Ostarine attenuates pyocyanin in Pseudomonas aeruginosa by interfering with quorum sensing systems

Limin Dong1,2 ● Lang Sun1 ● Xinxin Hu1 ● Tongying Nie1 ● Jing Pang1 ● Xiukun Wang1 ● Xinyi Yang1 ● Congran Li1 ●
Kaihu Yao2 ● Youwen Zhang 1 ● Xuefu You1

Received: 1 May 2021 / Revised: 15 July 2021 / Accepted: 16 August 2021
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2021

Abstract
Antimicrobial resistance has been an increasingly serious threat to global public health. Anti-virulence strategies are being developed to manage antibiotic resistance because they apply a lower selective pressure for antimicrobial-resistant pathogens than that created using traditional bactericides. We aimed to discover novel small molecules that can reduce the production of virulence factors in Pseudomonas aeruginosa and determine the mechanism of action underlying these effects. A clinical compound library was screened, and ostarine was identified as a potential anti-virulence agent. The effects of ostarine were studied via antimicrobial susceptibility testing, bacterial growth assays, pyocyanin quantitation assays, transcriptomic analysis, quorum sensing signal molecule quantification, and real-time PCR assays. Ostarine treatment significantly decreased the synthesis of pyocyanin without any bactericidal action. Besides, ostarine treatment did not affect the relative growth rate and cell morphology of bacteria. Treatment with ostarine interfered with quorum sensing by decreasing the transcription of genes associated with quorum sensing systems and the production of signalling molecules. The inhibition of ostarine on pyocyanin production and gene expression can be alleviated when signalling molecules were supplemented externally. Overall, ostarine may act as a novel anti-virulence agent that can attenuate P. aeruginosa pyocyanin by interfering with quorum sensing systems.

Introduction

Antimicrobial resistance is a serious threat to global public health. According to the global priority list of antibiotic-resistant bacteria to guide research, discovery and development of new antibiotics published by the

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41429- 021-00469-4.

⦁ Youwen Zhang ⦁ [email protected]
⦁ Xuefu You ⦁ [email protected]

1 Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
2 Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, China
World Health Organization in 2017, the microorganisms for which antibiotics are required at high priority are carbapenem-resistant Acinetobacter baumannii, Pseudo- monas aeruginosa, and Enterobacteriaceae [1]. P. aeru- ginosa is a human pathogen that can cause both community-acquired and hospital-acquired infections, which present serious threats to burn trauma victims, patients with cystic fibrosis, immunocompromised indi- viduals, and individuals with medical implants [2–4]. Traditional bactericidal approaches to combat bacterial infections can create a substantial selection pressure and lead to the selection of antimicrobial-resistant sub- populations [5]. Multiple novel strategies have been developed to manage antibiotic resistance, one of which involves targeting virulence factors. Anti-virulence stra- tegies indirectly kill bacteria, which presumably leads to a lower evolutionary pressure for bacteria than that cre- ated using traditional bactericides [6]. By affecting the virulence, bacteria become less efficient in colonising the host, and such phenomena can allow the host immune system to treat any established infections [6]. We screened a clinical compound library (Target Molecule

Fig. 1 Effect of ostarine treatment on the synthesis of pyocyanin in Pseudomonas aeruginosa. a Chemical structure and CAS number of ostarine, also referred to as 3-(4-cyanophenoxy)-2-hydroxy-2- methylpropanamide. CAS Chemical Abstracts Service. b Quantitation of pyocyanin in P. aeruginosa PAO1, P. aeruginosa ATCC 27853, P.
aeruginosa 16-2, and P. aeruginosa 16-17 treated with and without 4 μg ml−1 ostarine. c Production of pyocyanin by strain PAO1 treated with and without 4 μg ml−1 ostarine. Data were analysed using the Student’s t-test, in comparison to those of the control group; *P < 0.05,
**P < 0.01. Con control

