Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.690097
Title: Caf1R-mediated regulation of the F1 surface antigen of Yersinia pestis
Author: Kumar, D.
ISNI:       0000 0004 5921 9533
Awarding Body: University of Reading
Current Institution: University of Reading
Date of Award: 2016
Availability of Full Text:
Access through EThOS:
Full text unavailable from EThOS. Thesis embargoed until 20 Jun 2018
Access through Institution:
Abstract:
This thesis investigates the fundamental aspects of egulation of expression of the surface antigen, Fraction 1 (F1), of the plague pathogen Yersinia pestis. F1 contains a single subunit, Caf1, polymerised into a flexible fibrillar structure. It is a key diagnostic tool and a primary component of older whole cell plague vaccines and newer subunit ones under development. Caf1 is encoded by the caf gene cluster, comprising caf1R, an AraC/XylS family regulator and the chaperone/usher caf1MA1 operon. Despite considerable research on its assembly and role as a vaccine constituent there is virtually no information on how the expression of this key antigen is regulated. Availability of a spontaneous Caf1R point mutation (E98G) that virtually abolished F1 assembly stimulated initial interest in regulation. Modelling of Caf1R revealed location of E98 within the DNA binding helix-6 and its involvement in a novel ‘bridge-type’ DNA-protein interaction linking DNA backbone bound R62 (helix-4) to helix-6. It is proposed that this interaction may be critical for the correct spatial orientation of the base-binding residues Q93 and R97 within the major DNA groove. Site-specific mutagenesis supported this model and defined the requirement of other Caf1R residues modeled as interacting with DNA backbone or specific nucleotides. Promoter-lacZ fusions identified a Caf1R dependent class II promoter (PM) controlling expression of the caf1MA1 operon and 2 potential promoters upstream of caf1R (PR2 and PRK). PR2 promoter, also a class II promoter, was autoactivated by Caf1R. Provision of Caf1R in trans from a PBAD promoter, identified thermosensing within the caf locus is via 5′ UTR of Caf1R. Potential RNA thermometer (RNAT) structures were predicted from the 5′ UTR of transcripts from both promoters. The longer and more stable 5′ UTR from PRK and the higher level of Caf1R independent transcription from this promoter would be consistent with an RNAT within this transcript controlling early expression of caf1R in response to temperature. Five potential Caf1R binding sites (repeat motifs) were identified within the caf1R-caf1M intergenic region, R1-3 upstream of caf1R and R3′ and R4′ upstream of caf1M, with the consensus sequence TGCRCBS1RAMWAGCWARDBS2. An electrophoretic mobility shift assay (EMSA) confirmed specific binding of Caf1R to R4′. Several tagged approaches were taken for purification of Caf1R. Low levels of soluble hCaf1R were recovered using the PBAD promoter. Using the pET28a+ vector, hCaf1RT was mainly recovered in inclusion bodies but low levels of soluble hCaf1RT were recovered using codon optimisation and induction with glucose plus IPTG. Solubility of Caf1R was greatly enhanced with MBPCaf1R expressed in E. coli-K12 2508. While isolated Caf1R showed some activity in EMSA, comparison with activity in crude lysed cell fractions suggested either inactivation of Caf1R during isolation or loss of an activating factor. Thus this study has identified the fundamental features controlling transcription within the caf locus for subsequent expression of F1. This is the first example of AraC/XylS type regulator, directly linked to a CU system of ϒ3-fimbriae/pili family. Working models are presented that can be used as a basis to further clarify autoregulation of Caf1R including activation, the possibility of repression and thermoregulation. This study has provided the fundamental information and tools for in-depth understanding of expression of F1 in Y. pestis and its applications in vaccine development.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.690097  DOI: Not available
Share: