Bacterial sphingolipids - revealing hidden biosynthetic pathways of key players in host-microbe interactions.

Animal and bacterial cells have a protective, water-resistant outer shell that is composed of molecules with a water-loving (hydrophilic) head group and a long, water-hating (hydrophobic) tail. This large family of molecules are called lipids and include common things like saturated/unsaturated fats and cholesterol. One particular sub-family of lipids is called sphingolipids (SLs) and their more complex ceramide versions (which have two fatty tails). The SLs not only play structural roles in the outer shell that allow the cell membrane to resist water and let nutrients in and waste out; they are also able to stimulate the human immune system. SL levels are dynamic but also tightly controlled - any increase or decrease in the cellular SL levels is a sign that something has gone wrong. Changes in SL levels are strongly linked with old age and diseases such as Alzheimer's, diabetes, asthma, cancer and nerve-wasting diseases. An exciting area of research with direct implications for human health is the discovery that humans are hosts for many different types of bacteria - collectively these are known as the microbiota/microbiome. Current estimates are that for every human cell in our body, there is a bacterial one. These bacteria can be "bad" and cause disease (e.g. superbugs) but most are "good" bacteria and are beneficial to our well being. These bacteria live in our mouths, on our skin and in our gut and help us metabolise our food and are also thought to play protective roles. A surprising discovery was that the bacteria that live with us produce molecules that allow bacterial and human cells to communicate. One such family of molecules are the SLs - it is highly unusual that human and bacterial cells both make the same molecule and this suggests some sort of evolutionary link. Moreover, it has been calculated that we have several grams of SLs in our gut at any one time and they are making a vital contribution to our health. Recent studies have linked the microbiota to diseases such as diabetes, obesity and cancer.
All cells make SLs by a multi-step pathway using simple building blocks - the steps are catalysed (sped up) by molecular machines called enzymes. Research has focussed on the enzymes involved in human SL biosynthesis but very little is known about SL biosynthesis in the microbiota. To fully understand the relationship between us and bacteria we must learn how bacteria make and transport such complex molecules as well as understanding how we metabolise them. We will study how gut and mouth bacteria make SLs with world experts in America and Germany with a collaborator from the UK. We will begin with a study of the enzyme serine palmitoyltransferase (SPT) that uses two main building blocks - an amino acid called L-serine and a long chain fatty acid, to make the first SL intermediate. We will determine the 3D structure of the SPT in each bacterium and compare their shapes and evolution. Of special interest, the structure of the bacterial SLs is unusual and contains distinctive chemical fingerprints and we will investigate their origins by feeding the bacteria heavy versions of the proposed building blocks and tracking their incorporation. Nothing is known about how the microbiota makes unusual branched chain SLs so we will study enzymes that convert can branch-chain amino acids into specific building blocks. Bacteria contain ceramides with an unsusual inositol sugar so we will purify and characterise the enzyme myo-inositol phosphate synthase (MIPS) that uses glucose phosphate as a substrate. At the end of our study we will have begun to define the biosynthetic blueprint of the microbiota. Our results will be of interest to academic microbiologists and chemists as well as those interested in human health. Moreover, a number of drug and healthcare companies are also interested in the microbiome and they could use our knowledge to develop therapies that may have impact on disease and long term well being.

