Activation of Peptidoglycan Synthesis by Outer Membrane Proteins.
Lead Research Organisation:
Newcastle University
Department Name: Inst for Cell and Molecular Biosciences
Abstract
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.
Technical Summary
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.
Planned Impact
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.
Organisations
- Newcastle University (Collaboration, Lead Research Organisation)
- California Institute of Technology (Collaboration)
- University of California, San Francisco (Collaboration)
- University of Liege (Collaboration)
- Technical University Kaiserslautern (Collaboration)
- European Molecular Biology Laboratory (Collaboration)
- University of Amsterdam (Collaboration)
- IBS Grenoble (Collaboration)
- Fred Hutchinson Cancer Research Center (FHCRC) (Collaboration)
- Utrecht University (Collaboration)
- UNIVERSITY OF BRITISH COLUMBIA (Collaboration)
- University of Washington (Collaboration)
Publications
Biboy J
(2013)
In vitro peptidoglycan synthesis assay with lipid II substrate.
in Methods in molecular biology (Clifton, N.J.)
Chou S
(2015)
Transferred interbacterial antagonism genes augment eukaryotic innate immune function.
in Nature
Denapaite D
(2012)
Biosynthesis of teichoic acids in Streptococcus pneumoniae and closely related species: lessons from genomes.
in Microbial drug resistance (Larchmont, N.Y.)
Derouaux A, Terrak M, Den Blaauwen T, Vollmer W.
(2013)
Bacterial Membranes: Structural and Molecular Biology.
Egan AJ
(2014)
Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B.
in Proceedings of the National Academy of Sciences of the United States of America
Egan AJ
(2013)
The physiology of bacterial cell division.
in Annals of the New York Academy of Sciences
Frirdich E
(2014)
Peptidoglycan LD-carboxypeptidase Pgp2 influences Campylobacter jejuni helical cell shape and pathogenic properties and provides the substrate for the DL-carboxypeptidase Pgp1.
in The Journal of biological chemistry
Jean NL
(2014)
Elongated structure of the outer-membrane activator of peptidoglycan synthesis LpoA: implications for PBP1A stimulation.
in Structure (London, England : 1993)
Jean NL
(2015)
Solution NMR assignment of LpoB, an outer-membrane anchored Penicillin-Binding Protein activator from Escherichia coli.
in Biomolecular NMR assignments
Pilhofer M
(2013)
Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ.
in Nature communications
Description | Bacteria have a complex cell envelope that contains the peptidoglycan layer, also called 'sacculus', as the stress-bearing and cell shape-maintaining structure. Peptidoglycan biosynthesis is targeted by some of our most successful classes of antibiotics, such as the beta-lactams and glycolipids. During growth and cell division Gram-negative bacteria activate their major peptidoglycan synthases, the bifunctional glycosyltransferase-transpeptidase penicillin-binding proteins (PBPs), by outer membrane-anchored lipoproteins (LpoA and LpoB) for sacculus growth. In this project we collaborated with the groups of Jean-Pierre Simorre (Grenoble, France) and Rick Lewis (Newcastle University) to determine the structures of LpoA and LpoB. Our structural data explained how both proteins span the periplasm to interact with their cognate peptidoglycan synthase, PBP1A and PBP1B, respectively. We found that LpoA has an N-terminal tetratricopeptide (TPR)-like domain and a C-terminal PBP1A-interaction domain, and we provided a model of the elongated structure of LpoA based on our experimental data. LpoB has a different structure than LpoA and spans the periplasm with its disordered N-terminal stretch of amino acids, which is followed by the globular PBP1B-interaction domain. We also showed at molecular level how LpoB interacts with PBP1B and determined key amino acid residues that form the interface of the PBP1B-LpoB complex. We established that the interaction with LpoB is essential for the activation of PBP1B in the test tube and its functioning in the cell. Our combined results allowed us to propose a new model for cell wall growth regulation in Gram-negative bacteria. According to this model, the Lpo activators penetrate the pores in the sacculus to stimulate their cognate peptidoglycan synthases and, hence, the extent of stimulation depends on the physical properties of the pores. This mechanisms allows for stronger activation of peptidoglycan synthesis in fast growing cells in which a high turgor stretches the sacculus causing larger pores. We also used our experience in the analysis of peptidoglycan composition and enzyme assays within other research