Membrane steps in bacterial cell wall synthesis

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Bacteria are surrounded by a highly complex cell envelope which contains the essential peptidoglycan layer or sacculus, a net-like molecule made of glycan chains connected by short peptides which surrounds the cell membrane. The sacculus provides mechanical strength to resist the cell's turgor (osmotic pressure) of several atmospheres, protecting the cell from bursting and maintaining its cell shape. Growth and division of a bacterial cell requires the controlled enlargement of the peptidoglycan layer, which involves more than 60 known enzymes and proteins but the precise mechanisms of how they work together to grow the cell wall have remained largely unknown. Peptidoglycan synthesis starts in the cytoplasm with the formation of soluble building blocks. These are equipped with the carrier lipid C55-P (undecaprenol phosphate) leading to the final precursor lipid II, which is transported across the cell membrane and polymerized for sacculus growth. During the polymerisation reaction, the carrier lipid is released, and it gets recycled for further rounds of precursor transport. These membrane steps are poorly understood: the required transporter for C55-P is unknown and the identity of the lipid II transporter (flippase) and some of the lipid II polymerases are currently hotly contested in the field; different integral membrane proteins have been suggested to perform these functions. The proposal aims to decipher the membrane steps in the model bacterium Escherichia coli by determining the activities and interactions of all proteins involved using a combination of biochemical and cellular techniques. We aim to reconstitute all membrane steps alone or in combination in proteoliposomes for functional analysis, and we will back biochemical experiments up with cellular studies on mutant phenotypes and in vivo interactions. We have acquired a large amount of preliminary data for this project. We can already purify all the 'difficult' integral membrane proteins from E. coli in sufficient quantity and reconstitute them in proteoliposomes, and we show that all the enzymes are active.The expected results will substantially expand our knowledge on the molecular mechanisms of peptidoglycan synthesis in the model bacterium Escherichia coli, which is an important pathogen and, according to the Health Protection Agency (HPA), the most common cause of bacteraemia in the UK with ca. 20,000 cases per year. The WHO priority pathogens list for Research&Development of new antibiotics published in February 2017 includes 10 Gram-negative species. Our expected results will be relevant to other Gram-negative pathogens like Haemophilus, Salmonella, Klebsiella, Enterobacter, Serratia and Citrobacter, and to Gram-positive bacteria.The biosynthetic pathway of peptidoglycan assembly is the target of our most important antimicrobials, the beta-lactams (e.g. penicillin) and glycopeptides (e.g. vancomycin). Because peptidoglycan is essential and specific for bacteria, and is not present in humans, it represents an ideal target for antimicrobial therapy. Our research may generate knowledge that could be used to develop novel antibiotics that are urgently needed for the treatment of antibiotic-resistant bacteria the spread of which is increasingly seen as a threat to public health.