Quercetin was supplied as a purified compound (code No. NPFL10051.1) from the Styphnolobium japonicum (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.
All strains and plasmids used in this study are listed in Supplementary Table 1. Chromosomal mutants were all derived from the same parental PAK (P. aeruginosa strain K; wild-type strain) strain, as indicated in Supplementary Table 1 [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).
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 (Supplementary Table. 2, Bioneer, Daejeon).
Western blot analysis
For the ExoS-FLAG detection, the p30 and p137 strains (Supplementary Table 1) 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.