Corp., Boston, MA, USA), a collection of 688 clinical compounds, and discovered that ostarine (Enobosarm, MK2866, or GTx-024) (Fig. 1a) could act as a potential anti-virulence agent against P. aeruginosa.
Ostarine is a non-steroidal selective androgen receptor modulator (SARM) that has been studied in clinical trials for treating cancer cachexia, sarcopenia, breast cancer, and stress urinary incontinence [7–10]. In this study, we aimed to evaluate the effect of ostarine on the production of pyocyanin and other virulence factors. Pyocyanin is an important virulence factor produced by P. aeruginosa, which can generate reactive oxygen species and increase oxidative stress owing to its redox-active properties [11, 12]. In addition, pyocyanin can regulate the activity of chloride ion channels in host cells by inhibiting cellular respiration and depleting intracellular cyclic adenosine monophosphate and adenosine triphosphate levels [12]. Furthermore, pyocyanin enables the survival of P. aeru- ginosa under oxygen-deprived conditions by accepting and transporting electrons produced during respiration [13]. In our previous study, we found that the lack of pyocyanin was associated with a decreased pathogenicity in P. aeruginosa PAO1 [14]. The secondary metabolite pyocyanin is synthesised via multiple gene products encoded by two phzABCDEFG operons and the phzH, phzM, and phzS genes [15] and regulated by quorum sensing (QS) systems [16–18]. QS system involves in cell- density dependent accumulation of signal molecules, which plays a key role in the regulation of bacterial virulence and biofilm formation [19]. In P. aeruginosa hierarchy quorum sensing network, las governs expression of both rhl and pqs systems, and rhl system is under the control of both las and pqs system [19]. Signal molecules could bind to the corresponding transcriptional regulator and subsequently regulate biofilm formation and virulence factor production, such as pyocyanin. In this study, we
evaluated the effects of ostarine treatment on P. aerugi- nosa-derived pyocyanin and determined the mechanisms underlying these effects.

Materials and methods

Bacterial strains and culture conditions

The bacterial strains used in this study were obtained from the American Type Culture Collection (ATCC) and the Chinese Academy of Medical Sciences Collection Center of Pathogen Microorganisms (CAMS-CCPM-A). All isolates were frozen at −80 °C until use. The strains were routinely cultured in Luria-Bertani (LB) broth or Mueller-Hinton (MH) broth at 37 °C.

Antimicrobial susceptibility testing

Levofloxacin was purchased from the National Institutes for Food and Drug Control (Beijing, China) for quality control, and ostarine was purchased from Target Molecule Corp. (Boston, MA, USA). The minimal inhibitory concentrations (MICs) of levofloxacin and ostarine were determined via the agar dilution method according to the Clinical and Laboratory Standards Institute guidelines (M100-S30). The agar plates were incubated at 37 °C for 16–20 h. The experiments were performed in triplicates on different days. For checkerboard analysis, 1–64 μg ml−1 meropenem or 16–1024 μg ml−1 methicillin were mixed with 0.5–32 μg ml−1 ostarine in 96-well plates to test the combination effects, followed by the addition of a bacterial suspension at a final concentration of 5 × 105 CFU ml−1 in cation-adjusted MH (CAMH) broth. The results were observed without any equipment after incubation for 18 h at 37 °C. The experiments
were performed in triplicates on different days.

Scanning electron microscopy

Overnight P. aeruginosa PAO1 cultures (1 ml) with and without 8 μg ml−1 ostarine were centrifuged at 6000 × g for 3 min. The supernatants were discarded, and bacterial pellets were washed twice and re-suspended in 1 ml of phosphate-buffered saline. The pellets were fixed in 2.5% glutaraldehyde overnight at 4 °C, and fixatives were removed via centrifugation at 6000 × g for 3 min. The images were observed using a scanning electron micro- scope (Hitachi SU8020; Hitachi High-Tech, Tokyo, Japan).

Growth curve analysis

P. aeruginosa strains treated with and without 4, 16, 64, or 256 μg ml−1 ostarine were cultured in CAMH broth at 37 °C, and the optical density at 600 nm (OD600) was measured every 10 min using a Bioscreen C reader (FP-1100-C; Oy Growth Curves Ab Ltd.). Four independent culture samples per group were grown overnight until saturation was attained. The cultures (0.3 ml) were 1000-fold diluted by CAMH broth, and aliquots were added to a Bioscreen C plate in duplicate. The growth curves were evaluated after measuring the OD600 values over a period of 24 h.

