F.nucleatum (ATCC 25586) and P. aeruginosa PAO1 strain (ATCC BAA-47) were obtained from American Type Culture Collection (ATCC). F. nucleatum was grown in tryptic soy broth (TSB. Becton, Dickinson and Company, Sparks, MD, USA) supplemented with 5% defibrinated sheep blood, 10mg/ml hemin and 5mg/ml menadione in anaerobic atmosphere (10% H2, 10% CO2, 80% N2) at 37 ℃. P. aeruginosa was cultured in TSB under aerobic conditions at 37 ℃.
Bacterial aggregation assay.
P. aeruginosa and F. nucleatum were standardized in sterile coaggregation buffer (150 mM NaCl, 1 mM Tris, 0.1 mM CaCl2 and 0.1 mM MgCl2) to give a final cell density of 1×109 colony-forming units per mL (CFUs/mL). Equal numbers of single- or dual-species bacterial cells were suspended and vortexed for 30 s in a reaction tube (T=0 h). The tubes were incubated at room temperature for 1 h to allow aggregation (T=1 h). A visual rating scale of 0-4 was used to grade the reaction. “0” indicates an evenly turbid suspension with no visible aggregates, meaning no coaggregation; “1+” indicates turbid supernatant with finely dispersed coaggregates; “2+” indicates definite coaggregates that do not precipitate immediately; “3+” indicates slightly turbid supernatant with formation of large precipitating coaggregates; and “4+” indicates complete sedimentation with a clear supernatant[28, 29]. In addition, a spectrophotometric assay was employed to determine the percentage of bacterial auto- and coaggregation[19, 28]. The optical density of bacterial suspensions at 600 nm wavelength (OD600) were measured at the time points of 0 and 1 h. Percentage aggregation was calculated using the following equation: % autoaggregation or coaggregation =(OD600(T0)-OD600(T1))/ OD600(T0) ×100. Percentage aggregation were classified as high (more than 40%), intermediate (30-40%) and low aggregation (less than 30%).
Human pulmonary epithelial cell line A549 was obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM. HyClone Laboratories, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS. HyClone Laboratories, Logan, UT, USA) in a humidiﬁed 37℃ incubator with 5% CO2.
Bacterial infection of pulmonary epithelial cells.
A549 cells were incubated with F. nucleatum and P. aeruginosa alone or together at multiplicity of infection (MOI) of 10, 50 or 100. For bacterial simultaneous infection model, A549 cells were incubated with both F. nucleatum and P. aeruginosa at the same times. For bacterial sequential infection model, A549 cells were treated with F. nucleatum or P. aeruginosa for 12 h, and then infected with the other bacteria for 12 h.
Antibiotic protection adhesion and invasion assay
Assays of bacterial adherence and invasion were performed using methods previously described. Briefly, bacteria were harvested, washed, and resuspended in DMEM without antibiotics. A549 cells were infected with bacteria for 3 h, and washed with phosphate-buffered saline (PBS). For determining total adhesion and invasion levels (attachment levels), cells were lysed with sterile distilled water for 30 min, then dilutions of the lysate were plated and cultured anaerobically on TSB agar supplemented with 5% defibrinated sheep blood, 10mg/ml hemin and 5mg/ml menadione to determine CFUs for F. nucleatum, or cultured aerobically on TSB agar to determine CFUs for P. aeruginosa. For invasion assay, extracellular bacteria were killed with 200 μg/mL gentamicin (Sigma, St. Louis, MO, USA) and 300 μg/mL metronidazole (Sigma, St. Louis, MO, USA) for 1 h. Cellular lysate were diluted and cultured to determine CFUs. The efficiency of bacterial attachment or invasion is equal to CFUs divided by the number of cells.
Scanning electron microscopy (SEM)
A549 cells were infected with P. aeruginosa and F. nucleatum alone or together for 3 h, washed with PBS, and fixed with 2.5% glutaraldehyde (BioChemika, Fluka, Switzerland). A549 cells were gradually dehydrated with ethanol and smeared onto copper plates followed by gold sputtering, and images were acquired using SEM (Inspect F50, FEI Company, USA) to observe the bacterial adhesion and cell morphology.
CCK-8 cell viability assay
Cell counting kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to assess cell proliferation according to the manufacturer’s manual. Briefly, A549 cells were infected with P. aeruginosa and F. nucleatum alone or together for 4, 8, 16 and 24 h. 10 μL of CCK-8 solution was added to 100 μL of medium solution and incubated for 1 h at 37℃. OD450 was measured with a microplate reader (Infinite M200, Tecan, Austria) to reflect the cell proliferation.
Cytotoxicity assessment by Lactate dehydrogenase (LDH) leakage assay
LDH activity was monitored by the PierceTM LDH cytotoxicity assay kit (Thermo Scientific, USA) in accordance with the manufacturer’s recommendations. A549 cells were exposed to P. aeruginosa and F. nucleatum alone or together for the desired time, the culture supernatant was harvested and centrifuged at 12000 g for 5 min, and 50 μL supernatant was mixed with 50 μL Reaction mixture and incubated at room temperature in the dark for 30 min. 50 μL stop solution was added to stop reaction. OD490 and OD680 were measured with a microplate reader (Infinite M200, Tecan, Austria). LDH activity = OD490-OD680.
Live/dead assay by calcein acetoxymethyl ester/propidium iodide (Calcein-AM/PI) staining
The effects of P. aeruginosa and F. nucleatum on the viability or cytotoxicity of pulmonary epithelial cells were evaluated using the Calcein-AM/PI Double Stain Kit (Molecular Probes, Eugene, OR, USA). In brief, A549 cells were exposed to P. aeruginosa and F. nucleatum alone or together for the desired time, washed with PBS. Cells were stained with 200 μL Calcein-AM/PI stain working solution at 37 ℃ for 15 min. Living cells (green cytoplasmic fluorescence) and dead cells (red nucleus fluorescence) were immediately observed by fluorescence microscope.
Enzyme-linked immunosorbent assay (ELISA)
A549 cells were exposed to P. aeruginosa and F. nucleatum alone or together for the desired time, the culture supernatant was harvested and centrifuged at 12000 g for 5 min. IL-1β, IL-6 and TNF-α levels in the culture supernatant were determined via ELISA kits (Invitrogen, Cambrillo, USA) according to the manufacturer’s protocol. After the procedure, plates were read on the spectrometer at 450 nm wavelength. The results were converted to numeric values using standard curves.
All experiments were performed in triplicate and repeated three times. All values were presented as mean ± standard deviation (SD) and analyzed by analysis of variance followed by Dunnett’s multiple comparisons test using GraphPad Prism version 7.00 (GraphPad Software, La Jolla, CA, USA). Differences were considered statistically significant at P<0.05.