collaborations. In collaboration with Joseph Mougous (Seattle, USA) we discovered that several ancestors of ticks and mites had acquired and functionalized bacterial peptidoglycan hydrolase genes and that current species of some of the modern lineages express these genes to control bacterial colonization. With Joseph Mougous we also determined the structure and activity of a type VI secretion system effector peptidoglycan hydrolase. With Grant Jensen (Caltech, USA) we discovered the presence of peptidoglycan in chlamydia, which are intracellular bacteria that have long been thought to lack peptidoglycan. With Nina Salama (Seattle, USA) and Erin Gaynor (Vancouver, Canada) we reported the effects of peptidoglycan hydrolase gene deletions on cell shape in Helicobacter pylori and Campylobacter jejuni, respectively. With Regine Hakenbeck (Kaiserlautern, Germany) we proposed a model for teichoic acid biosynthesis pathway in Streptococcus pneumoniae. In summary, the project generated knowledge about how bacteria grow and divide. We discovered molecular mechanisms underlying bacterial cell wall growth and how cell wall growth is regulated by outer membrane proteins, which led to a novel mechanism of cell wall growth regulation. We also advanced the knowledge in cell wall biosynthesis and the roles of cell wall targeting enzymes in other bacterial species. |
Exploitation Route | 1. Researchers working on bacterial growth benefit from the basic knowledge generated. 2. Researchers searching for novel antibacterial compounds benefit from the advance in basic microbiology. 3.The public benefits from the results by information obtained from the media and University website. |
Sectors | Pharmaceuticals and Medical Biotechnology |
Description | Carol Gross |
Organisation | University of California, San Francisco |
Department | Radiology and Biomedical Imaging at UCSF |
Country | United States |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Carol Gross on bacterial cell envelope. We contributed our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise bacterial envelope and stress response, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 25951518 PMID: 22203377 PMID: 21183073 |
Start Year | 2009 |
Description | Eefjan Breukink |
Organisation | Utrecht University |
Department | Rudolf Magnus Institute |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Eefjan Breukink on bacterial cell wall proteins. We contribute our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributes their expertise in peptidoglycan enzymes and precursor synthesis, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 30504892 PMID: 30233559 PMID: 30093673 PMID: 30046664 PMID: 30044025 PMID: 28233869 PMID: 27709766 PMID: 27257764 PMID: 26370943 PMID: 25951518 PMID: 24821816 PMID: 22606933 PMID: 22487093 PMID: 21472954 PMID: 21183073 PMID: 17938168 PMID: 16840781 PMID: 16154998 |
Description | Erin Gaynor |
Organisation | University of British Columbia |
Department | Department of Forest Resources Management |
Country | Canada |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Erin Gaynor on cell walls synthesis in Campylobacter. We contributed our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in Campylobacter biology, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 31070821 PMID: 28316093 PMID: 27474744 PMID: 24394413 PMID: 22457624 |
Start Year | 2011 |
Description | Grant Jensen |
Organisation | California Institute of Technology |
Department | Caltech Astronomy |
Country | United States |
Sector | Academic/University |
PI Contribution | Expertise in bacterial cell wall analysis. Performed cell wall analysis. |
Collaborator Contribution | Expertise in Chlamydia biology, electron microscopy. |
Impact | Scientific publications: PMID: 24292151 PMID: 20435724 |
Start Year | 2009 |
Description | Jean-Pierre Simorre |
Organisation | IBS Grenoble |
Country | France |
Sector | Private |
PI Contribution | This is a long-term scientific collaboration with Jean-Pierre Simorre on bacterial cell walls and proteins. We contributed our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in NMR spectroscopy for the analysis of bacterial cell walls and protein structure determination, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 30713527 PMID: 30046664 PMID: 30044025 PMID: 30031884 PMID: 25951518 PMID: 24954617 PMID: 24821816 PMID: 24691651 PMID: 24493340 PMID: 22192687 PMID: 20681725 PMID: 18393418 |
Description | Joe Gray |
Organisation | Newcastle University |
