Cell surface display of bacterial proteins

The proteins exposed at the cell surface of bacteria play a pivotal role in a multitude of fundamental processes such as cell growth and division. In pathogens, surface proteins promote host invasion and later during infection, evasion from the host immune response. Most surface proteins are anchored to a scaffold molecule called peptidoglycan, an essential component of the bacterial envelope. Peptidoglycan is a bag-shaped giant molecule surrounding the cell that made plays a protective role. Its synthesis is unique to bacteria and represents the target of the most important antibiotics ever discovered such as penicillin. To date, a plethora of surface proteins has been described but how they interact with the bacterial envelope remain poorly understood. We have recently described the molecular basis for peptidoglycan recognition by a ubiquitous protein domain called LysM in vitro using synthetic ligands. We are now ready to take the next step to actually understand how LysM proteins bind to the complex peptidoglycan molecule in live bacteria and are targeted to specific subcellular localizations. To achieve this aim, we will use a multidisciplinary approach combining bacterial genetics, state-of-the-art microscopy and biophysics.
Binding of LysM domains to the PG molecule represents a key bacterial mechanism for controlling the location, synthesis, degradation and decoration of cell wall features. The understanding generated by this project will give us the tools to dissect how bacteria grow, divide and interact with their environment, to develop new therapeutic strategies, and to learn how to apply synthetic biology to bacterial cell walls.

The proteins displayed at the bacterial surface play key roles in fundamental processes such as cell growth and division. In pathogens, surface proteins promote host invasion and evasion from the host immune response. Core to all these functions is the ability of the bacterial proteins to bind to their own cell wall and thus be appropriately displayed for interaction with the environment. This project will focus on a ubiquitous domain (LysM) that mediates non-covalent binding of proteins to peptidoglycan (PG), the essential component of the bacterial cell wall which is the target of most important antibiotics ever discovered, penicillin. I recently elucidated the molecular basis underpinning LysM-PG interaction in vitro using the multimodular LysM domain from a PG hydrolase in the opportunistic pathogen Enterococcus faecalis.
Our unpublished data indicate that LysM proteins are targeted to distinct sites at the cell surface. The aim of this project is to understand how this is achieved. We will undertake a multidisciplinary approach combining microbial genetics and super-resolution microscopy to study the subcellular targeting of LysM-GFP fusions in live bacteria. We will explore the impact of LysM domains properties (e.g., modularity, charge), secretion signals and cellular factors (membrane potential and PG-associated polymers) to the subcellular localization of proteins. We will also investigate the functional diversity of naturally occurring LysM domains to understand how PG structure modulates binding. Finally, we will characterize the role of newly identified components in LysM-mediated surface display.
This project will show how different bacteria use their LysM domains to recognise and target different features of peptidoglycan. Enzymes linked to LysM are used by bacteria to remodel the cell wall, build and destroy septa, and in pathogenesis. The project will produce a toolkit to map bacterial surface features, and target novel features for drug design.

The aim of this project is to understand how ubiquitous LysM domains mediate non-covalent cell surface display of proteins in bacteria. This project will show how different bacteria use their LysM domains to recognise and target different features of peptidoglycan. Enzymes linked to LysM are used by bacteria to remodel the cell wall, build and destroy septa, and in pathogenesis. The project will produce a toolkit to map bacterial surface features, and target novel features for drug design, an area of research of immense public concern. There will be a variety of impacts over a range of timescales and in different arenas.
(i) Impact on academic researchers (short to medium term). The work will directly benefit UK and international microbiologists. Due to the multidisciplinary nature of the project, we also expect this work to benefit a wide range of researchers including structural biologists, biophysicists, cell biologists and scientists working on the development of novel antimicrobials. We will communicate our findings through open access publications in peer-reviewed journals, presentations at symposia, seminars, and national/international conferences. All the protocols and biological materials generated during this work will be made available through the PI's website that will be regularly updated in a timely manner.
(ii) Impact on RA and technician employed (short term). Both RA and technician will acquire a wide range of skills to improve their employability, thereby having a direct impact on the knowledge-led economy of UK Plc. The skills will be transferred to students working on related projects, as well as to visiting scientists. The RA will also benefit from the support offered through the action plan for continued implementation of the Concordat to support the career development of researchers. The RA will be encouraged to undertake training offered for personal professional development by the Researcher Personal Development Team at the University of Sheffield.
(iii) Biotechnology and pharmaceutical companies developing and commercialising antimicrobials (short to medium term) -translational fusions between acidic/basic LysM motifs and the catalytic domain of colicin M wich cleaves peptidoglycan lipid precursors at the outer face of the cytoplasmic membrane will provide the proof of concept that LysM domains can be used as a means to target therapeutic molecules to pathogens. We will have a strong commitment to secure any intellectual property (IP) associated with this work and maximise the opportunities to exploit translation of research findings into commercial applications. Once IP has been secured, we will consider the opportunity to create a spin-out company with the support of the Commercialisation team at the University of Sheffield or licensing out. This project will provide the opportunity to initiate collaborations with established companies to seek funding in partnership with the support of Sheffield Science Gateway which offers several schemes. A contact has been established with Lisando GmbH to explore potential partnership.
(iv) The public, as potential users of the research (short to long term). The expected impact of this project (long term) is to increase the effectiveness of the health service offered by the NHS and enhance the quality of life and health in the UK and worldwide. This will be achieved through commercialisation of novel therapeutic molecules generated, with the PI and RA also being involved in various outreach activities (school visits and promotion of our research during work and family focussed scientific events).