Pyocyanin quantification assay

P. aeruginosa strains were cultured in glycerol-alanine medium [20] with and without 4 μg ml−1 ostarine for 24 h. In the signal molecules supplemented assay, P. aeruginosa PAO1 were cultured with and without 8 μg ml−1 ostarine, or ostarine combined with QS signal molecules (10 μg ml−1 3- oxo-C12-HSL, 10 μg ml−1 C4-HSL, and 20 μg ml−1 PQS). Pyocyanin was extracted and quantified as previously described [21]. Briefly, 4 ml of cell culture was mixed with 3 ml of chloroform, followed by centrifugation at 6000 × g for 5 min. The chloroform layer (2 ml) was then retrieved and mixed with 2 ml of 0.2 mol l−1 HCl. The A520 of the pink pigment was measured, and the concentration of pyocyanin was determined using an extinction coefficient of 2460 M−1 cm−1. The experiments were performed in tri- plicates on different days.

Transcriptomic analysis

P. aeruginosa PAO1 was grown in LB broth with and without 8 μg ml−1 ostarine at 37 °C with shaking (180 × rpm) for 6 h, and then, 1 ml of cells was harvested via centrifugation (12,000 × g) at 4 °C for 2 min. RNA extraction from the bacteria was performed using the RNAprep Pure Cell/Bacteria Kit (Tiangen, Beijing, China), and transcriptomic analysis was performed by
Novogene (Beijing, China) as described previously [14]. Briefly, a total of 3 μg RNA was used to generate sequencing libraries after the integrity was assessed. After cluster generation, the library preparations were sequenced, and differential expression was analysed using the DESeq R package (1.18.0). P-values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate, and genes with an adjusted P- value of <0.05 were considered differentially expressed. Three biological replicates were used in each group.

Relative gene expression analysis via real-time PCR

Total RNA extraction from PAO1 strains treated with and without ostarine was performed using the RNAprep Pure Cell/Bacteria Kit (Tiangen) as described for transcriptomic analysis. Subsequently, the mRNA was reverse- transcribed to generate cDNA using the FastQuant RT Kit (Tiangen). Forward and reverse primer mix (2 μl, 10 μ mol l−1 each; sequences are listed in Supplementary Table S1), template cDNA (1 μl), 2 × Power SYBR Green PCR Master Mix (10 μl; Applied Biosystems, Foster City, CA, USA), and nuclease-free water (7 μl) were added to the real-time PCR reaction mixture. The cycling condi- tions were 50 °C for 2 s, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min using a 7500 Fast Real-Time PCR System (Applied BiosystemsTM). The experiments were performed in triplicates on different days.

Quantitation of quorum sensing signalling molecules via liquid chromatography-tandem mass spectrometry

P. aeruginosa PAO1 was grown in LB broth with and without 8 μg ml−1 ostarine at 37 °C with shaking (180 × rpm) until an OD600 of ~1.2 was obtained. Quantitation of acyl homoserine lactones (AHLs; 3-oxo-C12-HSL: N-(3- oxo-dodecanoyl)-L-homoserine lactone, C4-HSL: N- butanoyl-L-homoserine lactone) was performed as pre- viously described [22, 23]. Samples were centrifuged at 10,000 × g using a Thermo Fresco 21 tabletop centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min at 4 °C. The liquid supernatant was extracted using the same volume of acid ethyl acetate, and the organic phase was dried using a vacuum freeze dryer (Alpha 2–4 LD plus; Martin Christ, Osterode am Harz, Germany). AHLs were reconstituted in methanol and filtered using a 0.22-μm millix filter (Merck, Kenilworth, NJ, USA). 3- Oxo-C12-HSL (Sigma Aldrich) and C4-HSL (Cayman Chemical, Ann Arbor, MI, USA) were used as external standards. Three biological replicates were used in each group.

