Activation of Peptidoglycan Synthesis by Outer Membrane Proteins.

The bacterial cell wall peptidoglycan forms a continuous layer, a so-called 'sacculus', in the envelope of most bacteria and it is essential to maintain cell integrity and cell shape. Gram-negative bacteria like Escherichia coli have a very thin and mainly single-layered sacculus, which is sandwiched between the two cell membranes, the inner, cytoplasmic membrane and the outer membrane. Growth and division of a bacterial cell requires the controlled enlargement of the peptidoglycan layer, which involves more than 50 known enzymes and proteins but the precise mechanisms have remained largely unknown. Data from our own and other laboratories favour a model in which the sacculus is enlarged by multi-enzyme complexes made of peptidoglycan synthases and hydrolases, which are controlled from inside the cell by components of the bacterial cytoskeleton. This model has now been revised based on our recent, exciting results: our work has shown that peptidoglycan synthesis is also controlled from outside the sacculus by novel lipoproteins, LpoA and LpoB, which are anchored to the outer membrane and which interact with, and activate, the major peptidoglycan synthases. The discovery of these outer membrane activators has dramatically changed our view on peptidoglycan growth and, according to our hypothesis, suggests a mechanism by which bacteria regulate the surface density of their peptidoglycan layer during growth. There are many unanswered questions on the Lpo-mediated activation of peptidoglycan synthases which will be addressed in this research proposal. Within the proposed project we aim to clarify the physiological role of the new peptidoglycan synthesis activators, the importance of their localization to the outer membrane and their effect on the activities of peptidoglycan synthases and of peptidoglycan synthesis complexes. We will further study the interactions between the activators and their docking domains in the peptidoglycan synthases, and we aim to determine the co-crystal structures of Lpo-docking domain complexes. This part will provide insights into the mechanisms of Lpo-mediated activation of peptidoglycan synthases. Finally, we will explore the possibility that outer membrane peptidoglycan synthase activators also interact with and affect the activities of peptidoglycan hydrolases, an appealing hypothesis for which we have preliminary data. The project will involve a variety of molecular biology, biochemistry and structural biology techniques. 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. Our expected results will be relevant to other Gram-negative bacteria including pathogens like Haemophilus influenzae, which is known to have an essential lpoA gene, Salmonella, Klebsiella, Enterobacter, Serratia and Citrobacter. The biosynthetic pathway of peptidoglycan assembly is the target of our most important antimicrobials, the beta-lactams (like penicillin) and glycopeptides. 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. However, preliminary data show that peptidoglycan synthases and hydrolases form membrane-anchored multi-enzyme complexes which are, by an unknown mechanism, controlled by cytoskeletal elements that form dynamic, intracellular filaments. This proposal aims to follow up an important aspect of peptidoglycan synthesis in Gram-negative bacteria recently discovered by our laboratory, the activation of the major peptidoglycan synthases from the outside of the sacculus by the new outer membrane lipoproteins LpoA and LpoB. The identification of outer-membrane activators of peptidoglycan synthases has led us to propose a hypothetical mechanism by which Gram-negative bacteria constantly adjust peptidoglycan growth rate to overall cell growth rate to maintain a homogeneous peptidoglycan surface density. The proposed research aims to establish the physiological role of the Lpo proteins in cell elongation and division by assaying the essentiality of outer membrane-localization of these activators at various growth conditions. We will further investigate the molecular and structural details of the interactions between Lpo proteins and peptidoglycan synthases, and we will explore if Lpo proteins interact with and affect peptidoglycan hydrolases. Our expected results will significantly advance our understanding of the molecular mechanisms of peptidoglycan growth, a fundamental, yet poorly understood process in microbiology.

Scientists working on peptidoglycan synthesis will benefit from this research as they will receive new knowledge about important, yet unanswered questions in microbiology. The mechanisms by which bacteria enlarge their cell wall during growth and cell division is poorly understood, and the proposed project will significantly expand the knowledge of these essential processes. The expected results are also of great interest to other scientists working on analogous systems in plants and fungi. The methodologies and technologies used in this proposal will be of interest for scientists who work on the biosynthesis of biological macromolecules and multi-enzyme complexes. The project aims to follow up the recent discovery of outer-membrane peptidoglycan synthase activators 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 pharma companies who are searching for novel antibacterial compounds to treat infections caused by multi-resistant Gram-negative pathogens. We have already 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 (letter attached). Thus, in the longer term, the UK health system and the society in general could benefit from this research which could lead to the development of novel antibiotics against drug-resistant strains. The knowledge generated by the proposed research will be disseminated by publication in high impact, peer reviewed scientific journals, making it available for the scientific community. In the case of a major discovery, the Press Office of Newcastle University will produce a press release to inform the general public via the media, and the information will be given on the University's website. The results will also be presented on national and international conferences and in seminars at other institutions. Before public presentation and publication, any result that could be commercially exploited will be evaluated by the experienced commercial development team of Newcastle University which will develop an appropriate protection and commercialisation strategy.