Molecular and Immunological Analysis of Recombinant Bacteroides forsythus Heat Shock Protein 60

Reid, Helen Isobel (1999) Molecular and Immunological Analysis of Recombinant Bacteroides forsythus Heat Shock Protein 60. PhD thesis, University of Glasgow.

Abstract

This study addresses the putative role of Bacteroides forsythus heat shock protein 60 (hsp60) as a pathogenicity factor in periodontal disease by defining the molecular identity and antigenicity of the protein. The gene encoding the heat shock protein 60 (hsp60) of B. forsythus was identified by PCR using degenerate hsp60 oligonucleotide primers, and by Southern blot hybridisation using probes derived from the E. coli hsp60 DNA sequence. The DNA sequence of the B. forsythus gene was determined, and was predicted to encode an amino acid of 544 amino acids with a molecular weight of 62kDa. The DNA and protein sequences displayed extremely high levels of conservation with hsp60 homologues from a range of other species, including those from other oral bacterial pathogens and human hsp60. The hsp60 gene of Prevotella intermedia was similarly identified, and the DNA sequence of the C terminal portion of the gene determined. Bacterial hsp60 from a range of different species has been implicated in the pathogenic mechanisms of a wide range of diseases, and it is suggested that they may instigate autoimmune reactions in the host as a result of the high level of molecular conservation shared between bacterial and human hsp60 homologues. With the aim of evaluating the potential role of B. forsythus hsp60 in periodontitis, recombinant B. forsythus hsp60 was expressed in E. coli and it's immunoreactivity determined by screening with 13 healthy adult and 11 chronic adult periodontitis serum samples. Both patient groups displayed a low level of IgG antibody reactivity with B. forsythus hsp60. The antibodies recognising the B. forsythus protein were cross-reactive with E. coli hsp60, and may have been initially raised against this species. These results indicate that B. forsythus hsp60 is immunogenic, but do not suggest that this protein has an important role in the pathogenesis of adult periodontitis. A B. forsythus genomic library was constructed in pUC18 and screened using a variety of techniques with the aim of identifying further immuno-reactive proteins of this species. Of the clones analysed, periodontitis patient serum did not display any reactivity. Random clones were selected for further analysis by DNA sequencing, and a clone carrying part of the lac operon of B. forsythus identified. This thesis constitutes an original report on the molecular characterisation of part of the genome of the putative periodontal pathogen Bacteroides forsythus, and a preliminary investigation of host immune responses to a recombinant antigen of this species.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Marcello Riggio
• Keywords: Molecular biology, Microbiology, Immunology
• Date of Award: 1999
• Depositing User: Enlighten Team
• Unique ID: glathesis:1999-74891
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 15:29
• Last Modified: 27 Sep 2019 15:29
• URI: https://theses.gla.ac.uk/id/eprint/74891