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

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.

Our aim is to displace resistance plasmids from bacteria by delivering genetic cassettes composed of loci or parts of loci that can block replication of the targeted plasmids and neutralize their addiction systems. We have explored delivery of the cassette on a promiscuous conjugative IncP-1 plasmid as the best way to spread through a complex mix of microbiota. We found that wild type RK2 does not function well as a vehicle for this but is made very effective by a small deletion adjacent to the essential replication origin that removes a control element, causing the copy number to rise slightly (< 2-fold).

However, we discovered that raised copy number is not sufficient - the korB gene is also needed intact - raising fundamental questions about how the potentiation works. Understanding this phenomenon will help our knowledge of ParB proteins which are essential for stable inheritance of many plasmids and chromosomes and help in the design of plasmid displacement tools in many organisms. We will mutate the korB gene to explore which functions are needed for potentiation and then use biochemical analysis to determine what korB does to the plasmid and the genes it carries that causes potentiation. Obvious possibilities include changing supercoiling or recruiting other host proteins that modulate function.

This plasmid spreads efficiently in bacteria growing on surfaces even without selection but at a lower rate in liquid or semi-liquid environments like the gut and this correlates with its pilus type. Some plasmids encode both short rigid pili and long flexible pili that should stabilised bacterial mating pairs. We will use recombineering to insert genes for a flexible pilus into RK2 under control of its global regulatory circuits. We will use forced evolution to isolate mutants that spread more efficiently and also mutate the genes targeted by fertility inhibition genes to optimize these hybrids for spread through liquid and semi-liquid cultures.

The development of the pCURE plasmid displacement technology with the University of Birmingham spinout company Plasgene has been driven by an urgent need to tackle the crisis of multiply antibiotic resistant superbugs. That development process has uncovered the fundamental questions about the genetic elements involved that is the basis of this proposal and which need to be answered to underpin the further development of this technology or reveal alternative approaches to plasmid displacement. Therefore, the most immediate impact of the work will be on the further development of the pCURE technology. For example, understanding the basis of the potentiation of curing may allow us to plan new pCURE development more efficiently, perhaps at the level of modifying promoters for genes to be inserted into low copy number conjugative plasmids, thus avoiding the need for potentiation. Additionally, understanding how possession of two types of conjugative pilus might improve plasmid spread in different environments could immediately be tested in animal trials on the effectiveness of plasmid displacement in the gut as well as providing basic understanding of why some plasmids have just one pilus type while others have two.

The technology has applications in animal husbandry, aquaculture and human healthcare where there many worries about the escape of the genetic elements we have constructed. Therefore, this work will will impact on the drive to find solutions to the issues of how to prevent spread outside of the target context and block transposition and recombination that might allow resistance genes to move to other genetic locations and thus avoid the displacement process.

In addition to this very specific technology the work will help to increase general understanding of the spread of antibiotic resistance. Possible solutions which are not simply developing new antibiotics and that provide a real prospect of reversing the accumulation of resistance will be news-worthy. The applicant has a track record of working with the London Science Media Centre and undertaking other Public Engagement activities and will continue to do so. I also have strong links with the Microbiology Society which has active programmes of public engagement on the issues surrounding antibiotic usage and will ensure these provide a vehicle for dissemination of information about the project and how it addresses these problems.

The proposed work also has significant implications in terms of public acceptability of GM and Synthetic Biology. The lead applicant has discussed this with Dr Claire Marris (now at Centre for Food Policy, Sociology Department, City, University of London) to explore the social science side of this research but I also have links with Sheelagh McGuinness at the University of Bristol Law School, Bristol Population Health Science Institute. This will provide engagement with stakeholder groups to identify key difficulties and how education and improved presentation can help to ensure that worries about the technology are not founded on misconceptions. This will also lead to identification of real issues that will need to be addressed for the technology to be acceptable. Discussions with Professor Janet Bainbridge, former Chair of the UK Scientific Advisory Committee on Genetic Modification (Contained Use), raised the possibility of this being used as an exemplar technology for engagement with the public, and this will be explored if the project is funded. Colleague Peter Hawkey and Sheelagh McGuinness visited the Medicines and Healthcare products Regulatory Agency (MHRA) to discuss the status of such probiotic substances and were encouraged by their response (letter attached).