Role of Putative ECF Sigma Factors in Porphyromonas Gingivalis

Objectives: The Gram-negative anaerobic bacterium Porphyromonas gingivalis (P. gingivalis) has been recognized as a major pathogen in the development and progression of chronic periodontitis. To colonize and survive at the gingival crevice, P. gingivalis must regulate gene expression in response to the various extracellular changes. Some bacteria utilize sigma factor proteins of the extracytoplasmic function (ECF) subfamily. In this study, we have investigated the ECF sigma factors to determine its role in this bacterium.
Methods: ECF sigma factor-defective mutants with the erythromycin resistance gene cassette were constructed by electroporation in P. gingivalis 33277. The biofilm quantity on the plates was evaluated by crystal violet staining.
Hemagglutination and proteolytic activities (Kgp, Rgp) were measured in the ECF sigma factor mutants. Real-time PCR analysis was used to assess transcription of kgp and rgp in the PGN_0274 mutant. In the PGN_0274 mutant, the localization and maturation of Kgp was investigated by SDS–PAGE/western immunoblotting analysis.
Results: Biofilm formation of the PGN_0274 and PGN_1740 mutants increased compared with the wild-type (p < 0.001). Kgp and Rgp activities of the PGN_0274 mutant decreased remarkably compared with the wild-type. In addition, the PGN_0274 mutant showed no hemagglutination activity. The results of real-time PCR analysis indicated that kgp was transcribed in PGN_0274 mutant as well as the wild-type. Immunoblotting analysis using anti-Kgp antiserum showed that Kgp was poorly secreted into an extracellular portion and the precursor forms of Kgp and adhesins were accumulated within the cells of the PGN_0274 mutant. Taken together, our findings indicated that the secretion of Kgp is defected in the PGN_0274 mutant.
Conclusions: These results suggest that PGN_0274 and PGN_1740 play a key role in biofilm formation and PGN_0274 is required for secretion of gingipains in P. gingivalis.