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. 2023 Jan 31;11(2):e0441122.
doi: 10.1128/spectrum.04411-22. Online ahead of print.

The Oxidative Stress-Induced Hypothetical Protein PG_0686 in Porphyromonas gingivalis W83 Is a Novel Diguanylate Cyclase

Alexia D Ximinies  1 , Yuetan Dou  1 , Arunima Mishra  1 , Kangling Zhang  2 , Champion Deivanayagam  3 , Charles Wang  1 , Hansel M Fletcher  1
Affiliations

The Oxidative Stress-Induced Hypothetical Protein PG_0686 in Porphyromonas gingivalis W83 Is a Novel Diguanylate Cyclase

Alexia D Ximinies et al. Microbiol Spectr. .

Abstract

The survival/adaptation of Porphyromonas gingivalis to the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. Several functional classes of genes, depending on the severity and duration of the exposure, were induced in P. gingivalis under H2O2-induced oxidative stress. The PG_0686 gene was highly upregulated under prolonged oxidative stress. PG_0686, annotated as a hypothetical protein of unknown function, is a 60 kDa protein that carries several domains including hemerythrin, PAS10, and domain of unknown function (DUF)-1858. Although PG_0686 showed some relatedness to several diguanylate cyclases (DGCs), it is missing the classical conserved, active site sequence motif (GGD[/E]EF), commonly observed in other bacteria. PG_0686-related proteins are observed in other anaerobic bacterial species. The isogenic mutant P. gingivalis FLL361 (ΔPG_0686::ermF) showed increased sensitivity to H2O2, and decreased gingipain activity compared to the parental strain. Transcriptome analysis of P. gingivalis FLL361 showed the dysregulation of several gene clusters/operons, known oxidative stress resistance genes, and transcriptional regulators, including PG_2212, CdhR and PG_1181 that were upregulated under normal anaerobic conditions. The intracellular level of c-di-GMP in P. gingivalis FLL361 was significantly decreased compared to the parental strain. The purified recombinant PG_0686 (rPG_0686) protein catalyzed the formation of c-di-GMP from GTP. Collectively, our data suggest a global regulatory property for PG_0686 that may be part of an unconventional second messenger signaling system in P. gingivalis. Moreover, it may coordinately regulate a pathway(s) vital for protection against environmental stress, and is significant in the pathogenicity of P. gingivalis and other anaerobes. IMPORTANCE Porphyromonas gingivalis is an important etiological agent in periodontitis and other systemic diseases. There is still a gap in our understanding of the mechanisms that P. gingivalis uses to survive the inflammatory microenvironment of the periodontal pocket. The hypothetical PG_0686 gene was highly upregulated under prolonged oxidative stress. Although the tertiary structure of PG_0686 showed little relatedness to previously characterized diguanylate cyclases (DGCs), and does not contain the conserved GGD(/E)EF catalytic domain motif sequence, an ability to catalyze the formation of c-di-GMP from GTP is demonstrated. The second messenger pathway for c-di-GMP was previously predicted to be absent in P. gingivalis. PG_0686 paralogs are identified in other anaerobic bacteria. Thus, PG_0686 may represent a novel class of DGCs, which is yet to be characterized. In conclusion, we have shown, for the first time, evidence for the presence of c-di-GMP signaling with environmental stress protective function in P. gingivalis.

