VIRULENCE OF A PORPHYROMONAS-GINGIVALIS W83 MUTANT DEFECTIVE IN THE PRTH GENE

In a previous study we cloned and determined the nucleotide sequence o f the prtH gene from Porphyromonas gingivalis W83. This gene specifies a 97-kDa protease which is normally found in the membrane vesicles pr oduced by P. gingivalis and which cleaves the C3 complement protein un der defined conditions. We developed a novel ermF-ermAM antibiotic res istance gene cassette, which was used with the cloned prtH gene to pre pare an insertionally inactivated allele of this gene. This genetic co nstruct was introduced by electroporation into P. gingivalis W83 in or der to create a protease-deficient mutant by recombinational allelic e xchange. The mutant strain, designated V2296, was compared with the pa rent strain W83 for proteolytic activity and virulence. Extracellular protein preparations from V2296 showed decreased proteolytic activity compared with preparations from W83. Casein substrate zymography revea led that the 97-kDa proteolytic component as well as a 45-kDa protease was missing in the mutant, In in vivo experiments using a mouse model , V2296 was dramatically reduced in virulence compared with the wild-t ype W83 strain. A molecular survey of several clinical isolates of P. gingivalis using the prtH gene as a probe suggested that prtH gene seq uences were conserved and that they may have been present in multiple copies. Two of 10 isolates did not hybridize with the prtH gene probe, These strains, like the V2296 mutant, also displayed decreased virule nce in the mouse model. Taken together, these results suggest an impor tant role for P. gingivalis proteases in soft tissue infections and sp ecifically indicate that the prtH gene product is a virulence factor.