Construction of a pepO gene-deficient mutant of Porphyromonas gingivalis: potential role of endopeptidase O in the invasion of host cells.

Porphyromonas gingivalis has been isolated from lesions of advanced adult periodontitis, and implicated as a periodontal pathogen. We have previously cloned a novel endopeptidase, designated PepO, from P. gingivalis 381, which exhibited significant homology to human endothelin-converting enzyme (ECE)-1. In order to determine the nature and function of the PepO in the host, a pepO gene-deficient mutant strain was constructed by allelic exchange mutagenesis using the ermF-ermAM cassette. No endopeptidase activity was detected in the pepO-deficient mutant. In addition, adherent HeLa (HEp-2) cells were infected with the mutant and the two wild-type strains for assessment of bacterial invasion by an antibiotic protection assay. The invasion efficiency of the mutant strain was about a quarter of the wild type strains. These results suggest that PepO is involved in the first step, i.e. invasion/lysis of mammalian cell membrane, which affects the kinetics of rate of invasion.