Membrane steps in bacterial cell wall synthesis

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.

The peptidoglycan sacculus is embedded in the bacterial cell envelope and is essential to maintain structural integrity of the cell and cell shape. Gram-negative bacteria like E. coli have a thin, mainly single-layered sacculus in their periplasm between the cytoplasmic and outer membrane. Peptidoglycan is made of glycan chains that are cross-linked by short peptides, and its biosynthetic pathway is the target of beta-lactam and glycopeptide antibiotics. The molecular mechanism by which the sacculus is enlarged during growth and division is largely unknown. Particularly poorly understood are the enzymatic and transport steps at the cytoplasmic membrane involving the carrier lipid undecaprenol (pyro)phosphate and the lipid-linked peptidoglycan precursors, lipid I and lipid II.

This proposal aims to build on our preliminary protein-protein interaction data to decipher the molecular mechanisms of these membrane steps. We will reconstitute all membrane steps in proteoliposomes and perform functional assays to test our hypothesis that some of the reactions at the membrane are coupled. We will test for interactions between the membrane proteins involved in the synthesis and transmembrane transport of lipid II, the synthesis and transmembrane transport of the carrier lipid, polymerization of lipid II and recycling of the carrier lipid by pyrophosphatases, and we test the binding of lipid II and carrier lipid to flippase candidates. The biochemical data will be complemented by cellular interaction studies and mutant phenotypes, aiming to decipher the molecular mechanisms of the membrane steps in peptidoglycan synthesis Our expected results will significantly advance our understanding of the molecular mechanisms of peptidoglycan growth, a fundamental, yet poorly understood process in microbiology.

PHARMACEUTICAL COMPANIES: Peptidoglycan biosynthesis is a validated target for antimicrobials such as beta-lactams and glycopeptides, which are amongst the most successful classes of antibiotics. The project aims to follow up extensive preliminary work, in which we have prepared all the known proteins involved in carrier lipid synthesis, utilization and recycling, and peptidoglycan synthesis in the Gram-negative model organism Escherichia coli. Peptidoglycan synthesis is an ideal and established target for antimicrobial therapy. The outcome of this research will become of interest for academic scientists and researchers in pharmaceutical companies who are searching for novel antibacterial compounds to treat infections caused by multi-resistant Gram-negative pathogens. We already have close contacts to the Newcastle biotech company Demuris Ltd. and they have expressed their support for the proposed work and their interest to develop and to commercialise discoveries coming out of the proposed project.

UK ECONOMY: The project involves training of a PDRA to bring the research skills to the pharmaceutical, medical, biotechnological and industries. Within the project the PDRA will receive transferable skills to the benefit of themselves and to the UK intellectual and fiscal economy, including skills important to a modern UK biotechnology industry (protein chemistry, microbiology, bioengineering).

NHS AND PUBLIC: Antimicrobial drug resistance has become an increasingly serious problem in the hospitals, in particular when treating patients suffering from infections caused by Gram-negative pathogens. In the longer term the proposed work could lead to the development of novel antibiotics against drug-resistant strains. The assays developed in this work are useful to either help verifying the precise target of new compounds targeting peptidoglycan biosynthesis, or they can be used in screening assays to identify active compounds. The UK health system and the society in general could therefore benefit from this research.