Quercetin was supplied as a purified compound (code No. NPFL10051.1) from the Styphnolobiumjaponicum (L.) Schott in The Korea Plant Extract Bank (https://extract.kribb.re.kr) at The Korea Research Institute of Bioscience & Biotechnology (Daejeon, Korea). The structural information of authentic compound was listed as a supplementary data. (Fig. 1A and Fig. S1)
All strains and plasmids used in this study are listed in Table S1. Chromosomal mutants were all derived from the same parental PAK (P. aeruginosa strain K; wild-type strain) strain [26, 38, 39] and were generated by allelic exchange.
Bacterial culture and growth curve
All bacterial strains were grown overnight in Luria-Bertani (LB) media at 37℃ on a rotary shaker if not otherwise specified. Bacterial cells were harvested at 12,000 g at 4°C for 10 min after overnight LB broth culture. Antibiotics were used at concentrations of spectinomycin (Sp; 200 μg/ml, Cat. No. S9007-5G), streptomycin (Sm; 200 μg/ml, Cat. No. S6501-5G), gentamycin (Gm; 150 μg/ml, Cat. No. G1264, Sigma-Aldrich, St. Louis, MO, USA) and carbenicillin disodium (Cb; 150 μg/ml, Cat. No. 2485-1G, BioVision, CA, USA) [39, 40]. Overnight cultures of untreated P. aeruginosa PAK strain and P. aeruginosa treated with a 20 μM concentration of quercetin were re-inoculated and diluted 1:1,000 in fresh LB broth. Growth of the bacteria was determined at OD 600 using an ultraviolet–visible (UV/VIS) spectrophotometer (Cat. No. Optizen 2120UV, Mecasys Co., Ltd., Daejeon, Korea) .
The H292 cell line (ATCC; American Type Culture Collection, Manassas, VA, USA) is human epithelial cells derived from human lung carcinoma. H292 cells were cultured in the RPMI 1640 medium (Cat. No. LM 011-01, Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Grand Island, New York) and antibiotics (1% penicillin-streptomycin; Invitrogen, Grand Island, New York) and incubated at 37℃ in a 5% CO2 incubator.
Cell viability was measured via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. H292 cells were seeded into 96-well plates (SPL, Gyeonggi-do, Korea) at a concentration of 1×104 cells/well. After incubation for 5 h, the cells incubated with various concentrations of quercetin for 20 h. In the infection cases, H292 cells were infected by MOI 100 of the PAK ExoS strain after a quercetin treatment for 1 h. Subsequently, 5 μl of 5 mg/ml MTT solution (Amresco, LLC, solon, OH, USA) was added to each well for 4 h. The supernatant was removed, and 100 μl of dimethyl sulfoxide (DMSO) was added to each well for dissolution of the formazan crystals.
ExoS-FLAG enzyme-linked immunosorbent assay (ELISA) assay
To detect ExoS secretion, the secreted ExoS-FLAG amounts in bacterial overnight culture supernatants supplemented with quercetin and 1 mM of EGTA were determined using an ExoS-FLAG sandwich ELISA system designed according to a previous study . An anti-ExoS antibody as a capture antibody commissioned by Koma Biotech (Seoul, Korea), Inc. and produced by Prosci, Inc. (CA, USA). The ExoS amino acid sequence was diluted to 1:5,000 in a carbonate-bicarbonate buffer (0.05 M, pH 9.6), coated into 96-well microplates at 4℃, and kept overnight. The plates were incubated with a blocking buffer (2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)) for 1 h, after which they were washed, and a bacterial supernatant was added to each well. After incubation for 2 h, the plates were washed and mouse anti-FLAG antibody (Anti-DDDDK antibody; Cat. No. ARG62342, Arigo Biolaboratories, Taiwan, ROC), a detection antibody diluted to 1:5,000 in 1% BSA (Bovine Serum Albumin; Cat. No. BSAS0.1, Bovogen Biologicals Pty. Ltd., AUSTRALIA) in PBS (assay diluent), was added to each well. After incubation for 1 h, the plates were washed, and horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) diluted to 1:2,000 in an assay diluent was then added to each well for 1 h. After incubation, the plates were washed, and a substrate solution was added to each well. Lastly, the color reaction of the plates was stopped with H2SO4 and the absorbance was determined at 450-570 nm using a plate reader instrument. All steps except for the incubation of the capture antibody were performed at room temperature.
