Identification of a Diguanylate Cyclase and Its Role in Porphyromonas gingivalis Virulence

Abstract

Porphyromonas gingivalis is a Gram-negative obligate anaerobic bacterium and is considered a keystone pathogen in the initiation of periodontitis, one of the most widespread infectious diseases. Bacterial bis-(3=-5=) cyclic GMP (cyclic di-GMP [c-di-GMP]) serves as a second messenger and is involved in modulating virulence factors in numerous bacteria. However, the role of this second messenger has not been investigated in P. gingivalis, mainly due to a lack of an annotation regarding diguanylate cyclases (DGCs) in this bacterium. Using bioinformatics tools, we found a protein, PGN_1932, containing a GGDEF domain. A deletion mutation in the pgn_1932 gene had a significant effect on the intracellular c-di-GMP level in P. gingivalis. Genetic analysis showed that expression of the fimA and rgpA genes, encoding the major protein subunit of fimbriae and an arginine-specific proteinase, respectively, was downregulated in the pgn_1932 mutant. Correspondingly, FimA protein production and the fimbrial display on the mutant were significantly reduced. Mutations in the pgn_1932 gene also had a significant impact on the adhesive and invasive capabilities of P. gingivalis, which are required for its pathogenicity. These findings provide evidence that the PGN_1932 protein is both responsible for synthesizing c-di-GMP and involved in biofilm formation and host cell invasion by P. gingivalis by controlling the expression and biosynthesis of FimA.

Fimbriae‐mediated outer membrane vesicle production and invasion of Porphyromonas gingivalis

“…How- ever, the invasive activities varied between vesicles derived from 33277 and W83, which is consistent with the invasive activities observed in their originating cells (Wayakanon et al 2013). Since FimA was far more abundant in 33277 vesicles than in W83 vesicles, we speculated that a decreased invasive activity would be found in vesicles from the fimA mutant, as we showed previously that the fimA mutant lost 90% of its invasive activity into HGFs (Chaudhuri et al 2014). Surprisingly, vesicles from this mutant showed a similar invasive activity as 33277, while vesicles from the fimR mutant showed a significantly decreased invasive activity, due to reduced expression of the fim locus that encodes minor components (FimC, FimD, and FimE) of long fimbriae.…”

“…Transmission electron microscopy of P. gingivalis vesicles was conducted as described previously (Chaudhuri S, et al. ). Porphyromonas gingivalis vesicles (0.5 ng) were suspended in 25 μ L ddH 2 O and applied to a Formvar‐coated copper grid (200 mesh, Electron microscopy Sciences, Hatfield, PA) for 1 min and were air‐dried.…”

“…Porphyromonas gingivalis vesicles that translocated through the monolayered HOKs were collected by centrifugation and resuspended in 50 μ L ddH 2 O. Vesicles were quantitated with modifications using an enzyme‐linked immunosorbent assay (ELISA) as described previously (Chaudhuri et al. ). Briefly, Nunc‐immuno modules (Nalgene, San Diego, CA, USA) were coated with vesicle proteins (50 μ L) for 16 h at 4°C.…”

Recombinant fimbriae protein of Porphyromonas gingivalis induces an inflammatory response via the TLR4/NF‑κB signaling pathway in human peripheral blood mononuclear cells

“…The innate immune system of the host responds to decrease inflammation by activating mechanisms including the mononuclear/macrophage system. Increased levels of inflammatory factors and release of matrix metalloproteinases may cause irreversible decomposition of collagen fibers in the periodontium, resulting in periodontal bone loss and organizational damage (29,30). Ogawa et al (31) demonstrated that the detection rate of P. gingivalis FimA-specific antibody-expressing cells increased with the development of periodontal disease.…”

Regulation of gingival epithelial cytokine response by bacterial cyclic dinucleotides

“…Information on cyclic dinucleotides in periodontal disease pathogenesis is limited and the presence and regulatory functions of the molecules in oral bacteria remain to be fully characterized. The presence of cdi-GMP, or its binding proteins, has been demonstrated in some periodontitis-associated bacteria, including Porphyromonas gingivalis, Treponema denticola, and Selenomonas noxia [6][7][8][9]. In addition, these secondary signaling molecules have been found to regulate the oxidative response, extracellular polysaccharide matrix production, and biofilm formation of cariogenic Streptococcus mutans [10,11].…”