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控制感染所施加的选择性压力，以及细菌本身的遗传可塑性。这使得耐药机制在细菌之间迅速进化和传播。一旦这种抵抗机制存在，就很难消除。细菌所拥有的这种遗传武器库的一个关键部分是质粒，其特征是相对于染色体较小，通常是环状的，能够在细菌细胞内独立于宿主染色体复制的DNA元件。许多质粒具有通过专门的纳米分子机制在细菌之间转移的能力，该机制在细菌和对接过程之间产生融合和隧道，该对接过程允许质粒的拷贝从一个细菌转移到另一个细菌。这个过程被称为接合或细菌性别，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，从而使耐药基因在不同的细菌物种和不同的生态位之间迅速传播。在选择性环境中，优势质粒倾向于携带抗生素抗性。主要在使用新的抗微生物剂治疗感染的临床背景下，质粒积累对其宿主已经暴露的多种抗生素的抗性决定簇，使得当它们移动时，其新宿主同时对多种抗生素产生抗性。因此，在菌株由于耐药基因在自我传播质粒上的积累而变得不可治疗的情况下，扭转这种情况的一种可能方法可能是取代耐药质粒本身。因此，如果携带多个抗性基因的质粒可以被置换，那么所有的抗性基因都将丢失，从而允许重新使用否则无效的抗生素。我们已经开发出一种方法来做到这一点，使用广泛的宿主范围质粒携带一个基因功能盒，阻止靶质粒繁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有很多了解，但我们发现它促进质粒置换的能力取决于一个特定的基因，该基因属于一个基因家族，该基因家族在不同的质粒上广泛存在。了解这种增强作用是如何起作用的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创建突变，看看哪些会影响这种增强。随后将进行生化分析，以了解质粒的哪些特性受到这些突变的影响。第二个工作包的重点是质粒在肠道中从一种细菌传播到另一种细菌的速度。这种传播依赖于细菌表面的一种称为“菌毛”的蛋白质“毛发”。不同种类的质粒有不同种类的菌毛，但长的柔性菌毛被认为在稳定配对方面更好，从而允许质粒转移。我们选择的质粒有一个短的刚性菌毛，在液体中不太好。有些质粒同时具有两种菌毛，因此以这个质粒为模型，我们将改造我们选择的质粒，使其编码一个长的柔性菌毛，使其具有两种菌毛。然后我们将使这种新的杂交体发生突变，并将其置于我们可以分离出传播更快的突变体的环境中。这些质粒将成为进一步研究的基础，包括动物试验，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.