌毛，在液体中不太好。一些质粒有两种类型的菌毛，所以用这个作为模型，我们将设计我们选择的质粒来编码一个长而灵活的菌毛，这样它就有两种类型的菌毛。然后，我们会对这个新的杂交品种进行变异，并把它放在我们可以隔离传播更快的变异品种的环境中。这样的质粒将为进一步的动物试验奠定基础，我们希望最终能进行临床试验。

Antibiotic resistance in bacteria is becoming an increasingly urgent problem that is recognised as one of the key global challenges to public health. The rise of resistance is due to the selective pressure imposed by the use of antibiotics and other antimicrobial agents to control infection combined with the genetic plasticity of the bacteria themselves. This allows resistance mechanisms to evolve and spread rapidly between bacteria. Once such resistance mechanisms exist it is very difficult to get rid of them. A key part of this genetic arsenal possessed by bacteria are plasmids that are characteristically small relative to the chromosome, often circular, DNA elements capable of replication within bacterial cells independently of the host chromosome. Many plasmids possess the ability to transfer between bacteria via specialised nano-molecular machinery that creates a fusion and a tunnel between bacteria and a docking process that allows a copy of the plasmid to be transported from one bacterium to another. This process, called conjugation or bacterial sex, provides a powerful mechanism for bacteria to acquire advantageous genes from elsewhere in a bacterial community. Indeed, many plasmids are able to transfer between and multiply in many different bacterial types, thus allowing resistance genes to spread rapidly between different species of bacteria and different ecological niches. In a selective environment the dominant plasmids tend to carry antibiotic resistance. Principally in clinical contexts where new antimicrobial agents are used to treat infections, plasmids accumulate resistance determinants to the multiple antibiotics that their host has been exposed to so that when they move their new host becomes resistant to many antibiotics simultaneously. Thus, in situations where strains have become untreatable due to the accumulation of resistance genes on a self-transmissible plasmid, a possible way to reverse the situation might be to displace the resistance plasmids themselves. Thus if the plasmid carrying multiple resistance genes can be displaced then all the resistance genes would be lost, allowing the re-use of antibiotics that would otherwise be ineffective. We have developed a way of doing this using a broad host range plasmid to carry a cassette of genetic functions that stop the target plasmids from multiplying and blocking their survival mechanisms. We know a lot about the plasmid we have used to carry this anti-plasmid cassette but we discovered that its ability to promote plasmid displacement depends on a specific gene that belongs to a gene family that is widespread on different plasmids. Understanding how this potentiation works may help us to design better ways to displace plasmids and this forms the first work package. We will first create mutations in the gene and see which ones affect this potentiation. This will be followed by biochemical analysis to see what properties of the plasmid are affected by these mutations. The output of this work package will underpin further curing plasmid development.The second work package focuses on the speed with which the plasmid can spread from one bacterium to another in the gut. The spread depends on a sort of protein "hair" on the bacterial surface called a "pilus". Different sorts of plasmids have different sort of pilus but the long flexible ones are thought to be better at stabilising the pairings that allow plasmid transfer. The plasmid we chose has a short rigid pilus that is not so good in liquid. Some plasmids have both sorts of pilus and so using this as a model we will engineer our chosen plasmid to encode a long flexible pilus so it has one of each kind. We will then mutate this new hybrid and put it into situations where we can isolate mutants that spread more rapidly. Such plasmids will form the basis of further work involving animal trials and we hope eventually clinical trials.