Bokhari, Syed Habib
Characterisation and secretion mechanism of Bordetella pertussis autotransporter proteins.
PhD thesis, University of Glasgow.
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The identification and characterisation of new virulence determinants of 5. pertussis is providing important information for understanding the colonisation and survival strategies of the microorganism. B. pertussis deploys a range of surface-associated components to enable its successful colonisation of the host. Bap-5 has been identified as a new member of the B. pertussis autotransporter family of proteins that includes PRN, BrkA, TCF and Vag-8, largely due to its homology at the C-terminus and some other similar regions such as the RGD (integrin-binding) and SGXG (glycosaminoglycan-binding) motifs. The bap-5 gene also exists in B. bronchiseptica and B. parapertussis. Characteristic upstream regulatory sequences such as a ribosome-binding site were not seen in bap-5, but a potential heptameric BvgA-binding motif was identified. The expression of Bap-5 was confirmed by RT-PCR and Western blotting and was shown to be bvg dependent. Although Bap-5 does not possess a typical signal sequence like pertactin (PRN), its surface localisation was confirmed by agglutination and immunofluorescence assays.
A potential role for Bap-5 in infection was studied by generating Bap-5 deficient mutants in two strains of B. pertussis. An allelic exchange procedure with the suicide vector pSS1129 carrying the bap-5 gene disrupted with a kanamycin-resistance cassette was used. PCR and Southern blotting confirmed the replacement of the wild-type bap-5 gene with the mutated version. Moreover, SDS-PAGE and Western blotting of outer-membrane preparations of B. pertussis Taberman wild-type and its Bap-5-deficient mutant showed a clear difference in their outer-membrane profile at ~79.9kDa presumably representing the unprocessed form and bands at ~65 kDa and ~16 kDa may represent the processed forms of the protein.
The Bap-5 characterisation studies showed that the Taberman Bap-5-deficient strain was less able than the parent strain to colonise the lower respiratory tract of mice and adhesion studies (in vitro) showed that the Taberman parent was better in adhering to certain cell types than the Bap-5-deficient mutant.
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