Results

Effect of ostarine treatment on phenotypes of bacteria

First, we investigated the effect of ostarine treatment on the synthesis of pyocyanin in P. aeruginosa. Ostarine treatment significantly inhibited pyocyanin synthesis compared with that in the control group (P < 0.05). At a concentration of 4 μg ml−1 ostarine, the production of pyocyanin decreased to 28% in P. aeruginosa PAO1, 9% in P. aeruginosa ATCC 27853, 49% in P. aeruginosa clinical isolate 16-2, and 14% in P. aeruginosa clinical isolate 16-17 compared with that in the control (Fig. 1b, c).
The MIC of ostarine against 38 strains belonging to 14 bacterial species was determined. The MIC values of ostarine were all above 512 μg ml−1, indicating no anti- bacterial activity (Table 1). For carbapenem-resistant P. aeruginosa 16-2 and methicillin-resistant Staphylococcus aureus 08–50, 0.5–32 μg ml−1 ostarine showed no syner- gistic effect when combined with meropenem or methicillin (Supplementary Table S2).
The growth curves of P. aeruginosa treated with and without ostarine were analysed. Compared with the OD600 values of the untreated control group, the values of P. aeruginosa PAO1, P. aeruginosa ATCC 27853, P. aeru- ginosa 16-2, and P. aeruginosa 16-17 showed no differ- ence at the concentration of 4–256 μg ml−1 ostarine (Fig. 2a). In addition, scanning electron microscopy (SEM) images showed no apparent changes in cell morphology of
P. aeruginosa PAO1 treated with 8 μg ml−1 ostarine (Fig. 2b).

Effects of ostarine treatment on gene expression of virulence factors produced by P. aeruginosa

Ostarine treatment significantly inhibited pyocyanin synth- esis compared with that in the control group. Pyocyanin is synthesised via a series of complex steps involving two phzABCDEFG operons and the phzM and phzS genes. We performed real-time PCR to verify the effect of ostarine treatment on pyocyanin biosynthesis genes and other viru- lence factor genes in P. aeruginosa. Compared with the expression levels in the PAO1 control group, in ostarine- treated cells, the expression levels of phzA1, phzA2, phzM, and phzS were significantly decreased to 14–59%, whereas that of phzH did not differ considerably (Fig. 3a).
Furthermore, ostarine treatment downregulated other virulence factor genes regulated by the QS system. In the ostarine-treated group, the expression levels of rhamnolipid, elastase, and lectin-related genes were decreased (78% for lasA, 37% for lasB, 51% for rhlA, 59% for rhlB, and 56% for lecA) (Fig. 3b).
Effects of ostarine treatment on QS systems

Transcriptomic sequencing was performed to explore the mechanism of action of ostarine-mediated virulence inhi- bition. The Kyoto Encyclopaedia of Genes and Genomes enrichment analysis using KOBAS 3.0 software demon- strated that the activation of pathways associated with QS was significantly altered in the ostarine treatment group compared with that in the solvent treatment group (Fig. 4a). Differential expression analysis confirmed that ostarine treatment downregulated the genes associated with QS systems, as well as virulence factor genes regulated by the QS system (Table 2). Ostarine treatment significantly reduced the expression of phzA1, phzB1, phzM, and phzS, as determined via real-time PCR. In addition, the expression levels of the aforementioned genes regulated by the QS system, including lasA, lasB, rhlA, rhlB, and lecA, were decreased after treatment with ostarine (Table 2).
QS systems enable bacteria to modulate the expression of virulence factors via cell density-dependent accumulation of signal molecules [16–18]. The expression levels of genes encoding signal molecule-catalysing enzymes and tran- scriptional activators were measured via real-time PCR. In the ostarine treatment group, the expression levels of rhlI, rhlR, and pqsE were decreased (65% for rhlI, 72% for rhlR, and 4% for pqsE), whereas those of lasR and pqsR were increased by 1.21–1.23 fold, compared with those of the control group; no obvious changes were observed in the gene expression of lasI and pqsH (Fig. 4b). We then quantified the production of QS signalling molecules 3-oxo- C12-HSL and C4-HSL via liquid chromatography-tandem mass spectrometry. Ostarine treatment decreased the pro- duction of 3-oxo-C12-HSL and C4-HSL (76% for 3-oxo- C12-HSL, 79% for C4-HSL) compared with those pro- duced by the control (Fig. 4c). In addition, when the signal molecules of AHLs and PQS were supplemented simulta- neously, the expression levels of phzA2, pqsE, and rhlR were significantly increased by 3.1–6.4 fold compared with those of the ostarine-treated group (Fig. 4d). Moreover, treated with the combination of ostarine and C4-HSL/PQS/ AHLs, the production of pyocyanin increased by 1.8–2.0 fold, compared with that of the ostarine-treated group. A significant increase (5.8 times) of pyocyanin production could be observed in the combination of ostarine, AHLs, and PQS treatment group (Fig. 4e). Therefore, ostarine treatment could have suppressed the expression of P. aer- uginosa pyocyanin by interfering with QS systems.

