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Title: The role of the Caf1A outer membrane usher in the production of F1 surface fibres of Yersinia pestis
Author: Mycroft, Zoe
Awarding Body: University of Reading
Current Institution: University of Reading
Date of Award: 2011
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Abstract:
The Fraction I antigen of Yersinia pestis forms an immunogenic capsule around the cell when invading a mammalian host. This capsule is assembled by the chaperone-usher pathway, one of many strategies Enterobacteriacea have evolved for protein translocation across the outer membrane. Chaperone-usher systems are involved in assembly of polymeric surface fibres such as fibrillae and fimbriae, many of which are virulence factors. These fibres comprise repeating subunits; FI being a polymer of Caf1 subunits. Subunits lack the information required to form a stable fold therefore each system has a specific chaperone, providing the missing information by donating a β-strand which promotes subunits to form an immunoglobulin fold. Chaperone- subunit complexes bind the N-terminal periplasmic domain of the usher and are translocated to the cell surface through the pore of the usher, a process coupled with polymerisation of the subunits and catalysed by the usher. Each subunit has an N-terminal extension which displaces the donor strand of the chaperone forming a highly stable polymer with a condensed immunoglobulin fold. This thesis investigates the role of the Caf1A usher using deletion and point mutagenesis together with functional studies to investigate the domain structure of Caf1A and functions of periplasmic domains. A soluble periplasmic domain was identified within the transmembrane β-barrel and was shown to be dispensable for assembly of the β-barrel but required for function. The role of this domain as a plug was investigated and the region in and around the predicted A-strand was shown to be critical for FI' assembly (particularly residues M237, P239 and Y241). The functional requirement for the predicted C-terminal domain of CaflA was confirmed and its periplasmic location deduced. A protease-sensitive 10 kDa region and a stable 10 kDa periplasmic domain were identified. Hydrophobic patches on the surface of the stable domain were shown to be critical for F1 assembly. The identity of the initial residue of Caf1A and its signal sequence cleavage site was investigated, but was unable to be ascertained. The successful purification of the components of the Caf system allowed attempts to create an in vitro polymerisation system, which would allow individual components to be controlled and investigated, but in vitro polymerisation was unable to be observed under conditions used.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.552985  DOI: Not available
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