Carbon monoxide and metal carbonyl CO-releasing molecules (CORMs) as novel antimicrobial agents - a systems approach to cellular targets and effects

This project is concerned with how carbon monoxide (CO) affects bacteria. CO is a gas that is well known as a fuel, pollutant, and respiratory poison. Numerous accidental deaths and suicides are the result of CO poisoning each year. However, CO also has surprising but very important essential roles in biology and medicine. It is used as an energy supply by some environmental bacteria, which are able to sense the presence of the gas and switch on appropriate ways to deal with it. CO is also produced naturally in the body by enzymes (haem oxygenases). This CO has potent biological effects, including improvement of vascular tone and other cell protecting functions. Surprisingly, CO has shown promising effects on treating bacterial infections in mice. For example, CO decreases counts of Pseudomonas aeruginosa in the spleen and increases survival of the animals. So, CO plays an important role in antimicrobial processes and recent data show that CO administration may be a clinically useful intervention. However, we do not understand how CO works. This project will make significant advances in our understanding of the mode of antibacterial activity of CO gas and newly designed molecules that can be used to deliver CO (CORMs). We will compare the inhibitory activities of CO applied as a gas and via administration of selected CORMs. In parallel, a computational model of the impact of CO on the bacterium Escherichia coli will be developed, capable of being refined when new experimental results become available. A major focus will be testing the hypothesis that CO is a competitive inhibitor with oxygen and that oxygen concentration around the bacterium is a prime determinant of CORM effectiveness. We will determine the sensitivity to CO of each of the bacterium's respiratory enzymes, prime targets for CO inhibition. For the first time, we will determine how CO and CORMs enter bacteria and try to understand the full extent of their effects on genes and proteins. This research will have long-term benefits in improving the control of bacterial infections and might be used to treat disease in humans, especially diseases caused by antibiotic-resistant bacteria.

CO is a colourless, odourless gas of fundamental importance as a fuel and anthropogenic pollutant, but also a carbon/energy source for some bacteria and a potent respiratory poison. CO is produced endogenously in mammals by haem oxygenases and the CO generated has potent biological effects, including modulation of vascular tone as well as cytoprotective and anti-apoptotic properties. Surprisingly, CO enhances phagocytosis of Escherichia coli by macrophages and CO shows promising effects on bacteremia in sepsis. For example, CO decreases counts of Ps. aeruginosa in the spleen and injection of a CO-releasing molecule (CORM) into wild-type mice increases phagocytosis of Enterococcus faecalis and rescues HO-1-deficient mice from sepsis-induced lethality. This project will make significant advances in our understanding of the mode of antibacterial activity of CO gas and CORMs. We will methodically compare the inhibitory activities of CO applied as a gas and via administration of selected CORMs and in parallel develop and test new, rigorous control molecules (inactive CORMs) for experimental use. In parallel, a predictive computational model of the impact of CO on E. coli metabolism and gene regulation will be developed, capable of being iteratively refined when new experimental data become available. A major focus of the data collection will be testing the hypothesis that CO is a competitive inhibitor with oxygen and that oxygen availability is a prime determinant of CORM effectiveness. We will determine the sensitivity of each of the respiratory chain terminal oxidases, prime targets for CO inhibition. We will determine the intracellular access and fates of CO and CORMs when applied to intact bacteria, and the kinetics and energetics of CORM uptake. Finally, we will test the hypothesis that, by virtue of its reactivity with haems, CO is an antagonist of haemoglobin-mediated, and other bacterial, responses to NO toxicity.

There is mounting published evidence that CORMs and/or CO gas are bactericidal in vitro. Furthermore, CORMs in particular are bactericidal in animal models and this activity is associated with reduced mortality. Therefore, we already have proof of principle for the potential use of CORMs as an antimicrobial therapy. In this work, we will obtain evidence for the mechanism of bactericidal activity in the expectation that key findings can be extended readily to other species of pathogenic bacteria. We envisage that the most useful application of CORMs will be against the bacteraemias of multidrug-resistant bacteria, especially Staphylococcus aureus, and Enterococcus. However, specifically, ESBL (Extended-Spectrum Beta Lactamase) E. coli appears to be the most promising target. ESBL-producing E. coli now infects about 30,000 people annually in England and Wales. ESBL-producing E. coli infections are growing globally We are ideally set up to commercialise any discovery. The University of Sheffield (Brian Mann) and Northwick Park Institute for Medical Research, London (Dr Motterlini's base before moving to Italy) founded a spin-out company, Hemocorm, which merged with Alfama last year. The joint company has extensive patent coverage of the use of CORMs in medicine and a pipeline agreement exists between the University of Sheffield and Alfama. All the CORMs to be used in the proposal are included in patents owned by Alfama and the bactericidal activity of CORMs is of interest to Alfama (see Alfama patent application, WO 2008/130261). This project is an excellent fit with several BBSRC priorities, particularly SYSTEMS APPROACH TO BIOLOGICAL RESEARCH and TECHNOLOGY DEVELOPMENT FOR SCIENCE. In the former, the research addresses 'systems biology of microorganisms, which may include signalling pathways and metabolic pathways' as well as 'systems biology applied to user needs such as in the basic biology underpinning healthcare' and 'tools and technology platforms for systems biology research'. In the latter, the most pertinent aspects are 'predictive modelling of biological systems', 'informatics for biology', 'chemical biology tools' and 'biomolecular characterisation'. In this project, new knowledge is the key and most powerful output. We expect the outcomes to drive new technologies, processes and products (e.g. treatment of infections caused by antibiotic resistant pathogenic bacteria) and to increase our understanding of complex biological systems especially host-pathogen interactions. Finally, we propose that the training of bioscientists will be a major impact. Established scientists will develop new skills, and be trained in new areas during the course of this research. Equally, two postdoctoral researchers will gain valuable experience in research at the borders of microbiology/biochemistry, systems modelling and chemistry. Both Universities have well-established procedures for ensuring optimal training in generic and transferable skills. This research will probably create new capabilities within the scientific community as skills acquired during training are transferred out of academia.