Full-length structure and heme binding in the transcriptional regulator HcpR.

HcpR is a CRP-family transcriptional regulator found in many Gram-negative anaerobic bacteria. In the perio-pathogen Porphyromonas gingivalis, HcpR is crucial for the response to reactive nitrogen species such as nitric oxide (NO). Binding of NO to the heme group of HcpR leads to transcription of the redox enzyme Hcp. However, the molecular mechanisms of heme binding to HcpR remain unknown. In this study we present the 2.3Å structure of the P. gingivalis HcpR. Interdomain interactions present in the structure help to form a hydrophobic pocket in the N-terminal sensing domain. A comparison analysis with other CRP-family members reveals that the molecular mechanisms of HcpR-mediated regulation may be distinct from other family members. Using docking studies, we identify a putative heme binding site in the sensing domain. In vitro complementation and mutagenesis studies verify Met68 as an important residue in activation of HcpR. Finally, heme binding studies with purified forms of recombinant HcpR support Met68 and His149 residues as important for proper heme coordination in HcpR.

Insertional Inactivation of Prevotella intermedia OxyR Results in Reduced Survival with Oxidative Stress and in the Presence of Host Cells.

One of the most abundant bacteria in the subgingival pockets of patients with bleeding following mechanical periodontal therapy is Prevotella intermedia. However, despite its abundance, the molecular mechanisms of its contribution to periodontal disease are not well known. This is mainly due to the lack of genetic tools that would allow examination of the role of predicted virulence factors in the pathogenesis of this bacterium. Here, we report on the first mutant in the P. intermedia OMA14 strain. The mutation is an allelic exchange replacement of the sequences coding for a putative OxyR regulator with ermF sequences coding for the macrolide-lincosamide resistance in anaerobic bacteria. The mutant is severely impaired in its ability to grow with eukaryotic cells, indicating that it is an important target for interventional strategies. Further analyses reveal that its ability to grow with oxidative stress species, in the form of hydrogen peroxide and oxygen, is severely affected. Transcriptome analysis reveals that the major deregulated genes code for the alkylhydroperoxide reductase system, AhpCF, mediating protection from peroxide stress. Moreover, genes coding for Dps, CydA and Ftn are downregulated in the mutant strain, as further verified using qRT-PCR analysis. In conclusion, we succeeded in generating the first P. intermedia mutant and show that the OxyR-deficient strain is unable to survive with a variety of host cells as well as with oxidative stress.

Non-human Primate Macaca mulatta as an Animal Model for Testing Efficacy of Amixicile as a Targeted Anti-periodontitis Therapy.

Periodontitis is an inflammatory condition triggered by selected oral microbiota; thus treatment strategies should be aimed at reducing the abundance of the pathogenic bacteria. An obstacle to preclinical testing of such strategies is the availability of reliable animal models. Here, a non-human primate (NHP), Macaca mulatta, was used to examine the effectiveness of a novel antimicrobial, amixicile, which inhibits pyruvate-ferredoxin oxidoreductase (PFOR) present in anaerobic bacteria. Animals were assessed for their periodontal health, including radiography, clinical attachment loss (CAL), presence of plaque (PI), bleeding on probing (BOP) and pocket depth (PD), and sampled for saliva, gingival crevicular fluid (GCF), and subgingival plaque to determine their baseline clinical status. Amixicile was then administered for 2 weeks (40 mg/kg/day) and the animals were monitored for periodontal health immediately after the antibiotic treatment, then at 1 month-, 3 months-, and 6-months posttreatment. Microbial species present in plaque and saliva were determined through 16S rDNA sequencing. Baseline assessment of the microbiome has shown a significant proportion of bacteria belonging to the Streptococcus, Haemophilus, Porphyromonas, Gemella, and Fusobacterium genera. The abundance of Porphyromonas and Fusobacterium was reduced following treatment with amixicile, whereas that of Escherichia, Haemophilus, and Gemella were elevated. CAL, PD, and BOP were also significantly reduced following the treatment. In conclusion, the NHP model proves useful for preclinical studies of strategies targeting selected members of the oral microbiome. We show that amixicile reduces the levels of anaerobic bacteria under in vivo conditions, correlating with a reduction in CAL, PD, and BOP, thus validating its usefulness as an antimicrobial strategy.

Treatment with Nd:YAG Laser Irradiation Combined with Sodium Hypochlorite or Hydrogen Peroxide Irrigation on Periodontal Pathogens: An In Vitro Study.

Objective: The purpose of this study was to evaluate the effect of neodymium-doped yttrium aluminum garnet (Nd:YAG) laser with low concentrations of hydrogen peroxide (H2O2) or sodium hypochlorite (NaOCl) on viability of oral bacteria. Materials and methods: Bacterial species Streptococcus gordonii, Porphyromonas gingivalis, and Fusobacterium nucleatum were grown in an anaerobic chamber at 37°C. Samples were irradiated with the Nd:YAG laser (1064 nm, 300 µm Varian tip) using parameters: 150 mJ, 20 Hz, 3 W, 50 sec, and 100 µs short pulse duration in contact mode. Treatment groups included (1) control, (2) Nd:YAG, (3) 0.5% H2O2, (4) Nd:YAG and 0.5% H2O2, (5) 0.5% NaOCl, and (6) Nd:YAG with 0.5% NaOCl. Viable colonies were counted, calculated into colony forming unit/mL, and converted into log form for statistical analysis using a two-tailed paired t-test. Results: The combined treatment with the Nd:YAG and H2O2 showed the greatest reduction in all bacterial viability compared with other treatment groups (p < 0.001). Antiseptic solutions and laser were most effective against P. gingivalis, least effective against S. gordonii but improved significantly in combination with laser irradiation (p < 0.001). Laser alone was effective against all of three bacterial species, however, it was not significant. Conclusions: Combination treatment with Nd:YAG laser and an oxidative disinfectant (0.5% NaOCl or H2O2) resulted in more effective reduction of bacterial viability than monotherapies.

