IS195, AN INSERTION SEQUENCE-LIKE ELEMENT ASSOCIATED WITH PROTEASE GENES IN PORPHYROMONAS-GINGIVALIS

Porphyromonas gingivalis is recognized as an important etiologic agent in adult and early-onset periodontal disease. Proteases produced by t his organism contribute to its virulence in mice. Protease-encoding ge nes have been shown to contain multiple copies of repeated nucleotide sequences. These conserved sequences have also been found in hemagglut inin genes. In the process of studying the genetic loci containing the conserved repeated sequences, we have characterized a prtP gene homol og from P. gingivalis W83 encoding a cysteine protease with Lys-X spec ificity. However, this prtP gene was interrupted by an insertion seque nce-like element which we designated IS195. Furthermore, IS195 and ano ther element, IS1126, were present downstream of prtP gene homologs (k gp) found in P. gingivalis H66 and 381. IS195, a 1,068-bp insertion se quence-like element, contained Il-bp inverted repeats at its termini a nd was bordered by 9-bp direct repeats presumed to be a transposition- mediated target site duplication. Its central region contained one lar ge open reading frame encoding a predicted 300-amino-acid protein whic h appeared to be a transposase. We isolated two naturally occurring va riants of P. gingivalis W83, one carrying IS195 within the coding regi on of the prtP gene and another containing an intact prtP gene. Bioche mical characterization revealed a lack of trypsin-like Lys-X specific proteolytic activity in the P. gingivalis W83 variant carrying the dis rupted prtP gene. Studies using a mouse model revealed a reduction of virulence resulting from insertion of IS195 into the coding region of the prtP gene. An allelic-exchange mutant defective in the prtP gene a lso was constructed and tested in vivo. It displayed intermediate viru lence compared to that of the wild-type and prtP::IS195 mutant strains . We conclude that the Lys-X cysteine protease contributes to virulenc e in soft tissue infections.