Bacterial strains and media
T. pyogenes TP13 isolated from the lung pus of forest musk deer [4] and wild-type (WT) P. aeruginosa strain PAO1 were preserved in the lab and used elsewhere [15]. All the strains were routinely cultured in brain heart infusion with 5% fetal bovine serum (BHI-FBS) or in lysogeny broth (LB) from a single colony.
Culture conditions
A total of 55 compounds (Additional file 1 Supplementary Table S1) with similar core structure to the Acyl-homoserine lactones (AHL) signals of P. aeruginosa QS system were selected and purchased from MedChemExpress (Shanghai, China). Overnight cultured T. pyogenes TP13 was diluted to optical density of 1.0 at wavelength of 600 nm (OD600=1.0) by sterile saline solution. Equal amount of T. pyogenes (10 µL) was inoculated in 200 µL of BHI-FBS medium containing different concentrations (0, 50, 100, and 200 µM) of compounds and culture at 37°C overnight, and then the cell densities were determined at OD600. Subsequently, T. pyogenes TP13 and P. aeruginosa PAO1 were mixed (1:9, 1:1, and 9:1) and co-cultured overnight on BHI-FBS agar containing 200 µM of the compounds with significant growth inhibition activities on T. pyogenes. The composition of T. pyogenes and P. aeruginosa in the co-culture were determined by counting the colony forming units (CFUs) of them on BHI-FBS agar plates after appropriate dilution, because the phenotypes of T. pyogenes and P. aeruginosa are significantly different and can be easily discriminated. Finally, the compounds that could inhibit the growth of T. pyogenes and P. aeruginosa were added (0, 50, 100, and 200 µM) to 200 µL of LB medium containing 10 µL of P. aeruginosa and cultured overnight at 37°C. The cell densities were determined at OD600. All the experiments above were independently repeated for three times.
Quorum-sensing inhibition assay
M9-adenisine (0.1%, wt/v) and M9-skimmed milk (0.5%, wt/v) agar medium were used to evaluate the inhibitory activity of compounds on P. aeruginosa QS regulation [19]. Overnight cultured P. aeruginosa PAO1 was adjusted to OD600=1.0 and inoculated (5 µL) on M9-adenisine agar and M9-milk agar containing different concentrations (0, 50, 100, and 200 µM) of compounds. The growth status of P. aeruginosa on M9-adenisine plates and the diameter of proteolytic circle formed on M9-milk plates were determined after 24 hours. The experiments were independently repeated for nine times.
Biofilm production assay
Equal amount (20 µl, OD600=1.0) of P. aeruginosa PAO1 was inoculated in glass tubes containing 2 mL of LB broth supplemented with different contractions (0, 50, 100, and 200 µM) of compounds, and overnight cultured at 37°C with shaking (220 rpm). The cell density was measured at OD600. After the culture solution and unadhered biofilm were gently removed, the adhered biofilm on the tube wall was stained with crystal violet (0.1%) for 30 minutes and washed with PBS buffer for three times. Subsequently, the stained biofilm was dissolved by 95% of ethanol solution and quantified at OD595. The experiments were independently repeated for three times.
Pyocyanin production assay
Equal amount (20 µl, OD600=1.0) of P. aeruginosa PAO1 was inoculated in glass tubes containing 2 mL of LB broth supplemented with different contractions (0, 50, 100, and 200 µM) of compounds, and overnight cultured at 37°C with shaking (220 rpm). After the cell density was equalized with fresh LB broth, 200 µL of bacterial solution was taken out to extract the pyocyanin by chloroform and 0.2 N HCl and measured at OD520 as described by Essar [31]. The experiments were independently repeated for three times.
Motility assay
For the swimming motility assay, 5 µl (OD600=1.0) of P. aeruginosa PAO1 was inoculated on the surface of LB plates containing 0.5% of agar supplemented with different contractions (0, 50, 100, and 200 µM) of compounds and cultured at 37°C for 24 h. For the twitching motility assay, 2 µl (OD600=1.0) of P. aeruginosa PAO1 was stabbed into the bottom of LB plates containing 1.0% of agar supplemented with different contractions (0, 50, 100, and 200 µM) of compounds and cultured at 37°C for 24 h. The motilities of P. aeruginosa PAO1 were determined by measure the diameters of colony on the surface (swimming motility) or the thin film region on the bottom (twitching motility). All the experiments were independently repeated for six times.
Transcriptomic analysis
Bacterial cells of furazolidone-treated and -untreated P. aeruginosa PAO1 were harvested for total RNA isolation using TRIzol reagents (Invitrogen), respectively. RNAs samples were conducted for library construction and RNA-sequencing (RNA-seq) by Novogene Bioinformatics Technology Company using prokaryotic strand-specific Illumina-based RNA-Seq technology (HiseqTM2500 platform). The obtained clean reads were mapped to the reference genome of PAO1 (NCBI accession number: AE004091) by the software Tophat2 [32]. SOAP2 program [33] and Cufflinks [34] were used to calculate the expected fragments per kilobase of transcript per million fragments (FPKM) sequenced, and the differentially expressed transcripts were presented and analyzed by EdgeR [35]. Differentially expressed gene with false discovery rate p < 0.05 was thought to be significantly different. The significantly differently expressed genes were mapped to the list of QS-activated genes reported by Schuster [20] by using VENNY 2.1 (http:// bioinfogp.cnb.csic.es/tools/venny/).
Quantitative PCR
Total RNAs of furazolidone-treated and -untreated P. aeruginosa PAO1 were isolated by using TRIzol reagents, and the cDNA was synthesized by reverse transcription using a high-capacity cDNA Reverse Transcriptase kit Specific with gDNA removal (Takara). Quantitative PCR was performed by using an iTaq™ universal SYBR® Green Supermix (Bio-Rad) and the CFX Connect Real-Time PCR Detection System to validate the expression of typical QS-activated genes including lasR, rhlR, pqsR, lasB, rhlA, pqsA, pqsD, pqsE, hcnA, and phzA (Additional file 1 Supplementary Table S2). Gene expression was calculated by the 2−ΔΔCT method using 16S rRNA as reference.
Caenorhabditis elegans assay
For the fast-killing assay, 20 µl (OD600=1.0) of P. aeruginosa PAO1 was spread on peptone-glucose-sorbitol (PGS) agar media with and without furazolidone, and cultured overnight at 37°C. The naturally cooled plates were seeded with 10 newly cultured adult C. elegans (L4 stage) and further incubated at 25°C for 96 hours. For the slow-killing assay, to prevent C. elegans from laying eggs, 40 µL of 5-fluoro-2'-deoxyuridine solution (40 µg/mL) was evenly coated on the surface of nematode growth medium (NGM). Subsequently, 20 µl (OD600=1.0) of P. aeruginosa PAO1 was spread on NGM plates with and without small molecule drugs and cultured overnight at 37°C. The naturally cooled plates were seeded with 10 newly cultured adult C. elegans and further incubated at 25°C for 10 days. The survival status of C. elegans in each experiment were observed and recorded. Growth of C. elegans on PGS agar plates or NGM plates feed with uracil auxotrophy Escherichia coli OP50 were set as controls.
Statistical analyses
Data analysis and statistical tests were performed by using Graphpad Prism version 9.0 (San Diego, CA, USA). Mean values of standard deviation were compared by using two-tailed unpaired t-test or One-way ANOVA. The survival curves of C. elegans were compared by using Log- rank (Mantel-Cox) test.