Cytokine ELISA assay
H292 cells were seeded into 12-well plates at a concentration of 4×106 cells/well and cultured overnight at 37℃. After incubation, the media in the plates were changed to RPMI 1640 without supplementation, and the cells were pretreated with various concentrations of quercetin for 1 h. The H292 cells were then infected by moment of infection (MOI) 100 of the PAK ExoS strain at 37℃ for 6 h. Cytokines of the culture supernatant of the infected cells were measured using ELISA kits (IL-18 set, Cat. No. DY318-05, R&D system, Minneapolis, USA ; IL-1β set, Cat. No. 557953; IL-6 set, Cat. No. 555220; BD biosciences, CA, USA). Each experiment was conducted according to the manufacturer’s protocol.
Real-time polymerase chain reaction (PCR) analysis
Briefly, H292 cells were seeded into 12-well plates at a concentration of 4×106 cells/well and cultured at 37℃ overnights. The cells were treated with quercetin and PAK ExoS for 6 h. In contrast, bacterial cells were grown overnight at 37℃ in a rotary shaker and re-inoculated in fresh LB broth supplemented with quercetin and 1 mM of EGTA for 4 h. After all of the steps, the H292 cells and bacterial cells were harvested. The total RNA was isolated using the TRIzol® Reagent (Invitrogen; Thermo Fisher Scientific, Inc., MA, USA). The synthesis of the complementary DNA was performed using a ReverTra Ace® qPCR RT Master Mix kit (Cat. No. FSQ-301, TOYOBO, Japan). cDNA was used for real-time PCR using primers (Table. S2, Bioneer, Daejeon). This experiment was conducted using SYBR Green PCR Master Mix (KAPA Bio-systems, Woburn, Massachusetts).
Western blot analysis
For the ExoS-FLAG detection, the p30 and p137 strains (Table. S1) were grown overnight in LB broth supplemented with quercetin and 1 mM of EGTA at 37℃ in a rotary shaker. Bacterial cells were then harvested into 1.5-ml tubes and lysed using a diluted 5× bacterial protein extraction reagent (Cat. No. AKR-180, Cell Biolabs, Inc., CA, USA) for protein extraction. The bacterial protein quantity was measured using a Pierce™ BCA Protein assay kit (Cat. No. 23225, Thermo Fisher Scientific Inc., MA, USA).
Equal amounts of protein were loaded and separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Loaded proteins were transferred to a hydrophilic polyvinylidene fluoride membrane (PVDF; EMD Millipore, Billerica, MA, USA). Each membrane was blocked with 5% skim milk in tris-buffered saline with Tween (TBS-T) for 1 h at room temperature. Primary antibodies used ExoS-FLAG (Anti-DDDDK antibody; Cat. No. ARG62342, Arigo Biolaboratories, Taiwan, ROC), β-actin (MA5-15739), IκBα (MA5-15132), p-IκBα (MA5-15087, Thermo Fisher Scientific Inc., MA, USA), p65 (8242S), p-p65 (3033S), caspase-1 (3866S), IL-1β (83186S), NLRC4 (124215S, Cell Signaling Technology, MA, USA) was diluted to 1:1,000 in 5% skim milk in TBS-T, added to the membrane rack, and then incubated overnight at 4℃. After incubation, the membranes were washed with TBS-T and incubated with secondary antibody HRP-conjugated anti-mouse IgG (Cat. No. sc-2005, Santa Cruz Biotechnology Inc., Texas, USA), anti-rabbit IgG (Cat. No. 111-035-003, Jackson Immune Research Laboratories, Inc, PA, USA) diluted to 1:5,000 in 5% skim milk in TBS-T for 1 h at room temperature. The protein bands were visualized using a chemiluminescence (ECL; Cat. No. 32106, Thermo Fisher Scientific Inc., MA, USA) kit and a luminescent image analyzer (LAS-4000, Fujifilm, Tokyo, Japan).
The data are expressed as mean ± SEM. Statistical significance was determined using two-tailed Student’s t-test. A value of p<0.05 was considered to indicate a statistically significant difference.