Noisy Strep

Summary. Streptococcus pneumoniae is a major pathogen causing invasive (pneumonia, meningitis, bacteraemia) and non-invasive (acute otitis media, sinusitis) disease in children and in elderly and/or immuno-compromised adults. The last decades have seen the emergence and spread of pneumococcal strains with multiple antibiotic resistance posing a serious threat to human health. The strategy of the pathogen development is regulated by so called bistability, and may lead to formation of drug resistant forms of bacteria, such as biofilms. Transcriptional noise is the main determinant of switching of bistable systems. Therefore, understanding of molecular mechanisms giving birth to the noise in transcription in S. pneumoniae is highly medically relevant. By using cutting-edge techniques in biochemistry and microbiology as well as mathematical modelling we will address the problem of origins of transcriptional noise in S. pneumonia. This will result in a high resolution systems-level understanding of the role of transcriptional noise in gene regulation of a human pathogen. The results will potentially provide a new target for intelligent drug design to manipulate differentiation and life-strategy decisions of pathogenic bacteria.

Technical Summary. Bistable switches are the key elements of the regulatory networks governing cell development, differentiation and life-strategy decisions. Transcriptional noise is a main determinant that causes switching between different states in bistable systems. By using the human pathogen Streptococcus pneumoniae as a model bacterium, we will investigate how transcriptional fidelity and processivity influence (noisy) gene expression and participate in bistability. To study this question, we will use both natural and synthetic S. pneumoniae bistable switches as a highly sensitive probe for transcriptional noise. We will screen for mutations of the transcriptional apparatus that display altered bistability. A pure in vitro transcription system for S. pneumoniae will be set up and used to quantitatively characterize effects of these mutations on transcription. Detailed single-cell analysis using time-lapse microscopy will yield quantifiable data on the effects of the mutations on switching times and probabilities. Mathematical models that take transcription fidelity and processivity into account will be used to pinpoint parameters which most strongly affect the switching probabilities of our bistable networks. A global model encompassing all our in vivo and in vitro data will yield a high resolution systems-level understanding of the role of transcriptional noise in gene regulation of a human pathogen. Genetic and biochemical characterization of mutant RNAPs and/or accessory factors will yield molecular insights into the fundamental mechanisms of transcription. Furthermore, our results might lead to novel drug discovery projects specifically aimed to reduce or increase transcriptional noise to prevent unwanted development of pathogenic bacteria such as S. pneumoniae.

Impact Summary Beneficiaries within the 'commercial private sector', are UK and Holland based pharmaceutical companies, named in the Impact Plan, (letters of support are available upon request). Both immediate and long term beneficiaries are in the sphere of investigation of infectious disease caused by bacteria and drug design. As a result, potential long term beneficiaries will be health organisations and consequently the wider public. Additional potential benefits for the 'wider public' will be in publicising the research via press releases, interviews, etc. S. pneumoniae is a major pathogen causing invasive (pneumonia, meningitis, bacteraemia) and non-invasive (acute otitis media, sinusitis) disease in children and in elderly and/or immuno-compromised adults. The last decades have seen the emergence and spread of pneumococcal strains with multiple antibiotic resistances posing a serious threat to human health. Research may lead to novel antimicrobial targets. It is foreseen that novel targets to tune noise and/or prevent unwanted differentiation processes such as competence development and biofilm formation, will be investigated for patent possibilities. Drugs that prevent S. pneumoniae from forming highly resistant biofilms for instance, will render the bacterium more sensitive to common antibiotics. Dual treatment with standard antibiotics and a compound that prevents initiation of competence development might result in reduced occurrence of multidrug resistant bacteria. Therefore, a potential benefit for the mentioned parties will be in novel targets for drug design and pathogen manipulation that will be investigated in our study. The drug design companies (mentioned in the Impact Plan) expressed considerable interest in our work. We agreed for a meeting half-way into the project for discussions of possible patenting and exploitation of our finding. Moreover, one of the companies is based in the same University as one of the Partners of the consortium, allowing intensive communication of our group with the researches of this company. The leading European laboratory that models Streptococcus infection in animal systems (see Impact Plan) is keen to test the mutant bacteria that will be obtained in our work in their experimental systems. This will be the next step of exploitation of our results bringing them to a more medical sphere, which may also lead to further integration with the 'commercial private sector'. The Impact activities will be managed by the members of the consortium and supported by the respective institutions. In the Newcastle University it will be supported by NU commercial development team and press office that are partly funded via this grant.