Bacterial capsules are important virulence factors for a number of different pathogens, including Escherichia coli, Neisseria meningitidis, Haemophilus influenzae, and Pasteurella multocida. Capsular polysaccharides (CPSs) synthesized via the ATP-binding cassette (ABC) transporter-dependent pathway protect these bacteria from complement-mediated killing and phagocytosis, and consist of long polysaccharide chains attached to the cell surface via a phospholipid. CPSs are synthesized on the cytoplasmic face of the inner membrane before transport to the cell surface. While the enzymes that synthesize the polysaccharide have been studied in detail, very little is known about the structure and biosynthesis of the phospholipid terminus. To determine the structure of the reducing terminal glycolipid, CPS from E. coli K1, K5, and N. meningitidis group B was purified using a novel strategy and its structure was determined by mass spectrometry, nuclear magnetic resonance and chemical methods. All three polysaccharides possess terminal lyso-phosphatidylglycerol, which is connected to the CPS repeat unit by a linker consisting of multiple 3-deoxy-D-manno-octulosonic acid (Kdo) residues, forming an alternating β-2,4/β-2,7-linked structure. In addition to describing its structure, the biosynthesis of the glycolipid terminus was also investigated. KpsC and KpsS are conserved proteins encoded in the capsule loci from different bacteria with ABC transporter-dependent capsule assembly pathways but have no previously assigned function. An in vitro assay was developed to characterize KpsSC activities, leading to the finding that they are the Kdo transferases responsible for synthesis of the poly-Kdo linker. This research has contributed significantly to the understanding of the structure and biosynthesis of capsular polysaccharides.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OGU.10214/7479 |
| Date | 07 September 2013 |
| Creators | Willis, Elizabeth |
| Contributors | Whitfield, Chris |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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