Quorum sensing and virulence in Gram positive pathogens: structure, function and inhibition of the agr system

The emergence, rapid spread and persistence of multi-antibiotic resistant bacteria is considered by the WHO as one of the three greatest global threats to human health. Antimicrobial resistance (AMR) threatens the treatment and outcome of even simple infections and common medical interventions (surgery, dentistry, obstetrics) that until recently were considered low-risk. Against this backdrop, the development of new classes of antibiotics has lagged far behind the urgent requirement for new drugs. This is in part because discovering safe and effective new antibiotics is scientifically challenging and because many major pharmaceutical companies withdrew from developing expensive new drugs likely to become rapidly obsolete through resistance. Consequently, novel antibacterial drugs with that do not succumb to conventional antibiotic resistance mechanisms, nor select for new forms of resistance, nor damage the host microflora, are desperately needed. The discovery of such drugs depends on a thorough understanding of the physiology and molecular biology of pathogenic bacteria and their strategies for colonizing host tissues and combatting host immune defences. These include the deployment of multiple virulence factors such as enzymes and toxins that cause host tissue damage and disease. Conventional antibiotics mostly act by killing bacteria and so exert enormous selective pressures leading to the emergence of resistant strains. If however, instead of killing bacteria, we simply prevent them deploying their virulence factors, infection should be attenuated with less pressure for resistance to emerge. Research on bacterial virulence factors and their control systems enables us to identify molecular target 'weak points' in bacteria so that methods for screening drug-like compounds active against such targets can be designed and new antibacterial drugs discovered. One promising target for anti-virulence agents is quorum sensing (QS). Although bacteria are single cell organisms, they can synchronize the activities of all the cells in a population through cell-to-cell communication. This is achieved through the production and sensing of signal molecules that inform the infecting bacteria that they are present in sufficient numerical strength to deploy their virulence factors and mount an attack. QS systems offer multiple molecular targets for anti-infective agents that include the production, export and response to QS signal molecules.

In problematic multi-antibiotic resistant pathogens such as Staphylococcus aureus (including MRSA) and Clostridium difficile, virulence factors including many major exotoxins are controlled by the agr QS system that employs autoinducing peptide signal (AIP) molecules. In this research project we are seeking to understand in depth the way in which S. aureus produces and exports AIP signal molecules via two transmembrane proteins called AgrB and 1984 since these are key to QS and hence virulence. We propose to use a multidisciplinary approach combining microbiology with chemistry, structural biology to elucidate the functions and 3D structures of the key enzymes involved in AIP generation. We also plan to discover how AIPs are exported out of the bacterial cell and to develop new drug-like molecules that block AIP production generation in staphylococci. These will be tested for efficacy alone and in combination with conventional antibiotics in laboratory media and by using novel infection imaging tools that will provide information on when and where agr-dependent QS is switched on or off. The work will focus primarily on S. aureus but promising compounds will also be tested against enterococci, clostridia and listeria and other. other staphylococcal species.

In Staphylococcus aureus and related Gram positive pathogens, virulence is regulated via the agr-dependent quorum sensing (QS) system that is an attractive target for novel anti-infective agents. With previous MRC funding, the successful development of in vitro assays for processing the AgrD pro-peptide by AgrB enabled us to uncover a new pathway for the generation and export of autoinducing peptide (AIP) signal molecules. In addition to AgrB/D, this pathway includes a novel CPBP transmembrane protein and an ABC transporter. In the present proposal we plan to adopt a multidisciplinary approach combining bacterial physiology and genetics with structural biology, biophysics and medicinal chemistry to: (1) elucidate the molecular basis and specificity of the interaction between AgrB and AgrD, (2) use NMR and crystallography to determine the high resolution structures of AgrB alone and in complex with AgrD, (3) employ medicinal chemistry approaches using ambuic acid as a structural template to develop novel AgrB inhibitors with activity against staphylococci, clostridia, enterococci and listeria, (4) elucidate the physiological function(s), selectivity, structure, contribution to virulence and regulation of the CPBP transmembrane protein, using techniques applied in (1) and (2). We discovered that mutations in a key ABC transporter operon resulted in the loss of extracellular AIP and we will therefore (5) elucidate the contribution of this ABC transporter to AIP export. By using super resolution microscopy, we propose to (6) define the cellular localization and spatial organization of agr system components and the impact of exogenously supplied agonists or antagonists. We have developed new tools for in vivo imaging that will be used to gain new insights into (7) the dynamics of agr expression in vivo and the efficacy of agr pathway inhibitors active against AgrB and AgrC.

The primary objectives of this project are the acquisition of new knowledge, scientific advancement, research capacity building and the development of new chemical entities for probing structure and function that may themselves prove to be of therapeutic value as antibacterial agents. Although for practical reasons, the project mostly focuses on S. aureus, the work is relevant to other important Gram positive pathogens that employ agr-type systems including other staphylococcal species, enterococci, clostridia and listeria. This group of organisms are particularly problematic in the context of antibiotic therapy and the emergence of antimicrobial resistance. This is not only a consequence of their virulence and their ability to become hyper-virulent but also because antibiotic usage increases resistance and infection rates. Hence there is an urgent need to better understand these pathogens and to develop new therapeutic and prophylactic agents that are not bactericidal and so do not kill the healthy human microbiome nor drive the selection of resistant strains.

The work described in the proposal although fundamental in nature is directly relevant to medicine and to the pharmaceutical industry in the context of novel antibacterial agent discovery and development. In addition to providing new potentially useful compounds for development as antibacterials, the project will provide structural information on protein targets useful for in silico structure-based design screening for novel inhibitors. It will also provide refined experimental animal models employing state of the art imaging for in vivo antibacterial agent evaluation.

The applicants have placed strong emphasis on the early exploitation of their research and have filed numerous patent applications between them to date and have been involved in the formation of spin-out companies. Exploitation of the research will continue to be delivered via the University of Nottingham technology transfer offices and through licensing agreements with commercial partners.

The project will generate both scientific tools and resources including strains, mutants, proteins and reporter gene fusions as well as novel compounds, structural and transcriptomic data that will be of research community in general. We will communicate our findings to users and beneficiaries through publication in international peer-reviewed scientific journals, international conferences, university web pages and the local and national press. We are also in the process of completely re-designing our 'quorum sensing' website (www.nottingham.ac.uk/quorum) which has proved to be very popular as a comprehensive site for scientists, journalists and the public with respect to bacterial cell-cell communication and its exploitation.

We will also continue our involvement in public engagement activities that have included television and radio contributions, provision of materials for museum displays, lay scientific presentations to local public groups and to schools as well as with academics outside our discipline (e.g in the Arts and Humanities) and invitations to the public to Open Days. At the time of publication of our results, we will engage with our University and the MRC press offices

The research staff involved will develop their skills in team working, project management, investigative planning and both written and oral communication which they can apply in all employment sectors. Importantly, there is considerable scope for multidisciplinary training in this project and unique opportunities for hands-on experience across the disciplines involved. Thus, the project will impact on the creation of human resources that could subsequently be employed in challenging multi-disciplinary projects in industry, academia and government and contribute to the UK science base.