Discussion

The long-term use of antibiotics has led to the emergence of multidrug-resistant bacteria. Anti-virulence therapies

Table 1 Antibiogram for ostarine
Species Isolate Phenotype MIC (μg ml−1)
Levofloxacin Ostarine
Staphylococcus epidermidis ATCC 12228 MSSE 0.25 >512
16-4 MSSE 0.25 >512
16-5 MRSE 8 >512
Staphylococcus aureus ATCC 29213 MSSA 0.12 >512
ATCC 33591 MRSA 0.25 >512
15 MSSA 0.12 >512
16-1 MSSA 0.12 >512
16-30 MRSA 64 >512
ATCC 43300 MRSA 0.12 >512
ATCC 700698 MRSA, VISA 16 >512
ATCC 700699 MRSA, VISA 16 >512
Enterococcus faecalis ATCC 29212 VSE 2 >512
ATCC 51299 VRE 1 >512
ATCC 51575 VRE 1 >512
16-6 VSE 1 >512
Enterococcus faecium ATCC 700221 VRE 128 >512
16-5 VSE 128 >512
12-1 VRE 128 >512
ATCC 51559 VRE 32 >512
Escherichia coli ATCC 25922
ATCC 35218
ATCC 2469 ESBLs (-)
ESBLs (+) NDM-1 (+) ≤0.03
≤0.03 8 >512
>512
>512
16-1 ESBLs (+) 0.5 >512
Klebsiella pneumoniae ATCC 700603
ATCC BAA-2146 16-2 ESBLs (+) NDM-1 (+)
ESBLs (-) 1
> 128
0.12 >512
>512
>512
16-14 ESBLs (+) 0.5 >512
Pseudomonas aeruginosa ATCC 27853 1 >512
PAO1 0.5 >512
16-2 CRPA 16 >512
Acinetobacter baumannii ATCC 19606 0.25 >512
16-33 CRAB 2 >512
Enterobacter cloacae ATCC 43560 ≤0.03 >512
Providentia rettgeri ATCC 31052 ≤0.03 >512
Serratia marcescens ATCC 21074 0.12 >512
Shigella flexneri ATCC 12022 ≤0.03 >512
Stenotrophomonas maltophilia ATCC 13636 4 >512
Proteus mirabilis ATCC 49565 0.06 >512
MIC minimum inhibitory concentration, MSSE methicillin-sensitive Staphylococcus epidermidis, MRSE methicillin-resistant Staphylococcus epidermidis, MSSA methicillin-sensitive Staphylococcus aureus, MRSA methicillin-resistant Staphylococcus aureus, VISA vancomycin- intermediate Staphylococcus aureus, VSE vancomycin-susceptible Enterococcus, VRE vancomycin-resistant Enterococcus, ESBL extended- spectrum beta-lactamase, NDM-1 New Delhi metallo-beta-lactamase 1, CRPA carbapenem-resistant Pseudomonas aeruginosa, CRAB carbapenem- resistant Acinetobacter baumannii

have been developed as a novel strategy to treat diseases caused by bacteria. For example, 5- aminosalicylic acid could downregulate the growth and virulence of
Escherichia coli associated with inflammatory bowel disease and colorectal cancer [24]. Dephostatin can repress the expression of intracellular virulence factors

