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. 2014 Jul;82(7):2728-35.
doi: 10.1128/IAI.00084-14. Epub 2014 Apr 14.

Identification of a diguanylate cyclase and its role in Porphyromonas gingivalis virulence

Swarnava Chaudhuri  1 , Siddharth Pratap  2 , Victor Paromov  2 , Zhijun Li  3 , Chinmay K Mantri  1 , Hua Xie  4
Affiliations

Identification of a diguanylate cyclase and its role in Porphyromonas gingivalis virulence

Swarnava Chaudhuri et al. Infect Immun. 2014 Jul.

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.

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Figures

FIG 1
FIG 1
Comparison of the growth curves and gene expression of P. gingivalis ATCC 33277 and its mutant 1932E. (A) P. gingivalis cells were grown in standard TSB for 64 h. Bacterial growth is indicated by the means of the optical densities (OD600s) of the bacterial cultures. Error bars represent SDs (n = 4 experiments). (B) The expression levels of the gene (pgn_1931) located immediately downstream of pgn_1932 were measured by real-time RT-PCR. Each bar represents relative gene expression in the pgn_1932 mutant compared to that in P. gingivalis ATCC 33277 (which was given a value of 1). Error bars represent standard deviations (n = 3).
FIG 2
FIG 2
Comparison of attachment ability of P. gingivalis ATCC 33277, the dgc mutant (1932E), and the fimA mutant (FAE). P. gingivalis strains were exposed to a saliva-coated surface (A), HPDLFs (B), and (C) HeLa cells for 1 h. The numbers of bacterial cells remaining on the surfaces were determined, and the relative binding abilities of the mutants were compared to the binding ability of wild-type strain ATCC 33277, which was given a value of 1. Error bar represents SDs (n = 4 experiments). *, a statistically significant difference between the wild type and the mutants (P < 0.05, t test,).
FIG 3
FIG 3
Comparison of invasive ability of P. gingivalis ATCC 33277 and the dgc (1932E) and fimA (FAE) mutants. HPDLFs (A) and HeLa cells (B) were infected with P. gingivalis strains at an MOI of 100 for 1 h, and the extracellular bacterial cells were eliminated by the use of antibiotics. The intracellular bacterial cells were plated on TSB blood agar plates. Each bar represents the average number of CFU of four independent experiments. *, a statistically significant difference between the wild type and the mutants (P < 0.05, t test).
FIG 4
FIG 4
Differential expression of genes in P. gingivalis ATCC 33277 and the dgc mutant (1932E). Total RNAs were extracted from the P. gingivalis strains. The expression levels of the genes were measured by real-time RT-PCR. Each bar represents the relative gene expression in the dgc mutant compared to that in P. gingivalis ATCC 33277 (which was given a value of 1). Error bars represent standard deviations (n = 3). *, a significant difference between the gene expression level in the dgc mutant and that in P. gingivalis ATCC 33277 (P < 0.05, t test).
FIG 5
FIG 5
Differential expression of FimA in P. gingivalis ATCC 33277, the dgc mutant (1932E), and the fimA mutant (FAE). Nunc immune modules were coated with equal amounts of the P. gingivalis surface protein from each strain. FimA levels were measured using ELISA with anti-FimA antibodies and horseradish peroxidase-tagged secondary antibodies. The absorbance was measured at 450 nm. The results represent the averages of three independent experiments. Statistical significance was measured by Student's t test. *, P < 0.05.
FIG 6
FIG 6
Transmission electron microscopic analysis of P. gingivalis fimbriae. P. gingivalis strains ATCC 33277 (left) and 1932E (right) were prepared by negative staining with ammonium molybdate. Fimbrial structures were visualized using transmission electron microscopy.
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