2012TawfickMMPhD.pdf (5.47 MB)
Characterisation of the Salmonella Stk Fimbrial Operon and Examination of Stkf, the Putative Adhesion Protein, As a Potential Diagnostic and Vaccine Candidate
thesisposted on 2012-09-11, 11:10 authored by Mahmoud Mohamed Tawfick Abd Raboh
The StkF protein of Salmonella enterica serovar Paratyphi A, a putative adhesion-associated protein, was used to develop a candidate adhesin-based vaccine against Salmonella infections, particularly S. Paratyphi A-associated enteric fever. Subcutaneous immunisation of BALB/c mice with recombinant StkF (rStkF) showed that this protein was strongly immunogenic as revealed by the presence of high-titre (1:50,000) anti-StkF antibody in sera from immunized mice. Furthermore, as IgG1 was the major antibody isotype induced, it would appear that rStkF generates a strong Th2-type humoral immune response. The induction of a Th2-dominant immune response was further confirmed by splenocyte-associated cytokine profile analysis which demonstrated greater up-regulation of IL-4 than that of IFN-γ or IL-12-p70. In terms of the protective potential of this antigen, in vitro assays demonstrated that StkF-specific murine antiserum markedly enhanced opsonophagocytosis-mediated uptake of StkF-expressing Salmonella bacteria by human neutrophils. Augmented antibody-specific complement-mediated lysis targeting StkF-expressing Salmonella specifically was also shown. These data suggest a likely direct contribution of StkF-specific antibodies to in vivo killing and clearance of Salmonella and strongly support further investigation of StkF as a potential Salmonella vaccine candidate. BlastN-based interrogation of the NCBI bacterial genome database and PCR investigation of a larger set of strains has shown that the S. Paratyphi A stkF gene and/or the whole stk fimbrial gene cluster is carried by ~1/3 of examined Salmonella serovars. Additionally, bioinformatics and phenotypic characterisation has revealed that the stk fimbrial operon belongs to the chaperone/usher-γ4-fimbrial clade and that it encodes a mannose-sensitive haemagglutinating fimbrial structure. The latter trait is typical of type 1 fimbriae. ELISAs based on rStkF, rStaF and rSipA showed variable sensitivity and specificity for the serodiagnosis of invasive Salmonella infections depending on the particular UK or region-specific cut off applied for each antigen. Further refinement and/or merging of these assays may allow for development of simple and cost effective tests with higher sensitivity and/or specificity than the Widal test. Importantly, despite some minor reactivity with serum from patients with other Gram-negative bacterial infections, the rStkF-based ELISA exhibited no reactivity with serum from patients with dengue fever supporting its potential as a discriminatory diagnostic tool between fevers caused by S. Paratyphi A and dengue virus.
Date of award2012-06-01
Awarding institutionUniversity of Leicester