Fig. 2 Effect of ostarine treatment on the phenotypes of Pseudomonas aeruginosa. a Growth curves of P. aeruginosa PAO1, P. aeruginosa ATCC 27853, P. aeruginosa 16-2, and P. aeruginosa 16-17 treated with and without 4–256 μg ml−1 ostarine. b SEM images of
Pseudomonas aeruginosa PAO1 treated with and without 8 μg ml−1 ostarine. The arrows show the integrity of the bacterial membranes. SEM scanning electron microscopy

Fig. 3 Effect of ostarine treatment on the synthesis of virulence factors in Pseudomonas aeruginosa. a Effect of ostarine on the expression of pyocyanin biosynthesis genes as determined via real-time PCR. b Effect of ostarine treatment on the expression of genes encoding other
virulence factors (rhamnolipid, elastase, and lectin). Data were ana- lysed via the Student’s t-test, in comparison to those of the PAO1 control group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. PCR polymerase chain reaction

and polymyxin resistance genes in serovars of Salmonella [25]. We found that ostarine could act as a novel anti- virulence agent against P. aeruginosa by attenuating pyocyanin production. Ostarine is a non-steroidal SARM

that induces conformational changes in the androgen receptor [26] and has been studied in clinical trials. Pyocyanin is a redox-active virulence factor produced by
P. aeruginosa during the stationary phase. In this study,

Fig. 4 Ostarine treatment attenuated pyocyanin production by inter- fering with quorum sensing systems. a KEGG enrichment analysis of differentially expressed genes in Pseudomonas aeruginosa PAO1 treated with ostarine. KOBAS was used to test the statistical enrich- ment of differential expression in KEGG pathways. b Effect of ostarine treatment on the expression of genes associated with quorum sensing systems as detected via real-time PCR. c Effect of ostarine on the production of quorum sensing-related signal molecules, AHLs (3- oxo-C12-HSL and C4-HSL). Data were analysed via the Student’s t- test, in comparison to those of the PAO1 control group; *P < 0.05,
**P < 0.01, ***P < 0.001, ****P < 0.0001. d Relative expression of pyocyanin biosynthesis and regulate genes determined via real-time PCR. P. aeruginosa PAO1 was treated with and without ostarine (8 μg
ml−1), or ostarine combined with AHLs (10 μg ml−1, 10 μg ml−1 C4- HSL) and PQS (20 μg ml−1). Data were analysed via one-way ANOVA and Bonferroni’s multiple comparisons, in comparison to ostarine-treated group; **P < 0.01, ****P < 0.0001. e Quantitation of pyocyanin in P. aeruginosa PAO1 treated with and without ostarine, or ostarine combined with QS signal molecules. Data were analysed via one-way ANOVA and Bonferroni’s multiple comparisons, in comparison to ostarine-treated group; *P < 0.05, ****P < 0.0001. KEGG Kyoto Encyclopedia of Genes and Genomes, PCR polymerase chain reaction, KOBAS KEGG Orthology-Based Annotation System, AHLs acyl homoserine lactones, 3-oxo-C12-HSL N-3-oxo-dodeca- noyl-L-homoserine lactone, C4-HSL N-butanoyl-L-homoserine lac- tone, PQS Pseudomonas quinolone signal

ostarine treatment significantly decreased the synthesis of pyocyanin without bactericidal action nor a synergistic effect combined with meropenem or methicillin. In addition, ostarine treatment showed no effect on the
growth curve or cell morphology. Without killing any pathogens, ostarine can apply a lower selective pressure for the development of antimicrobial resistance than that created using traditional bactericides.

