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Title: Analyses of the proteins KpsM, KpsE and KpsD in the group 2 capsular polysaccharide export complex of Escherichia coli
Author: Haas, Eva
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2012
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Abstract:
The expression of polysaccharide capsules is common in bacteria and associated with virulence in some pathogenic strains. Strains of the Gram-negative bacterium Escherichia coli express a structurally diverse range of capsular polysaccharides. E. coli strains expressing group 2 capsules are associated with a number of extra-intestinal infections, including sepsis, urinary tract infections, and neonatal meningitis. Group 2 capsular polysaccharides are synthesised on the cytoplasmic face of the inner membrane. Evidence from previous work suggests that export of polysaccharides across the Gram-negative membranes involves four transport proteins which interact to form a continuous membrane-spanning translocation complex (the KpsMTED translocon). Polysaccharide translocation across the inner membrane requires the ABC transporter KpsMT, in which KpsM is the integral inner membrane component and KpsT is the ATPase. Transport across the periplasmic space and outer membrane involves the integral inner membrane protein KpsE and the outer membrane protein KpsD, respectively. This thesis addressed some of the key areas in the study of group 2 polysaccharide transport by employing the K5 capsule as a model system. Using biochemical and molecular genetics approaches, the study focused on establishing functional and structural characteristics of the translocon members and analysing protein-protein interactions within the complex. This study demonstrated that KpsE can self-associate as dimers, tetramers and possibly higher order oligomers in the absence of other capsule gene products and the K5 substrate. A mutagenesis study of KpsE revealed that the periplasmic, membrane-associated C-terminus is essential for correct protein function. Work presented here confirmed previous data, which suggested a direct interaction between KpsE and KpsM, by alternative methods, and demonstrated that the C-terminal domain of KpsE is required for this interaction. Further experiments suggested that KpsE and KpsM can both form higher order oligomers when interacting as a complex. The C-terminus of KpsE is not required for an interaction between KpsE and KpsD, and the two proteins are thus more likely to interact via their respective periplasmic domains. Generation of a theoretical model of the secondary structure and topology of KpsD predicted that KpsD is made primarily of β-sheets with some interspersed α-helices, including a larger coiled coil region. The theoretical topology model proposed an N-terminal transmembrane domain made of eight membrane-spanning regions, and a large periplasmic domain. Substituted-cysteine accessibility method and myc-epitope insertion analysis were both assessed for their suitability for topology analysis of KpsD. Myc-epitope insertion was identified as the recommended approach for future topology study. Myc-epitope tagging of the periplasmic C-terminus of KpsD revealed that a native C-terminus is essential for correct KpsD function.In conclusion, this thesis contributes to the model of group 2 polysaccharide export in E. coli, and, more generally, provides clues about the transport of high-molecular weight molecules across Gram-negative membranes. It is hoped that a thorough understanding of polysaccharide transport might reveal therapeutic targets to block capsule export in pathogenic E. coli in the future.
Supervisor: Roberts, Ian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.564295  DOI: Not available
Keywords: E. coli ; Polysaccharide ; Capsule ; Membrane protein ; KpsE ; KpsT ; KpsM ; KpsD ; Transmembrane ; Export ; Translocon ; Group 2 ; Transport ; Protein complex
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