Abstract

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.