Table 2 Effect of ostarine treatment on the transcription of

Gene ID Gene name log2-fold
Changea

Padj Gene product

quorum sensing-related genes in

Pseudomonas aeruginosa PAO1
Quorum sensing system genes
PA1001 phnA −2.4481 1.18E−12 Anthranilate synthase component I PA1002 phnB −1.3066 0.003749 Anthranilate synthase component II PA0996 pqsA −4.2032 5.28E−30 PqsA
PA0997 pqsB −3.8067 6.00E−28 PqsB PA0998 pqsC −3.2601 2.13E−20 PqsC
PA0999 pqsD −3.001 3.22E−18 3-oxoacyl-[acyl-carrier-protein] synthase III PA1000 pqsE −2.7824 8.32E−16 Quinolone signal response protein
PA0649 trpG −0.661 0.47365 Anthranilate synthase component II PA3476 rhlI −0.88798 0.45419 Autoinducer synthesis protein RhlI PA3477 rhlR −0.79032 0.57566 Transcriptional regulator RhlR
PA4911 -- 0.70428 0.56683 Probable permease of ABC branched-chain amino
acid transporter
PA5568 -- −0.75368 0.43437 Conserved hypothetical protein PA4243 secY −0.93972 0.24074 Secretion protein SecY
PA4206 mexH −0.6122 0.61543 Probable Resistance-Nodulation-Cell Division (RND)
efflux membrane fusion protein precursor PA1874 -- 0.77477 0.46192 Hypothetical protein
PA0649 trpG −0.661 0.47365 Anthranilate synthase component II PA3299 fadD1 −0.81565 0.40537 Long-chain-fatty-acid--CoA ligase PA4276 secE −0.96254 0.58233 Secretion protein SecE
PA3822 -- −0.85471 0.53841 Conserved hypothetical protein
PA3319 plcN 0.60416 0.61543 Non-haemolytic phospholipase C precursor PA4747 secG −0.7933 0.58233 Secretion protein SecG
Virulence factor synthesis genes/operons
PA4210 phzA1 −3.9906 0.036681 Probable phenazine biosynthesis protein PA4211 phzB1 −3.7856 1.16E−17 Probable phenazine biosynthesis protein PA1899 phzA2 −2.8732 2.72E−05 Probable phenazine biosynthesis protein PA1900 phzB2 −2.8065 0.04832 Probable phenazine biosynthesis protein PA4209 phzM −0.95183 0.16772 Probable phenazine-specific methyltransferase PA4217 phzS −1.5987 0.000103 Flavin-containing monooxygenase
PA3479 rhlA −1.2931 0.002295 Rhamnosyltransferase chain A PA3478 rhlB −0.77651 0.39261 Rhamnosyltransferase chain B PA3724 lasB −0.88861 0.15582 Elastase LasB
PA2570 lecA −0.68848 0.61543 LecA
PA3361 lecB −2.3736 5.70E−11 Fucose-binding lectin PA-IIL PA0652 vfr −0.71674 0.58233 Transcriptional regulator Vfr

alog2-fold Change: log2 value of the gene expression level, calculated by comparing the values of the ostarine-treated group to those of the untreated control group. log2-fold change > 0, upregulated; log2-fold change < 0, downregulated

We explored the mechanism of action via which ostarine inhibited pyocyanin production. In the pyocyanin synthetic pathway, chorismic acid is converted to phenazine-1- carboxylic acid by PhzA-G proteins. Phenazine-1- carboxylic acid is subsequently converted to pyocyanin by PhzM and PhzS (Fig. 5) [15]. Ostarine treatment sig- nificantly downregulated phzA1, phzA2, phzM, and phzS, resulting in a decreased pyocyanin production.

Subsequently, transcriptomic sequencing was performed and the downregulation of QS systems was considered as the key reason for pyocyanin reduction caused by ostarine. Nowadays, several specific QS inhibitors were known to inhibit P. aeruginosa virulence. For example, S-phenyl-L- cysteine sulfoxide inhibits pyocyanin production in P. aeruginosa, and virulence expression operons regulated by the las, rhl, and pqs. QS systems are also highly

