Effects of estradiol on the virulence traits of Porphyromonas gingivalis

Bacterial and cell culture

P. gingivalis strain W50 kindly provided by Professor MA Curtis, (Molecular Pathogenesis Group, Queen Mary, University of London), was used in this study. P. gingivalis W50 was anaerobically cultured at 85% N25% CO2and 10% H2 at 37 °C in an anaerobic chamber (Concept 400-M Anaerobic Workstation; Ruskinn Technology Ltd., Leeds, UK) in tryptic soy broth (TSB) supplemented with yeast extract (1 mg/ml), hemin (5 μg/ml) and menadione (1 μg/ml). P. gingivalis W50 was grown for 48 h and then centrifuged for 10 min at 10,000 rpm, washed and resuspended with PBS.

The human gingival epithelial cell (GEC) line Ca9-22 (Tebu-bio, Paris, France) was grown in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza, Basel, Switzerland) with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM non-essential amino acids (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37 °C and 5% CO2 atmosphere. The culture medium was changed during experiments to DMEM with 2% FBS, 1 mM non-essential amino acids and 2 mM l-glutamine, as previously described2.24.

Bacterial growth assessment

P. gingivalis W50 (1 × 106 CFU/ml) was grown in TSB supplemented with yeast extract (1 mg/ml), hemin (5 μg/ml) and menadione (1 μg/ml) with or without the presence of 17β-Estradiol [1 pg/ml, 2 pg/ml, 5 pg/ml, 10 pg/ml, 100 pg/ml, 300 pg/ml and 1000 pg/ml (E2758, Sigma-Aldrich, St. Louis, MO, USA)] in a 96-well plate. The 96-well plate was then incubated at 37 °C anaerobically and the optical density (600 nm) was measured using a spectrophotometer (Cytation 3, Biotek Inc, Winooski, VT, USA), as previously described20,21.

Biofilm and gingipain measurement

After the growth assessment, the same 96-well plate was used for investigating biofilm formation. The plate was washed three times with sterile RO-water and 0.1% Crystal violet (Thermo Fisher Scientific) was then added to the wells for 20 min in order to stain the biofilm. The plate was then washed with RO water and left to dry overnight. Ethanol (95%) was used to dissolve the crystal violet and the absorbance (540 nm) was measured by a spectrophotometer (Citation 3).

After assessing the bacterial growth, the TSB was collected from the same 96-well plate and centrifuged at 10,000 rpm for 10 min in order to measure gingipain activity. The bacterial-free TSB was then transferred into a new 96-well plate and 100 µM Z-His-Glu-Lys-AMC (substrate for Lysine-Gingipain, PeptaNova GmbH, Germany) or Boc-Phe-Ser-Arg-AMC ( substrate for Arginine-Gingipain, PeptaNova GmbH) was added to the plate. The plate was incubated for 1 h at 37 °C and the gingipain activity was measured at 340/440 nm (Citation 3).

Cytokine release and viability assay

P. gingivalis W50 was grown in the presence or absence of estradiol for 48 h at 37 °C as previously stated. Estradiol was then washed away P. gingivalis W50 with PBS and the gingival epithelial cell line was infected with the respective treatment at Multiplicity of infection (MOI) of 100 for 24 h at 37 °C and 5% CO2. Supernatants were collected after the infection and centrifuged for 10 min at 10,000 rpm and stored at − 80 °C. An enzyme-linked immunosorbent assay (ELISA) was performed to measure Interleukin-1β (IL-1β), CXCL10 and TGF-β1 release from the GECs. The cytokine was measured with the IL-1β and CXCL10 kits (ELISA MAX Deluxe Sets, BioLegend, San Diego, CA, USA) and TGF-β1 kit (Duo set, ELISA, R&D Systems, Minneapolis, MN, USA) according to the kit’s instructions. The concentrations were determined by optical density at 450 nm. Cell viability after P. gingivalis W50 infection was assessed by optical density at 490 nm using the Pierce LDH cytotoxicity assay (Thermo Fisher Scientific) according to the kit’s instructions, as previously described20.25.

RNA isolation, cDNA generation and quantitative polymerase chain reaction

P. gingivalis W50 was grown in the presence or absence of estradiol for 48 h at 37 °C as previously stated. Estradiol was then washed away P. gingivalis W50 with PBS and the gingival epithelial cell line was infected with the respective treatment at MOI of 100 for 24 h at 37 °C and 5% CO2. Total RNA was isolated using the EZNA® Total RNA Kit I (Omega Bio-tek, Inc., Norcross, GA, USA) according to manufacturer’s instructions. RNA purity and concentration were measured using a spectrophotometer (Nano Drop 2000, Wilmington, NC, USA). 100 ng of total RNA was used for the cDNA synthesis (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems, CA, USA). For the RT-qPCR, 5 ng cDNA and 250 nM of primer (Table 1) (Eurofins MWG Synthesis GmbH, Ebersberg, Munich, Germany) was used with Maxima SYBR Green qPCR Master Mix (ThermoFisher Scientific). Amplification was done using the following protocol for 40 cycles: Denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s. A CFX96 Touch™Real-Time PCR Detection System (Biorad , CA, USA) was used. A dissociation curve between 60 and 95 °C was also done after the qPCR. CT values ​​were obtained and the ΔΔCt method (2−ΔΔCt) was used to calculate the fold difference. The results were normalized to the endogenous control GAPDH, as previously described20.25.

Table 1 Primers used for quantitative real-time PCR.

Colonization assay

P. gingivalis W50 was grown anaerobically in the presence or absence of estradiol (1 pg/ml and 100 pg/ml) for 48 h at 37 °C. Estradiol was then washed away P. gingivalis W50 with PBS. The bacteria were then FITC-labeled (Sigma-Aldrich) and the GECs were infected with the respective treatment at MOI of 100 for 2 h at 37 °C and 5% CO2 to measure bacterial colonization (adherent and intracellular bacteria). After which the cells were washed with PBS and the FITC-labelled P. gingivalis W50 were quantified with the Cytation 3 plate reader. Colonization is presented as % mean fluorescence intensity (MFI) of P. gingivalis W50, as previously described20.

Invasion assay

P. gingivalis W50 was grown anaerobically in the presence or absence of estradiol (5 pg/ml and 100 pg/ml) for 48 h at 37 °C. Estradiol was then washed away P. gingivalis W50 with PBS and the GECs were infected with the respective treatment at MOI of 100 for 2 h at 37 °C and 5% CO2. After which the cells were washed with PBS. DMEM supplemented with 2% FBS, 300 μg/ml gentamicin and 200 μg/ml metronidazole were then added to the GEC to kill remaining extracellular P. gingivalis W50 during 1 h. The plate was then washed again, and the cells were lysed with 0.1% Triton-X 100 in PBS (with calcium chloride 100 mg/l and magnesium chloride 100 mg/l) for 10 min under gentle rotation, as previously described with minor modifications26. Finally, P. gingivalis W50 were plated on agar plates [Tryptic soy broth supplemented with agar (15 mg/ml), yeast extract (1 mg/ml), hemin (5 μg/ml) and menadione (1 μg/ml)]incubated for 48 h anaerobically at 37 °C, and the colonies were then counted.

Statistical methods

All data shown are expressed as mean ± SEM. The differences between the groups were analyzed by student’s unpaired t test or one‐way ANOVA followed by Bonferroni multiple testing correction. Results were considered statistically significant at p < 0.05. n = number of independent biological experiments, as previously described20.

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