Understanding phopspholipid homeostasis in Gram-negative bacteria

Lead Research Organisation: University of Birmingham
Department Name: Immunity and Infection


For simplicity, all cells can be considered 'bags' of chemicals, where the chemicals encode the material required for the cell to replicate, to obtain and utilise nutrients, and essentially survive within its habitat. In all cases, the 'bag' that contains these chemicals is made up of fats called phospholipids; the correct term for the 'bag' is the cell membrane. Different organisms have different cell membrane properties. Some of the most dangerous bacteria have two cell membranes, simply termed an inner and an outer membrane. The outer membranes are studded with proteins which form gated holes in the membrane to allow water and nutrients to flow into the cell and waste products to be pushed out of the cell. The outer membranes of these bacteria are also decorated with a very specialised lipid called lipopolysaccharide that makes the membrane very strong and allows the bacteria to resist many antibiotics.
By understanding how bacteria make their outer membranes, and decorate it with proteins and lipopolysaccharide we can develop drugs that will either kill the bacteria or prevent it from growing. For several years we and other groups have study how the proteins and lipopolysaccharide is incorporated into the outer membrane. However, to date nobody has figured out how phospholipid, which is made by the chemicals inside the cell, can make its way across the inner membrane and into the outer membrane. Finding out how this happens is extremely important; if we can figure out how these phospholipids are trafficked to the outer membrane then we may be able to develop drugs that stop this process, ultimately killing the bacteria.
Fortunately, we have recently found a protein in the outer membrane that binds one of the phospholipids. We have good evidence that this protein receives phospholipid into the outer membrane and if you stop this protein from functioning the bacteria become very sensitive to antibiotics. We have shown that bacteria that lack this protein are unable to cause disease and interestingly this protein can be used safely and effectively in vaccines to help the body fight bacterial infections. This is the first time anyone has found a protein with such activity. Therefore, the overall aim of this project is to determine how bacteria recognize and organize lipids into their outer membrane.
We will tackle this problem in a number of different ways. We will investigate whether the protein is capable of recognising all of the different lipids found within the outer membrane. We will investigate if the protein organizes the lipids into specific places within the outer membrane that might be important for the outer membrane in resisting antibiotics. We will investigate if this protein requires partner proteins to be effective at its job. We will use many different techniques to address this problem including genetics, biochemistry and structural biology where we elucidate the 3-dimensional structures of proteins. Ultimately, this project will shed light on a pathway that many researchers have been looking for and it will allow industry to begin to exploit this knowledge to stop or prevent infections.

Technical Summary

Our data demonstrate YraP, a conserved Gram-negative bacterial protein, binds phosphatidylglycerol but not phosphatidylethanolamine, is required for phospholipid homeostasis in the outer membrane, is required for in vivo viability, and has a number of extragenic suppressors of synthetic lethality, most of which have known or predicted roles in maintaining lipid homeostasis in Gram-negatives. We predict that YraP is a component of the antegrade phospholipid trafficking pathway that specifically recognises anionic phospholipids.
In this project we will ask:
Does YraP interact directly with cardiolipin, another anionic phospholipid?
Does loss of YraP perturb levels of CL in the OM?
Does loss of YraP perturb levels of phospholipids present in the IM?
Can deletions in other genes suppress yraP sensitivity to vancomycin or SDS?
Do suppressor mutations restore the balance of phospholipids in the OM?
Does YraP show spatial organisation?
Does YraP co-localise with specific phospholipids?
Does YraP interact directly with proteins encoded by genes identified in the suppressor screening?
Can protein binding partners be identified through non-biased pull-down experiments?
Do other BON domain containing proteins in E. coli bind and traffic PE?
What genes are synthetically lethal with YraP?

Planned Impact

Academic Community: The scientific community will be engaged by primary journal articles, reviews, web sites, exchange visits and access to research products and services. Henderson, Viant and Overduin have produced over 50 manuscripts since 2008, including papers in EMBO R, Nature Str Mol Biol, PNAS USA and Science. Open access journals will continue to be the medium for communicating our results. The team will continue to presents at and organise conferences We will run a focused workshops each year to transfer knowledge and skills for metabolomics/lipidomics to the community. The project will be advertised via Henderson's active Twitter feed followed by hundreds of academics internationally. We will also promote the project online by establishing a webpage for the project within the Institute Research Pages.
Commercial Sector: The tools and methods being for lipidomics being used and developed during this project will be communicated to and shared with Pharmaceutical and biotech companies through our workshops. Henderson, Overduin and Viant have many mature collaborations with industry e.g. Novartis, GSK, AstraZeneca, Thermo. We will utilise these contacts to promote this project. Notably, Henderson has already established collaborations with Novartis on the topic of this project as we seek to understand the molecular basis of the adjuvanticity of YraP in te Bexsero vaccine.
Wider Public: The PIs will continue to play an active role in promoting the public understanding of science, and have contributed to stories in the Birmingham Post, Business Desk, Guardian, Information Daily, Telegraph, BBC-WM and Research TV in the past several years. Overduin is a member of the steering group of the British Science Association, which held a Science Festival in Birmingham in Sept 2010, and every four years thereafter, and runs events for this and University Open Days. Lab and facility tours will continue to given by Henderson, Viant and Overduin to the community at eh University open days and to secondary school students through our Microbiology summer schools.
Overduin also volunteers as Chair of the Science and Medicine Forum of the Lunar Society, a scientific body which was originally founded in the West Midlands in 1775. He serves on its Executive Committee, helping to organize monthly lectures and public events with attendances of up to 600 people. Speakers he has hosted include the Nobel Laureate Paul Nurse, President, Rockefeller University and Sir Liam Donaldson, Chief Medical Officer. This provides an avenue to present results on pharmaceutical research and genetics, proteomics and systems biology of disease, helping to overcome public anxiety about pathogenic diseases, and demonstrating the benefits of new technologies and drug discovery through academic - industrial collaborations.
Description YraP is a lipid binding protein conserved in all gram -negative bacteria that is associated with cell division
Exploitation Route novel antimicrobials
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description Lipid translocation 
Organisation National University of Singapore
Country Singapore 
Sector Academic/University 
PI Contribution We have provided microbial genetics support to the partner, identifying new candidates for lipid transport between Gram-negative bacterial membranes
Collaborator Contribution The partner has assisted in training the postdoctoral fellow in lipid analysis techniques
Impact multidiscipliniary
Start Year 2016
Description lipid translocation 
Organisation New York University
Country United States 
Sector Academic/University 
PI Contribution genetics experiments and lipid analysis
Collaborator Contribution cryo-EM and structural biology
Impact Paper in Cell describing the structures of the major lipid trafficking pathways in gram-negative bacteria
Start Year 2016