Bacterial cell wall architecture

The bacterial cell wall is essential for viability, shape determination and its production is the target of the most important types of antibiotics ever discovered (such as penicillin). The alarming spread of antibiotic resistance means it is crucial to understand more about this structure if we are to define new potential ways to control bacterial disease. The cell wall is like an exoskeleton (called the sacculus) that is able to withstand the considerable internal forces that would otherwise rupture the cell. The major structural element of the cell wall for most bacteria is a polymer called peptidoglycan (PG), which is unique to bacteria. PG is a single large, bag-like molecule that surrounds the cell and whilst very strong is also dynamic to allow the cells to grow and divide. Even though PG is chemically only made of relatively simple building blocks how these are assembled to produce an architecture able to fulfil the many functions of PG has remained largely elusive. The problem is that architecture has to be viewed in situ and bacteria are on the micron scale. To address this problem, in the last few years we have taken an interdisciplinary approach using a combination of biochemistry and high-resolution microscopy techniques. The new information gained has completely altered our views on PG architecture overturning previous models and revealing a hitherto unexpected complexity. We have now applied our approach to many different organisms and have discovered several different architectures from rings and knobbles in the human pathogen Staphylococcus aureus to cables in the rod shaped bacterium Bacillus subtilis and a heterogeneous architecture of pores and thicker regions in organisms such as Escherichia coli. In order to explain how such PG features allow the bacteria to maintaining cell integrity and yet be dynamic we have proposed new models for growth and division for several important bacterial species. To map sites of new PG synthesis we have a super-resolution fluorescence microscope and a totally new and unique machine capable of correlating different forms of microscopy. We are now ready to take the next step to actually solve the architecture of PG at the chemical level. This will give great insights into the fundamental biology of bacteria, how they are able to grow and divide and the action of important antibiotics. Amazingly, we know the target of penicillin but not how it kills bacteria. New understanding will require not only the development and use of ultra-resolution microscopy approaches, but also the synthesis of a suite of chemical probes such that we will be able to "see chemistry". Several of the proposed microscopy approaches have not been applied to biological samples before and so we will pave the way for their wider application.

Bacterial cell wall peptidoglycan (PG) is essential for the maintenance of cellular viability and shape determination for most eubacteria and its synthesis is the site of action of important antibiotics. PG is a polymer of glycan strands cross-linked via peptide sidechains, continuously being synthesised, modified and hydrolysed to allow for cell growth and division. Our recent work using a combination of atomic force microscopy (AFM) and latterly super resolution fluorescence microscopy has revealed PG to have a more complicated architecture than previously imagined. This has required new models for bacterial growth mode to be developed, enabling the accommodation of the observed architecture. In order to test these models and to solve how the chemistry of PG is organized to give such complex architecture we need to employ higher resolution microscopy approaches coupled with the use of novel chemical probes to be able to map the chemistry at the nanoscale. In order to facilitate this process we have built a STochastic Optical Resolution Microscope (STORM) capable of single molecule resolution and the first of a new class of microscopes combining STORM and AFM (Storm Force).
The project will take an integrated and thoroughly interdisciplinary approach to understanding PG architecture and its relationship to growth and division. It will involve a synergy between biology, chemistry and physics to capitalize on our novel findings. Using state-of-the-art biophysical and microscopic analysis PG will be studied from the chemical to the cellular level. PG architecture is all about finding engineering solutions to physical problems encountered by the bacteria. Our wide-ranging studies are at the forefront in this area and will address problems at the very heart of fundamental microbiology and the action of antibiotics.

The project will build on our recent findings and provide novel insight into how bacteria are able to maintain viability, grow and divide. There will be a variety of impacts over a range of timescales and in different arenas.

The Scientific Community
The project is specifically orientated to address fundamental questions about bacterial architecture.
- Development and extension of biophysical approaches (expected timescale: year 1 onwards): Following our initial successes, the project will use many state-of-the-art techniques to address fundamental biological questions.
- Establishment of Sheffield as a hub for interdisciplinary biophysical research (expected timescale: year 1 onwards): The project will enhance our standing in the area and lead to further inward investment.
- Publications (expected timescale: year 1 onwards): We will produce high quality data that will be published in leading international journals to provide maximum access to user communities.
- International collaborations in the field (expected timescale: year 1 onwards): Wider interactions will develop the area of cell wall research in the UK to maintain and enhance our international prestige in the area.
- Oral communications (expected timescale: year 1 onwards): We will participate in national and international meetings and conferences to publicise the work to a diverse audience.

Industry, Policy, the Public and UK-PLC
The project is fundamental underpinning science in this extremely important area and will have long-term impact.
- Intellectual property (expected timescale: year 2 onwards): The project team has strong histories in patent protection. Where appropriate IP will be secured to facilitate income generation in the long-term, helped by FusionIP, our partner commercialization company.
- New antibiotic targets (expected timescale: year 3 onwards): The project will provide data to inform the development of new antibiotic targets. SJF has many links with the pharmaceutical industry for exploitation of results.
- Novel technology development (expected timescale: year 1 onwards): Technology developments will be made available to imaging companies, we have links with, in order to direct the development of the next generation of imaging, cameras and techniques to become commercially available.
- Public Engagement (expected timescale: year 1 onwards): SJF and AC are coordinating a public exhibition, including super-resolution microscopy to be held in 2015. We have participated in radio and newspaper interviews to inform the public and have hosted school parties to inform the younger generation of science and due process. We will also enhance public understanding and engagement via students on our Science Communication taught MSc.

Training
Our interdisciplinary project should not be seen as stand-alone, or we will have missed a great opportunity.
- Training of project staff in interdisciplinary approaches to biology (expected timescale: year 1 onwards): The RAs will become experts in a diverse range of skills, integrating with existing projects in the research groups of the project leaders.
- The next generation of scientists (expected timescale: year 1 onwards): Both the RAs will be actively involved in transfer of their skills and knowledge to PhD students, MSc students and undergraduates who will be on projects in the applicant's laboratories.
- Dissemination of skills and expertise (expected timescale: year 1 onwards): Visitors from other laboratories in the UK and internationally will be trained in the new technologies.
- Teaching (expected timescale: year 1 onwards): The project will be key to the development of new models to explain the fundamental principles of bacterial shape, growth and division. Our initial work has overturned the textbook images of cell walls for several organisms. Thus the research will have implications at the level of teaching of microbiology to undergraduates.