P. gingivalis W83 was a gift from Professor RJ Lamont (now in Department of oral Immunology and Infectious Disease, School of Dentistry, University of Louisville) from College of Dentist, University of Florida. Freshly prepared brain heart infusion (BHI, Difco Laboratories, MI, USA) agar medium supplemented with 5% sterile defibrinated sheep’s blood, 1% hemin, and 0.1% menadione, was used to grow P. gingivalis W83 at 37 °C under anaerobic conditions (80% N2, 10% H2 and 10% CO2) for 5 to 7 days.
Antimicrobial peptide Nal-P-113, Ac-AKR-Nal-Nal-GYKRKF-Nal-NH2, was provided by Prof. Jiawei Cheng in National Tsing Hua University . H2O2 was purchased from Sigma Aldrich (CA).
P. gingivalis W83 was diluted to 5 × 105 CFU/mL (CFU, colony forming units). The bacteria were treated with Nal-P-113 in 100 μL culture medium for 24 h. Then an aliquot (50 μL) of the resulting bacterial cell suspension was cultivated on a brain heart infusion agar plate. The bacterial cells were enumerated after incubation at 37 °C for 7 days. All experiments were repeated three times.
Growth inhibition assay
P. gingivalis W83 culture was diluted to 5 × 105 CFU/mL. The bacteria were treated with Nal-P-113 at different concentrations (0, 5, 10, 20, 40, 80, 160 and 320 μg/mL respectively) in 100 μL culture medium for 48 h. The cell growth was measured by the absorbance at 600 nm in a microplate reader (Tecan Infini M200, Switzerland). All experiments were repeated three times.
Scanning electron microscopy (SEM) analysis on Biofilms
Biofilms formation was quantified on 6-well plates (Corning, Netherlands) which were coated with artificial saliva (Guangzhou Kodak Adhesives Co. Ltd., China). Five hundred microliter of P. gingivalis W83 (5 × 106 CFU/mL) with or without 6.25 μg/mL Nal-P-113 treatment was dropped on 6-well plates and cultured for 48 h to establish biofilms. Then, the samples were fixed with 2.5% glutaraldehyde (BioChemika, Fluka, USA), washed with PBS and gradually dehydrated with ethanol. The processed samples were smeared onto copper plates followed by gold sputtering, and images were acquired using scanning electron microscopy (Inspect F50, FEI Company, USA) at 20,000 × magnification.
The total RNA of the bacteria treated with or without 6.25 μg/mL Nal-P-113 was extracted, converted to cDNA, labeled with Cy3-dCTP (GE, Healthcare, CA) and followed by hybridization with P. gingivalis W83 chip (Agilent, CA). Array hybridization, washing, scanning and data analysis were performed at the CapitalBio Corporation (Beijing, China).
The array data were analyzed with the GeneSpring software V12 (Agilent). Significant genes were defined by following criterions: FDR < 5% and fold change > 2 [14, 15].
Quantitative PCR (qPCR)
The total RNA of P. gingivalis W83 treated with or without 6.25 μg/mL Nal-P-113 was reversed transcribed into cDNA with the M-MLV RTase cDNA Synthesis Kit (Takara, China). Real-time quantitative PCR reaction was performed using SYBR Premix Ex TaqTM II PCR Master Mix Reagents Kit (Takara). The primers for the RT-qPCR were listed in Additional file 1: Table S1. qPCR was performed three times for each sample. Relative quantification of the mRNA levels was performed using the comparative Ct method with the formula 2-ΔΔCt.
Hydrogen peroxide pre-treatment in P. gingivalis
P. gingivalis W83 was treated with H2O2 at different concentrations (0, 0.5, 1, and 3 mM, respectively) for 1 h. The H2O2 was washed away with phosphate buffer saline before subsequent experiments. Total RNA of the respective H2O2-treated P. gingivalis was extracted and followed by RT-qPCR to determine the mRNA expression of PG0841, PG0842, PG0872, PG0874, PG0875, PG1473, PG1474, PG1475, PG1478, PG1479, PG1482 and PG1485.
Biofilms susceptibility assay
3 mM H2O2 was used to change transposase genes expression of P. gingivalis W83 for 1 h. The effect of Nal-P-113 on P. gingivalis W83 biofilms formation was examined using the microdilution method . The resulting biofilms were fixed with 95% methanol and stained with 0.5% (w/v) crystal violet prior dissolving with 95% ethanol and subjected to microplate reader at absorption 590 nm. Percentage of inhibition was calculated using the equation [1-(A590 of the test/A590 of non-treated control)] × 100. All experiments were repeated three times.
Enzyme linked immunosorbent assay (ELISA)
Lipopolysaccharide, free hemin and hemoglobin were measured by commercial ELISA kit according to the respective manual. Free hemin ELISA kit was purchase from Abnova, Taiwan, and the hemoglobin ELISA kit was purchased from Leagene, China. The measurements were analysis by Curve Expert 1.3 (AL).
All experiments were performed in triplicate and repeated at least three times. Data were expressed as means ± standard deviations (SD). ANOVA and independent samples t-test were used to calculate the significance among the groups (SPSS Inc., IL, USA). P-value < 0.05 was considered statistically significant.