Er:YAG Laser Irradiation Reduces Microbial Viability When Used in Combination with Irrigation with Sodium Hypochlorite, Chlorhexidine, and Hydrogen Peroxide.

The erbium-doped yttrium aluminum garnet (Er:YAG) laser is used to treat periodontal disease; however, its effectiveness at killing oral bacteria is not well known. Furthermore, the compounding effect of the combination of a laser treatment and irrigation methods with antimicrobials on bacterial viability is yet to be determined. The purpose of this in vitro study was to evaluate the effect of the Er:YAG laser with irrigation using chlorhexidine (CHX), hydrogen peroxide (H2O2), or sodium hypochlorite (NaOCl) on the viability of oral bacteria. Three bacterial species were used in our study: Streptococcus gordonii, Fusobacterium nucleatum, and Porphyromonas gingivalis. Bacteria were grown in an anaerobic chamber in brain heart infusion broth and incubated at 37 °C. Bacterial samples with an OD of 0.5 were irradiated with the Er:YAG laser at 2940 nm using a 400-µm Varian tip. The experiment was repeated four times using these parameters: 40 mJ, 40 Hz, and 1.6 W for 20 seconds with the 300 µs short pulse duration in contact mode. Treatment groups consisted of the following: (1) no treatment, (2) 0.5% H2O2 alone, (3) 0.5% NaOCl alone, (4) 0.03% CHX alone, (5) Er:YAG irradiation alone, (6) Er:YAG irradiation with 0.5% H2O2, (7) Er:YAG irradiation with 0.5% NaOCl, and (8) Er:YAG irradiation with 0.03% CHX. Microbial viability was determined through plating and colony counts and calculated into CFU/ml. Statistical analysis was done using a two-tailed paired t-test. The use of the Er:YAG laser alone failed to show statistically significant antibacterial activity against any of bacteria. The most effective mono-treatment with irrigation solutions for all three bacteria were 0.5% H2O2 and 0.5% NaOCl (p < 0.001 for each solution). Irrigation with 0.03% CHX was most effective against F. nucleatum (p < 0.01) and less against P. gingivalis and S. gordonii and showed the least antibacterial action alone but improved significantly in combination therapy (p < 0.05). The combined treatment with the Er:YAG showed the greatest and most significant improvement in the reduction of bacterial viability compared to any other treatment group (p < 0.05 for each combined treatment). Irradiation with the Er:YAG laser with the addition of 0.5% H2O2, 0.5% NaOCl, or 0.03% CHX under a short working time (20 s) resulted in a significant reduction of bacterial viability for all three bacterial species compared with any single treatment option. The combination of irradiation with the Er:YAG laser with the addition of 0.5% H2O2, 0.5% NaOCl, or 0.03% CHX resulted in a larger reduction of bacterial survival when compared to monotherapies with antimicrobial solutions or laser. The combination of the Er:YAG laser with a low concentration irrigant solution of 0.5% H2O2, 0.5% NaOCl, or 0.03% CHX could be an effective treatment protocol for the reduction of periodontal pathogens and thus suitable treatment for non-surgical periodontal therapy.

Nitrosative stress sensing in Porphyromonas gingivalis: structure of and heme binding by the transcriptional regulator HcpR.

Although the HcpR regulator plays a vital step in initiation of the nitrosative stress response in many Gram-negative anaerobic bacteria, the molecular mechanisms that it uses to mediate gas sensing are not well understood. Here, a 2.6 Šresolution crystal structure of the N-terminal sensing domain of the anaerobic periodontopathogen Porphyromonas gingivalis HcpR is presented. The protein has classical features of the regulators belonging to the FNR-CRP family and contains a hydrophobic pocket in its N-terminal sensing domain. It is shown that heme bound to HcpR exhibits heme iron as a hexacoordinate system in the absence of nitric oxide (NO) and that upon nitrosylation it transitions to a pentacoordinate system. Finally, small-angle X-ray scattering experiments on full-length HcpR reveal that the C-terminal DNA-binding domain of HcpR has a high degree of interdomain flexibility.

The Porphyromonas gingivalis Hybrid Cluster Protein Hcp Is Required for Growth with Nitrite and Survival with Host Cells.

Although the periodontal pathogen Porphyromonas gingivalis must withstand high levels of nitrosative stress while in the oral cavity, the mechanisms of nitrosative stress defense are not well understood in this organism. Previously we showed that the transcriptional regulator HcpR plays a significant role in defense, and here we further defined its regulon. Our study shows that hcp (PG0893), a putative nitric oxide (NO) reductase, is the only gene significantly upregulated in response to nitrite (NO2) and that this regulation is dependent on HcpR. An isogenic mutant deficient in hcp is not able to grow with 200 µM nitrite, demonstrating that the sensitivity of the HcpR mutant is mediated through Hcp. We further define the molecular mechanisms of HcpR interaction with the hcp promoter through mutational analysis of the inverted repeat present within the promoter. Although other putative nitrosative stress protection mechanisms present on the nrfAH operon are also found in the P. gingivalis genome, we show that their gene products play no role in growth of the bacterium with nitrite. As growth of the hcp-deficient strain was also significantly diminished in the presence of a nitric oxide-producing compound, S-nitrosoglutathione (GSNO), Hcp appears to be the primary means by which P. gingivalis responds to NO2--based stress. Finally, we show that Hcp is required for survival with host cells but that loss of Hcp has no effect on association and entry of P. gingivalis into human oral keratinocytes.