Defining the O-antigen biosynthetic pathways in zoonotic Coxiella burnetii: studies of dTDP-sugar biosynthesis and LPS extraction

点击翻译按钮获取中文摘要

This project will examine the biosynthesis of the unusual sugars virenose and dihydrohydroxystreptose by the human and ruminant pathogen Coxiella burnetii. The experimental approaches will cover three main aims:1) To determine the pathway to synthesis for NDP-virenose and NDP-dihydroxystreptose2) To validate these pathways in vitro using purified enzymes to establish each proposed activity3) To probe the structure-function relationship of unusual enzymes in these pathwaysAim 1 will principally use mass spectrometry (MS). Coxiella will be grown at Dstl in defined conditions, and the cellular metabolites extracted for MS. Nucleotide sugars will be isolated using solid phase extraction with graphitised carbon, followed by ion-exchange chromatography. These will then be examined by LC-MS; or hydrolysed to release the sugars, which will then be silylated for analysis by GC-MS, with particular emphasis on searching for intermediates in putative pathways (Flores-Ramirez et al. 2012 Proteome Sci. 10, 67). These experiments will be supplemented by studies using isotopically labelled growth substrates to verify the proposed pathways.For aim 2, the relevant genes will be synthesised with codon optimisation for E. coli, and cloned into a set of vectors offering a range of solubility tags (vectors obtained from the Oxford Protein Production Facility). These will be expressed in small scale, and optimal constructs taken forward for high level expression and purification. Purification is likely to proceed through nickel affinity chromatography and size exclusion chromatography, followed by removal of the tags and a final purification to remove the tag and cleavage enzyme. Nucleotide binding will be determined using differential scanning fluorimetry or tryptophan fluorescence, and validated by more accurate methods (e.g. isothermal titration calorimetry, microscale thermophoresis) to determine dissociation constants. The reaction of each enzyme will be assessed using appropriate enzymatic assays, using either continuous, stopped, coupled assays, or a mixture of these, to obtain best results. Nucleotide-sugar precursors for the likely reaction paths are readily commercially available. To verify reaction outcomes, the products will be purified where stable using HPLC and confirmed with mass spectrometry; for less stable intermediates, coupled reactions will be used, with the products again verified as before.Aim 3 will use protein purified for the work in aim 2. Crystallisation will be performed using our Douglas crystallisation robot, with a set of four standard crystallisation screens. Crystallisation will be performed in complex with relevant nucleotides or nucleotide sugars; and with other cofactors, substrates/substrate analogues or products where appropriate. For experimental phasing, proteins will be grown selenomethionine labelling where appropriate; or heavy atoms will be added (preferably through halide soaking, following which conventional heavy atom compounds will be used).Milestones:12 months: identify metabolic intermediates using GC-MS and use isotopically labelled substrates to confirm metabolic pathways; clone enzymes (expect that time at Dstl will be spent in year 1).24 months: enzymatic verification of virenose biosynthesis; initial crystallography.36 months: enzymatic verification of dihydrohydroxystreptose biosynthesis; crystallography of enzyme-substrate complexes.48 months: final experiments, completion of thesis.