Identification and Testing of Porphyromonas gingivalis Virulence Genes with a pPGIVET System

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Abstract
An in vivo expression technology (IVET) system was designed to identify previously unknown virulence genes of Porphyromonas gingivalis. Fourteen ivi (for in vivo induced) genes that are induced during infection in a mouse abscess model were identified in our study. Of these, seven had homology to genes in the NCBI database, and the rest had no homology to reported DNA sequences. In order to determine virulence-related properties of these genes, three mutant strains, deleted of ivi 8 (no homology to genes in the database), ivi 10 (homologous to a putative TonB-dependent outer membrane receptor protein), and ivi 11 (an immunoreactive 33-kDa antigen PG125 in P. gingivalis ), were created. The mutants were tested in a mouse abscess model for alterations in virulence relative to the wild type by a competition assay in BALB/c mice. After 5 days we observed the enrichment of the wild-type strain over mutant strains Δ ivi 10 and Δ ivi 11, which indicated that mutant strains Δ ivi 10 and Δ ivi 11 are less able to survive in this model than the wild-type strain, while Δ ivi 8 survives as well as the wild-type strain. We propose that knockout of these ivi genes reduced the ability of the mutated P. gingivalis to survive and cause infection compared to the wild-type strain at the site of injection. Also, in separate experiments, groups of mice were challenged with subcutaneous injections of each individual mutant strain (Δ ivi 8, Δ ivi 10, and Δ ivi 11) or with the wild-type strain alone and were then examined to assess their general health status. The results showed that knockout of these ivi genes conferred a reduction in virulence. The ability of the mutants to invade KB cells compared to the wild type was also determined. Interestingly, the CFU counts of the mutant strain Δ ivi 10 recovered from KB cells were eight times lower than those of the wild type, indicating that this mutant has a lower capacity for invasion. These results demonstrate that IVET is a powerful tool in discovering virulence genes and the significant role that ivi genes play in the pathogenesis of this species.

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