Department | Newcastle University Medical School |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Joe Gray on bacterial cell walls and proteins. We contributed our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in mass spectrometry for the analysis of bacterial cell walls and proteins, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 31916938 PMID: 31209025 PMID: 30713527 PMID: 30102748 PMID: 30066424 PMID: 29311645 PMID: 28974693 PMID: 28581639 PMID: 27506799 PMID: 26537571 PMID: 26010014 PMID: 25424554 PMID: 24292151 PMID: 18996994 |
Start Year | 2007 |
Description | Joseph Mougous |
Organisation | University of Washington |
Department | Division of Medical Genetics |
Country | United States |
Sector | Academic/University |
PI Contribution | Expertise in bacterial cell wall. Experiments to determine peptidoglycan composition and enzymatic assays of peptidoglycan enzymes. |
Collaborator Contribution | Expertise in a the molecular biology of a variety of bacterial species, and in bacterial secretion systems. Experimental work on bacterial secretion systems. |
Impact | Scientific publications: PMID: 28696203 PMID: 25470067 PMID: 23878199 PMID: 22813741 PMID: 22607806 PMID: 21776080 |
Start Year | 2011 |
Description | Mohammed Terrak |
Organisation | University of Liege |
Department | Liege Space Center |
Country | Belgium |
Sector | Academic/University |
PI Contribution | This is a scientific collaboration with Mohammed Terrak on the biochemistry of cell wall enzymes based on our common interest in the bacterial cell wall. We contributed our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in enzymology, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 28233869 PMID: 22606933 PMID: 17938168 |
Start Year | 2006 |
Description | Nassos Typas |
Organisation | European Molecular Biology Laboratory |
Department | EMBL-Grenoble |
Country | France |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Nassos Typas on bacterial cell envelope biogenesis. We contributed our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in bacterial cell envelope, stress responses and high throughput techniques, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 25951518 PMID: 24821816 PMID: 22203377 PMID: 21183073 |
Start Year | 2009 |
Description | Nina Salama |
Organisation | Fred Hutchinson Cancer Research Center (FHCRC) |
Country | United States |
Sector | Academic/University |
PI Contribution | Expertise in bacterial cell wall biochemistry and analysis. We performed peptidoglycan analysis in Helicobacter pylori, and measured the activities of cell wall enzymes. |
Collaborator Contribution | Expertise in the biology of Helicobacter. Performed experiments with Helicobacter cells. |
Impact | Scientific publications: PMID: 20510929 PMID: 22457625 PMID: 24112477 PMID: 31916938 PMID: 31036730 |
Start Year | 2009 |
Description | Regine Hakenbeck |
Organisation | Technical University Kaiserslautern |
Department | Department of Computer Science |
Country | Germany |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Regine Hakenbeck on bacterial cell wall synthesis and antimicrobial resistance. We contributed our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in bacterial cell wall synthesis and antimicrobial resistance, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 28483958 PMID: 26010014 PMID: 25676688 PMID: 22432701 PMID: 18621904 PMID: 16885447 |
Start Year | 2015 |
Description | Rick Lewis |
Organisation | Newcastle University |
Department | Newcastle University Medical School |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Rick Lewis on bacterial cell wall proteins. We contributed our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in x-ray crystallography for the analysis for protein structure determination, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 27862967 PMID: 27329754 PMID: 27257764 PMID: 25403286 PMID: 24954617 PMID: 24909784 PMID: 22432711 PMID: 21964069 PMID: 19074383 |
Start Year | 2007 |
Description | Tanneke den Blaauwen |
Organisation | University of Amsterdam |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | This is a long-term scientific collaboration with Tanneke den Blaauwen on cell envelope biogenesis in E. coli. We contribute our expertise in bacterial cell wall structure and biosynthesis, provided research tools and performed experiments. |
Collaborator Contribution | The partner contributed their expertise in bacterial cell biogenesis and cellular localization of proteins, provided research tools and performed experiments. |
Impact | Publications in scientific journals: PMID: 30233559 PMID: 26124755 PMID: 25951518 PMID: 23387922 PMID: 22606933 PMID: 21472954 PMID: 21183073 PMID: 20545860 PMID: 16803586 |