Role Of Cas3 In Porphyromonas gingivalis Virulence

Objectives: Porphyromonas gingivalis is a major pathogen of severe adult periodontal disease (PD). In a previous study, we showed that most of the CRISPR-associated proteins in P. gingivalis were up-regulated at sites that progressed during PD but not in sites that did not progress. Among the most highly up-regulated genes was the nuclease cas3. To assess its role in virulence, we constructed a P. gingivalis cas3 mutant. The mutated Δcas3 strain showed an increase in virulence when tested in an animal model, and it altered the expression of cytokines in macrophages.
Methods: Given these results, we hypothesized that the mechanism by which CRISPR-Cas controls virulence in P. gingivalis involves controlling levels of specific mRNAs that can modulate the immune response in the host. Thus, to start testing our hypothesis, we wanted to: 1) Demonstrate that Cas3 has specific mRNAse activities, and 2) that mRNAs from the most highly up-regulated genes in the Δcas3 strain are the targets of Cas3.
Results: We cloned the cas3 gene in the expression vector pET-28b and started the process of purification to demonstrate that Cas3 of P. gingivalis has specific mRNAse activities against spacers with homology to proteins identified as highly up-regulated in the Δcas3 mutant. We also used the shuttle vector pT-COW to clone PGN1547. This gene was selected as a model because it was one of the most highly up-regulated in the Δcas3 strain when growing intracellularly.
Conclusions: The cas3 gene has been shown to be essential for fitness survival in cells and a mouse model. We are now in the process of transforming the wild type and the Δcas3 strains with the vector carrying PGN1547. The transformants will allow us to discern whether PGN1547 is one of the targets of the CRISPR-Cas systems in P. gingivalis.