Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487475
Title: Role of the Usher N-terminal Domain in Assembly of Fl Polymeric Antigen of Yersinia pestis
Author: Pudney, Alexander F.
ISNI:       0000 0001 3502 6567
Awarding Body: University of Reading
Current Institution: University of Reading
Date of Award: 2006
Availability of Full Text:
Access through EThOS:
Abstract:
The fraction 1 antigenic capsule of Yersinia pestis is a homopolymer assembled by the two-component chaperone-usher system; a terminal branch of the general secretory pathway. A periplasmic chaperone (CaflM) is responsible for folding and capping of monomeric Cafl, whilst preserving it in an energy competent conformation for fiber formation. Translocation of Cafl polymer to the cell surface occurs via the outer membrane usher CaflA, and circumstantial evidence suggests that polymerisation is catalysed by the usher. Recovery of OlT-sensitive CaflT10CT139C polymer at the surface of recombinant E. coli demonstrated that donor strand alignment corresponding to the register T10/T139 (donor strand/F strand) was used throughout the fiber. Sucrose density gradient centrifugation was used to show that periplasmic CaflM :Cafl fiber preassembly complexes specifically target CaflA within the outer membrane. CaflM bound to polymerisation-deficient subunit was shown to be degraded in the presence of usher, consistent with a secondary binding event upon interaction with CaflA, resulting sensitivity to periplasmic OegP. Deletion mutagenesis within the N-terminus of CaflA (CaflAN) showed that this domain is essential for capsular Fl biogenesis and that loss of even the 27 N-terminal residues of CaflA led to drastic reduction in assembly. Fl biogenesis was completely abolished in this mutant when periplasmic CaflAN was coexpressed, suggesting that the domain was able to compete for periplasmic Fl preassembly complexes. The usher N-terminus was overproduced as a soluble periplasmic domain and purified. This has recently been used for collaborative crystallographic studies of both the Nterminus alone and in complex with chaperone and subunit. Histagged CaflAN was purified and used for in vitro Biacore binding studies with purified CaflM:Cafl~3 binary complex and Cafl:CaflA9R2 ternary complex. Qualitative analysis was obtained for an interaction between CaflAN and the preassembly complexes, consistent with similar observations made in related chaperone/usher systems for the usher N-terminus.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.487475  DOI: Not available
Share: