sRNA-based therapeutics for disease caused by A. pleuropneumoniae

The bacterium Actinobacillus pleuropneumoniae (APP) causes is a highly contagious lung disease in pigs which affects herds in the UK and worldwide. Infection results in either rapid mortality or slow growth, breathing problems and suffering. APP is responsible for substantial economic losses to the worldwide pig industry.

After good husbandry practices (provision of good ventilation, all in/all out facilities, avoidance of temperature fluctuations and good hygiene) are taken into account there are two basic methods used to limit APP infection: namely vaccines and antibiotics. However, current vaccines have severe limitations and antibiotic resistance is of increasing concern, a recent survey suggesting that 70% of APP isolates in the UK are now resistant to one or more antibiotics. Consequently, there is an urgent need for effective therapeutic approaches to combat APP. Addressing such issues directly aligns to the key BBSRC strategic priorities of 'Animal Health', 'Welfare of Managed Animals' and 'Combating Antimicrobial Resistance'.

Traditional antibiotics seek to simply kill bacteria, but a novel therapeutic alternative is to target functions that are essential for the bacteria's ability to infect the host. Such an approach effectively disarms the bacteria, rather than killing them, thereby avoiding the selective pressure from which traditional antibiotic resistance originates. Bacterial cells, such as APP, have important switching molecules (sRNAs) which are responsible for turning on the mechanisms that enable host infection to occur. Our proposal plans to identify these crucial sRNAs in APP and then inhibit their function using compounds called peptide nucleic acids (PNAs). Targeting the sRNAs in this manner will lead to a new therapeutic approach for treating APP disease. More broadly, this research also provides the basis for exploring sRNA inhibition as an alternative therapeutic approach to bacterial diseases caused by other major pathogens of animals and man.

This work is innovative, timely and multi-disciplinary, building on recent work and employing the latest molecular tools and technological advances to deliver results. This is a joint collaborative venture which benefits from the synergy between world leading expertise in APP (Imperial College London) and an outstanding track record in sRNA research (University of Portsmouth). Within this context, the project has a substantial chance of success, standing to offer a step change in capability for treating APP, but with far reaching impact for the therapeutic treatment of bacterial diseases more broadly.

Actinobacillus pleuropneumoniae (APP) causes acute and chronic lung disease in pigs and is of substantial economic importance to the worldwide swine industry. There is an urgent need for improved vaccines and therapeutics as antibiotic resistance is an increasing problem. The focus of this proposal is bacterial small non-coding RNAs (sRNAs) which, facilitated by Hfq, play a key role controlling mRNAs responsible for virulence. Our work has shown that the hfq-mutants of APP are severely attenuated and rapidly cleared from pigs. While Hfq could be targeted directly as a therapeutic approach, the presence of homologous eukaryotic proteins suggests that targeting the associated sRNAs will be a more effective strategy. In particularly, this approach will bring about a therapeutic effect without the risk of developing bacterial resistance, since no survival pressure, from which traditional antibiotic resistance originates, is imposed.

To date, our work has experimentally identified 15 Hfq-associated sRNAs in APP, and computationally a further 30 have been predicted. Our recent bioinformatic analysis confirms a number of these sRNAs target mRNAs with relevance to virulence. This proposal represents a major collaboration between Imperial College London and the University of Portsmouth, utilising the extensive experience in APP and Hfq-sRNA interactions, respectively. We plan to ascertain the identity of Hfq-associated sRNAs that are key in turning on virulence genes and determine whether inhibiting these sRNAs using peptide nucleic acids (PNAs) provides a novel therapeutic approach to treating APP disease. Using a combination of in vitro biochemical and biophysical molecular characterisation approaches, coupled with in vivo reporter and/or phenotypic assays, we will determine the viability of this therapeutic approach. Success would offer a step change in capability for combating APP, but with far reaching impact for the treatment of bacterial diseases more broadly.

There is an urgent need for effective therapeutics to combat the highly contagious, and economically important, disease caused by Actinobacillus pleuropneumoniae (APP), which affects the UK and worldwide swine industry. Our research aims to meet this need through the development of a novel therapeutic approach which targets bacterial sRNAs that control virulence. Success would offer a step change in capability for combating APP, but would have far reaching impact for the treatment of bacterial diseases more broadly. This work therefore closely addresses the key BBSRC strategic priorities of 'Animal Health', 'Welfare of Managed Animals' and 'Combating Antimicrobial Resistance' and the beneficiaries of this research and how they will benefit are as follows:

*Academic Researchers: through broad academic advancement and instigation of new research programmes.
Briefly, our findings will allow a greater understanding of post-transcriptional gene regulation in APP, identify sRNA targets for therapeutic intervention and demonstrate the validity of inhibiting target sRNAs using PNAs. The findings will therefore be of immediate benefit to researchers with interests in bacterial pathogenicity, antimicrobial research, and gene regulation mechanisms, as well as those interested in molecular microbiology and biomolecular research more broadly. In particular, an immediate avenue of investigation will be to confirm whether the findings and approach within this proposal have broader applicability to other pathogenic bacterial species, leading to a potentially rapid evolution of related therapeutic treatments for bacterial infections. Additionally, this research is also valuable to biological chemists who are involved in the development of synthetic ligands (e.g. PNAs), in particular those keen to enhance the uptake efficiency of such molecules by bacteria for therapeutic purposes.

* The pharmaceutical industry: through exploitation and commercialisation within the antimicrobial arena.
Our research aims to provide the basis for a novel therapeutic approach which should be readily exploitable by the pharmaceutical industry. In the longer term, successful translation of our research into a drug has the potential to create wealth and economic prosperity through increased turn-over, profit and exports, and creating and safeguarding jobs for the company and workers involved. Additionally, whilst research into antibiotics is currently not favoured by pharmaceutical companies, due to the low return on investment, providing a new approach to treating bacterial disease that overcomes the issue of antibiotic resistance could rejuvenate interest in this area.

* The swine industry: through the ability to effectively treat APP.
In the longer term, successful translation of this research into a therapeutic treatment for APP will reduce medication costs, reduce pig suffering and mortality and overall reduce the significant economic impact on the swine industry of APP.

* The general public: through increasing public engagement with research.
Throughout it will be possible to engage with the general public and local schools to raise the profile of this project. This will encourage interest in biosciences research and UK innovation and provide a context to local bioscience education.

* Researchers working directly on the project: thereby contributing to a highly trained workforce.
The skills gained by the researchers working on this innovative, multidisciplinary project will be highly transferable; providing an excellent training experience to enable them to embark on a successful future career in any setting. For example, working on a joint research project provides experience in collaboration and team working, participation in outreach activities gives experience in communicating effectively with non-specialists, and through publishing manuscripts and presenting work at conferences they will hone their verbal and written skills.