Sphingolipids (SLs) and ceramides play essential roles in membrane structure and cell signalling. They are found in yeast, plants, mammals and some bacteria (e.g. human microbiota). The microbiota has gained attention since be linked to maintaining human health. Recently, members of the microbiota (Bacteroides, P. gingivalis) have been shown to produce SLs that mediate interactions with host cells. In contrast to higher eukaryotes, very little is known about microbial SL biosynthesis, regulation and transport. To fully understand the molecular details of the SL-mediated microbiota/human interaction we must first decipher the mechanisms that govern the biosynthesis and metabolism of these molecules. Our hypothesis is that the microbes of the human microbiota make sphingolipids by a novel biosynthetic pathway that shares common elements derived from prokaryotes and eukaryotes.
The chemical structure of a mature bacterial SL (such as galactosylceramide, GSL) suggests a concise biosynthetic pathway that draws precursors (amino acids, fatty acids and sugars) from primary metabolism. The microbiota GSL contains a diagnostic iso-Me branched fingerprint in the fatty acid moiety of the SLs. This suggests a number of key steps catalysed by a suite of interesting enzymes which will be the focus of our study. Specifically we will target three key steps in the pathway; firstly, the crucial enzyme serine palmitoyltransferase (SPT) that combines L-serine and fatty acids to form the SL backbone; secondly, identify the origin of the branched-chain fingerprint of microbiota SLs; and thirdly, characterise the enzyme that converts glucose 6-phosphate to inositol phosphate which generates complex inositol SLs in certain bacteria. In collaboration with experts in microbiology and structural biology we will use protein chemistry, enzyme assay, analytical chemistry, X-ray crystallography, genetic screens and mutagenesis as tools to deliver our aim to map bacterial SL biosynthesis.

Wider beneficiaries
This project will have far reaching impacts with a wide group of beneficiaries. As well as the training opportunities for our team of UK-based scientists in microbiology, protein biochemistry and structural biology, we will interact closely with our European (Ley) and USA project partners (Davey & Dunn) to transfer knowledge and expertise between our research groups. Our link with Lipid Maps will spread our results to >65000 members.
The key role of the microbiota in human and animal health and wellbeing is undisputed, and there is growing evidence that bacterial sphingolipids have a significant impact on host-microbiota tolerance and the development and maturation of the immune system. The basic understanding generated in this project will have the following wider beneficiaries and resulting impacts:

Healthcare and Medicine
Understanding the pathway for the production of bacterial sphingolipids will benefit clinicians and dentists interested in the effect of the oral microbiota on the progression of periodontitis. The role of bacterial sphingolipids in human and animal health will be explored with our project partners Davey and Ley, and this work will have impacts for clinicians working on the interplay between the gut microbiota and the development of the host immune system. This research may inform policy on recommendations for diet and supplementation to mitigate the role of the microbiota on periodontitis and auto-immune gut diseases, such as Crohn's disease.
Patient advocacy groups could benefit through direct access to knowledge and expertise generated in this project to inform clinical trials and treatments for diseases related to sphingolipid biosynthesis and autoimmune conditions. An example of the type of group who would benefit is the Deater Foundation. This charity seeks to research a neuropathy caused by mutations in the human sphingolipid biosynthesis pathway and access to research into this, has directly led to clinical trials of amino-acid supplementation as a targeted therapy against the clinical presentations of their specific mutations in this pathway.

Pharmaceutical Companies
The identification of the unique enzymes responsible for the various stages of the bacterial sphingolipid biosynthesis pathway and their structural characterisation will present novel drug targets for the development of new compounds to interfere with their action. Companies and researchers working in the pharmaceutical industry and in medicinal chemistry will therefore benefit from the knowledge created in this project.

Industrial biotechnology
Sphingolipids and their precursors have a high value as research tools in academic, clinical and industrial laboratories. But due to the difficulty associated with producing and isolating them from their native organisms they are extremely costly. Through understanding the bacterial sphingolipid pathway, we will produce knowledge that will allow the development of industrial strains optimised for the production of large quantities of sphingolipids and ceramides. The development of robust production hosts and lipid isolation methods by industrial biotechnology companies (e.g. Avanti) will have significant economic and scientific impacts.

Microbiome/Microbiota Industry
The biotechnology industry that has grown around microbiome/microbiota research is in an embryonic stage at present, but by 2025 is estimated to be worth up to $10bn. While there are currently no directly marketed microbiome/microbiota modifying drugs, there are many companies actively working in the pre-clinical and clinical stages of development. Furthermore, many companies are working in the neutraceutical and probiotic spaces. Our research will open possibilities to develop drugs to manipulate the production of sphingolipids in the human and animal microbiota; it will also inform the development of neutraceuticals and probiotics to stimulate, or inhibit, sphingolipid biosynthesis.