Keywords: PG_0686; Porphyromonas gingivalis; c-di GMP; diguanylate cyclase; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
The induction of PG_0686 gene in P. gingivalis W83 under different types of environmental stress. P. gingivalis W83 cultures were treated with (A) H2O2 (hydrogen peroxide stress, HPS), (B) O2/Air, or (C) NO (from NO donor diethylamine (DEA) NONOate), and total RNA was extracted from the samples and subjected to RT-PCR. (A, B, and C) Lane 1: PG_0686 gene from untreated control samples; Lane 2: PG_0686 gene from environmental stress-treated samples; Lane 3: 16S rRNA gene from untreated control samples; Lane 4: 16S rRNA gene from environmental stress-treated samples. (D) qRT-PCR confirmed PG_0686 induction under environmental stress. The results represent 3 independent experiments. Error bars indicate standard error of the mean (SEM). The PG_0686 gene was induced under multiple types of environmental stress.
FIG 2
FIG 2
The sensitivity and survival of the P. gingivalis PG_0686-deficient mutant, FLL361, under multiple environmental stress, compared to the W83 wild type. Strains were grown in BHI broth, from overnight cultures, to OD600 ~ 0.2. The cultures were treated with H2O2 (HPS), Air/O2, or NO (DEA NONOate), and left to grow over a 24-h period. For percent survival, at a 12 h time point, H2O2-treated culture portions were removed from the broth, diluted, plated on BHI agar plates, and grown 5 to 7 days. Colonies were enumerated and expressed as a percentage of the untreated controls for the respective strains. Untreated controls were included in the experiments, and the results represent 3 independent experiments. (A) W83 and FLL361 sensitivity to HPS. (B) W83 and FLL361 survival in the presence of HPS. (C) W83 and FLL361 sensitivity to O2/Air. (D) W83 and FLL361 sensitivity to NO. Compared to W83, the FLL361 mutant was significantly more sensitive to different stresses, except NO (A to D). Error bars indicate standard error of the mean (SEM). (*, P < 0.1; **, P < 0.05; ***, P ≤ 0.001).
FIG 3
FIG 3
Transcriptome analysis of FLL361. The genes that were differentially expressed were ≥ 2-fold up- or downregulated (P ≤ 0.05). (A) Volcano plots of differentially expressed genes between P. gingivalis PG_0686-deletion mutant FLL361 and W83 wild-type under: (A, panel i.) AN conditions and (A, panel ii.) HPS conditions. (B) Distribution of functions for P. gingivalis FLL361 genes that were: (B, panel i.) downregulated (blue) or (B, panel ii.) upregulated (red) under AN conditions, HPS conditions, or both conditions. Differentially expressed genes were grouped by functional class according to the KEGG P. gingivalis genome database (https://www.genome.jp/kegg-bin/show_genome?org=pgi). (C) The top enriched pathways in P. gingivalis FLL361 under AN (C, panel i.) and HPS (C, panel ii.) conditions. Enrichment plots were generated by ShinyGO 0.76 (31).
FIG 4
FIG 4
Proteolytic activity in P. gingivalis W83 and FLL361 strains. The whole cell culture Rgp and Kgp activities for W83 and FLL361 strains at log (OD600 ~ 0.6) and stationary (OD600 ~ 1.4) phases were measured using BPNA and ALNA substrates, respectively. Rgp and Kgp proteolytic activities were reduced in the FLL361 mutant compared to W83 wild type. Results shown represent at least 3 independent experiments, each in triplicate. Error bars indicate standard error of the mean (SEM). (*, P < 0.1; **, P < 0.05; ***, P < 0.005).
FIG 5
FIG 5
Architecture and in silico protein modeling of the hypothetical PG_0686 protein. (A) The domain architecture of the PG_0686 protein, a protein 517 amino acid residues in length, constructed based on BLAST analysis from NCBI and MOTIF Finder databases. PG_0686 does not contain the classic GGD(/E)EF motif found in DGCs. However, the leucine heptad repeat and RxxD inhibitory binding site, typical of DGCs, are observed. (B) In silico protein modeling and analysis of PG_0686, (B, panel i.) I-TASSER model of PG_0686, showing protein structure containing helix (blue), coil (gray), and beta sheet (pink) structures. (B, panel ii.) PG_0686 is predicted to be a dimer by SWISS-MODEL. (B, panel iii.) Putative chloro diiron-oxo binding residues (cyan) found in the predicted bacterial hemerythrin (BHr) domain. (B, panel iv.) Putative phosphate binding residues (yellow) found in the predicted PAS 10 domain.
FIG 6
FIG 6
The presence of iron in the rPG_0686 protein. (A) Iron staining of rPG_0686 shows the presence of iron in the protein. Three, 15, or 30 μg of rPG_0686 or BSA was incubated with 0.1 mM Fe(NH4)2SO4•6H2O, and then resolved on a native protein gel. (A, panel i.) The gels were then stained with 1 mM 3-(2-pyridyl)-5,6-bis(2-[5furyl sulfonic acid])-1,2,4-triazine (Ferene S), and 15 mM thioglycolic acid in 2% (vol/vol) acetic acid. (A, panel ii.) A duplicate gel was stained with SimplyBlue Safestain to determine equivalent protein loading. rPG_0686 protein not incubated with iron, and BSA were used as controls. (B) EDTA determination of iron content shows the presence of iron in rPG_0686 protein. Cytochrome c was used as the positive control, and lysozyme was used as the negative control. (C) A change in the UV-Vis spectra of rPG_0686, due to the reaction with potassium ferricyanide, indicated a change from oxy-hemerythrin to met-hemerythrin in the protein. Reduction of the mixture with sodium dithionite did not restore the as-isolated spectra, but changed the spectra, likely due to deoxy-hemerythrin in the protein. (D) No change in the UV-Vis spectra of rPG_0686 when incubated with potassium ferrocyanide. Reduction of the mixture with dithionite changed the spectra to the deoxy-hemerythrin profile. (**, P < 0.05).
FIG 7
FIG 7
Diguanylate cyclase activity of rPG_0686. (A) C-di-GMP levels in the PG_0686-deficient mutant are reduced compared to the W83 wild type. (B) rPG_0686 shows diguanylate cyclase activity similar to the known diguanylate cyclase, PleD. Bars indicate c-di-GMP or pyrophosphate levels, and error bars indicate SEM. (*, P < 0.05; ***, P < 0.001). (C) LC-MS/MS analyses of DGC assay reactions indicated that rPG_0686 produced c-di- GMP in the presence of GTP. A total of 3 μg/mL cXMP was added to each sample as an internal standard. Results show MRM transitions m/z 347 > 153 and 690.9 > 152 that correspond to cXMP and c-di-GMP in the reaction matrix, respectively. (C, panel i.) Commercial c-di-GMP, (C, panel ii.) PG_0686/GTP reaction, and (C, panel iii.) PleD/GTP reaction showed c-di-GMP peak. No c-di-GMP peak was observed in the (, panel iv.) no protein/GTP reaction and (C, panel v.) BSA/GTP reaction.
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