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Export of Bacterial O-antigen Polysaccharides by ATP-Binding Cassette Transporters

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Title: Export of Bacterial O-antigen Polysaccharides by ATP-Binding Cassette Transporters
Author: Mann, Evan
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Whitfield, Chris
Abstract: Bacteria coat their surfaces with diverse polysaccharides that provide survival advantages, such as protection against the mammalian immune system. Despite this diversity, the general strategies used for their assembly are limited. In one of the major processes found in both Gram-positive and Gram-negative bacteria, the surface glycan is polymerized in the cytoplasm on a polyprenol lipid carrier and exported through the cytoplasmic membrane by an ATP-binding cassette (ABC) transporter. Here, the ABC transporter-mediated export of O-antigenic polysaccharides (O-PS) was investigated. In one established prototype O-PS synthesis system, characterized in Escherichia coli O9a, the polysaccharide is capped with a methylphosphate group at the non-reducing terminus. The capping enzyme contains a ‘molecular ruler’ and modifies the O-PS at the site of elongation when it reaches a minimal length to halt further extension. The ABC transporter, Wzm-Wzt, contains a carbohydrate-binding module (CBM) appended to the C-terminus of Wzt which binds the O-PS and ensures only fully-extended polysaccharides are transported. The CBM is required for export of the glycan and provides a level of substrate specificity to the transporter that is not observed in related transporters that lack a CBM. In this thesis, the Klebsiella pneumoniae O12 biosynthesis system was investigated heterologously in an E. coli K-12 strain to identify conserved features. Results are presented demonstrating that the O12 glycan is susceptible to phage-mediated glucosylation, which had not been previously observed in ABC transporter-dependent polysaccharide biosynthesis systems. Additional evidence confirms that the WztO12 C-terminal domain is required for glycan presentation on the cell surface and comprises a CBM. The CBM can only bind to Kdo-capped O-PS and this ensures that only capped (and fully extended) O-PS is exported. These properties were re-examined in the E. coli O9a model system, which is capped with a methylphosphate group. It was demonstrated that the initial phosphorylation is sufficient for chain-length control, but subsequent methylation is required for CBM-binding and display of O-PS on the cell surface. Finally, additional polysaccharide ABC transporters containing CBMs were identified using a bioinformatics approach. These candidate systems imply additional terminator chemical diversity beyond that observed in available structural investigations.
Date: 2019-05
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