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Chapter 29 : Campylobacter Metabolomics
Category: Bacterial Pathogenesis
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The ultimate goal in metabolomics is to achieve unbiased identification and quantification of all the metabolites in a defined biological system. Much of the work in bacterial metabolomics has involved the study of well-established metabolic pathways such as the tricarboxylic acid cycle, glycolysis, and specific metabolic pathways of microorganisms used in industrial applications. In contrast, the field of Campylobacter metabolomics is very much in its infancy, and considering the lack of information on many of the novel glycoconjugate biosynthesis pathways in Campylobacter, there is much scope to use targeted metabolomics approaches to further define the substrates and genes involved in these metabolic pathways. The main challenges associated with the study of sugar nucleotide metabolites by nuclear magnetic resonance (NMR) have been the instability of the sugar nucleotides and their presence at low concentrations within the bacterial cells. UDP-α-D-QuiNAc4NAc is an important metabolite in the 2,4-diacetamido-bacillosamine biosynthesis pathway, and its accumulation in pseC had not been expected because it has been thought that the inactivation of pseC would lead to an accumulation of a novel precursor directly related to Pse5Ac7Ac biosynthesis. The focused metabolomics studies of flagellin glycosylation in Campylobacter jejuni 81-176 and Campylobacter coli VC167 were extensive and examined unknown gene functions, characterized novel biosynthetic substrates and novel flagellar glycans, and elucidated poorly understood metabolic pathways.
Prokaryotic biosynthetic pathway of N-acetylneuraminic acid (Neu5Ac) and the corresponding putative biosynthetic pathway of pseudaminic acid (Pse5Ac7Ac).
CE-ESMS and precursor ion scanning for fragments related to CMP (m/z 322) or UDP (m/z 385) activated sugars in cell lysates. (a) Wild-type C. jejuni 81-176. (b) Isogenic mutant pseC. (c) Isogenic mutant pseA.
Comparison of the ratio of signal to noise (S/N) for a UDP-6-deoxy-α-d-GlcNAc4NAc metabolite sample isolated from C. jejuni 81-176 showing the advantages of the use of a higher-field NMR spectrometer and cold probe. (a) 1H NMR spectrum acquired at 600 MHz using a cold probe (S/N = 100:1). (b) 1H NMR spectrum acquired at 500 MHz using a conventional 3-mm probe (S/N = 39:1). For (a) and (b), the metabolite sample was analyzed in a 3-mm tube (200 μl 99% D2O) with 128 scans.
NMR spectroscopy of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-l–glycero–l-manno-nonulosonic acid (CMP-Pse5-NAc7Am, I) purified from cell lysates. (a) 1H NMR spectrum (256 transients). (b) One-dimensional TOCSY of I H3ax (80 ms). (c) One-dimensional TOCSY of I H7 (80 ms). (d) One-dimensional TOCSY of I H9 (80 ms). (e) One-dimensional TOCSY of I NAc NH (30 ms). (f) One-dimensional TOCSY of I Am NH (30 ms). (g) The 13C HSQC spectrum (165 transients, 128 increments, 1JC,H_150 Hz, 24 h). From McNally et al. (2006a) .
HILIC-MS and precursor ion scanning for fragment ions related to CMP (m/z 322). (a) Cell lysate from C. coli VC167. (b) Cell lysate from C. jejuni 81-176. (c) Isogenic mutant ptmG. Peak 1, CMP-Leg5Ac7Ac; peak 2, CMP-Pse5Ac7Ac; peak 3, CMP-Leg5AmNMe7Ac; peak 4, CMP-Leg5Am7Ac; peak 5, CMP-Neu5Ac; peak 6, CMP-Pse5Ac7Am.
First steps of the pseudaminic acid biosynthetic pathway in C. jejuni. From McNally (2006b) . Pyranose rings are shown as their predominant chair conformation determined from nuclear Overhauser effects (NOEs) and J (H,H) coupling constants. Step 1, UDP-α-d-GlcNAc; step 2, UDP-2-acetamido-2,6-dideoxy-β-l–arabino-hexos-4-ulose; step 2′, gem-diol form of 2; 3, UDP-2-acetamido-2,6-dideoxy-α-d–xylo-hexos-4-ulose; step 3′, gem-diol form of 3 (from McNally et al., 2006b ); step 4, CMP-Pse5Ac7Ac.
PseB reaction monitored directly with 1H NMR spectroscopy (500 MHz, 1H) in aqueous reaction buffer (25°C, 10% D2O, 25 mM UDP-α-d-GlcNAc, 225 μg of PseB, 25 mM NaPO4, 50 mM NaCl, pH 7.2). From McNally et al. (2006b) . (a) Integrals for anomeric protons plotted over time. (b, c) 1H NMR spectra of the PseB reaction showing the anomeric region acquired at 0.08 and 6 h, respectively (from McNally et al., 2006b ).
Comparison of N-linked protein glycosylation in (a) eukaryotes and (b) the bacterium, C. jejuni. In the eukaryotic system, the oligosaccharide is assembled on the cytoplasmic face of the endoplasmic reticulum (ER) on the polyprenyl carrier dolichol phosphate. It is then flipped to the luminal side of the ER, where it is transferred to acceptor proteins with the Ser/Thr-X-Asn sequon by the oligosaccharytl transferase (OST) complex. In C. jejuni, the heptasaccharide is assembled on the cytoplasmic face of the cytoplasmic membrane on the polyprenyl carrier undecaprenyl phosphate. The completed heptasaccharide is then flipped to the periplasmic face of the membrane by PglK, where it is transferred to acceptor proteins with the extended sequon Ser/Thr-XpAsn-Glu/Asp by PglB.
Metabolomic analysis of the N-linked protein glycosylation pathway in C. jejuni. (a) CE-MS precursor ion scans for undecaprenyl-phosphate (Und-P-H2O, m/z 907.6) of affinity-captured lipid extracts of pglK indicating accumulation of Und-PP-heptasaccharide 1. (b) CE-MS-MS analysis of m/z 1168.0 in the negative ion mode, confirming that the N-glycan heptasaccharide is assembled on undecaprenyl-phosphate. (c) CE-MS spectrum demonstrating accumulation of UDP-diacetamido-trideoxyhexose (m/z 631.5) in pglJ cell lysates.
Metabolomic analysis of mutants of Neu5Ac biosynthesis gene homologues in C. jejuni 81-176
Results of N-glycan intermediate analyses in C. jejuni