Molecular characterisation of PgaA, an antigen from periodontopathogen Porphyromonas gingivalis

• Main Author: Rigg, Gordon P.
• Published: University of Leicester 1995
• Subjects: 570
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.737554

Molecular cloning techniques were used to investigate antigenic determinants of periodontopathogen P. gingivalis in an attempt to begin to explore the cell surface of this organism. A genomic library generated in Escherichia coli was probed with polyclonal antiserum specific for P. gingivalis whole cells. Clone pGPR1, which contained a 307 bp fragment of P. gingivalis DNA sequence, was found to react with both polyclonal antiserum and a monoclonal antibody (mAb) specific for the trypsin-like protease of this organism (Brick190). This fragment was used to probe a second P. gingivalis genomic library. Clone pGPR2 was shown to hybidise with the 307 bp probe. DNA sequence analysis revealed pGPR2 contains a 5,653 bp insert with seven open reading frames one of which shows significant DNA homology with the rnhB gene of E.coli. The 307 bp probe sequence was found to reside within an ORF predicted to encode a 455 amino acid (50 kDa) protein and this ORF was designated pgaA (P. gingivalis antigen). The predicted amino acid sequence of PgaA contains a putative ABC signature for binding NTPs as well as a predicted transmembrane domain. Subclones of pgaA in expression vector pTTQ18* were found to be reactive with a mAb specific for a 46 kDa species-specific antigen of the cell envelope of P. gingivalis (LDS28) as well as Brick190. One such subclone, pGPR7 was also shown to express a 46 kDa protein reactive in western blots with mAb LDS28. Minicell labelling of pGPR2-encoded proteins using pGPR2 subclones revealed that pgaA directs expression of protein of multiple molecular weights (31-46 kDa) from its own promoter in E. coli, and that some of these forms may be caused by proteolysis of a 50 kDa precursor which itself shows a reduced apparent molecular weight (46 kDa) on SDS PAGE.