Quorum sensing system
Biofilm formation
Virulence factor expression

OH COOH
N N
COOH
N
COOH

CH2

N CH3

N N

CH3

O COOH
OH

pyocyanin
5-methylphenazine-1 carboxylic acid betaine

CONH2
N
phenazine-1-carboxylic acid
chorismic acid

N
phenazine-1-carboxamide
Fig. 5 Biosynthesis and signalling system of pyocyanin. Bacteria use the quorum sensing system for the regulation of processes involved in their interaction with each other. When cells reach a threshold density, the las quorum sensing system is induced. The las system activates and regulates the pqs and rhl systems. The signal molecules 3-oxo- C12-HSL, C4-HSL, and PQS bind to the corresponding transcriptional regulators to regulate virulence factor production and biofilm

formation. The quorum sensing cascade interacts and controls phe- nazine production by PqsE and RhlR. Chorismic acid is converted into phenazine-1-carboxylic acid by the PhzA-G proteins. Phenazine-1- carboxylic acid is converted to pyocyanin by PhzM and PhzS [15, 17, 30]. 3-oxo-C12-HSL N-3-oxo-dodecanoyl-L-homoserine lac- tone, C4-HSL N-butanoyl-L-homoserine lactone, PQSPseudomonas quinolone signal

downregulated determined via RNA-seq transcriptomic analysis [27]. The RNA polymerase-binding transcriptional regulator, DksA1, inhibits QS-mediated virulence demon- strated by RNA-Seq analysis and may help control P. aeruginosa infection [28].
In P. aeruginosa, the QS network is organised in a multi- layered hierarchy consisting of several interconnected sig- nalling mechanisms [19] (Fig. 5). P. aeruginosa possesses three main QS systems (las, rhl, and pqs) that release small chemical signals: 3-oxo-C12-HSL, C4-HSL, and Pseudo- monas quinolone signals (PQS). These signal molecules can be synthesised by LasI, RhlI, or PqsH, which then interact with the transcription factors LasR, RhlR, and PqsR, respectively. When cells reach a threshold density, the las system can be induced which regulates the expression of both pqs and rhl systems by forming the 3-oxo-C12-HSL/ LasR complex [17]. The rhl system is regulated by both las and pqs, and the interaction of C4-HSL with the tran- scription factor RhlR regulates the production of pyocyanin, rhamnolipids, LasB elastase, and cytotoxic lectins [29]. In
the ostarine treatment group, the expression levels of rhlI, rhlR, and pqsE, as well as the production of 3-oxo-C12- HSL and C4-HSL were all decreased. The inhibition of formation of the signal-transcriptional activator complexes might have resulted in the reduction in pyocyanin produc- tion. Furthermore, the transcription of phzA1 and phzA2 can be induced by RhlR and PqsE [30]. The decrease in the levels of rhlR and pqsR mediated via ostarine treatment might have further led to the downregulation of phzA1 and phzA2. Besides, the inhibition of ostarine on pyocyanin production and gene expression of phzA2, pqsE, and rhlR could be alleviated when signal molecules were supple- mented externally. And the change of pyocyanin production treated with ostarine recovered significantly when AHLs and PQS supplemented at the same time. These results indicated that ostarine treatment could have attenuated pyocyanin production in P. aeruginosa by interfering with the whole QS systems. It is noteworthy that other virulence factor genes regulated by the QS system, such as lasB (elastase), lasA (protease), rhlAB (rhamnolipids), and lecA

(lectin) [19, 31, 32] were all significantly downregulated via treatment with ostarine, indicating the downregulation of the QS system.
In conclusion, we found that the non-antibiotic pharma- ceutical, ostarine, may act as an anti-virulence agent against
P. aeruginosa. Ostarine has no bactericidal effect but can significantly reduce pyocyanin production by interfering with QS systems. Ostarine can not only reduce the virulence of P. aeruginosa but also apply a lower selective pressure for the development of bacterial resistance than that created using traditional bactericides. This study may enable the discovery of additional anti-virulence agents against multidrug-resistant bacteria.

Acknowledgements This research was funded by the National Natural Science Foundation of China (grant numbers 81573475, 82104248), CAMS Initiative for Innovative Medicine (grant number 2016-I2M-3- 014), National Mega-project for Innovative Drugs (grant number 2019ZX09721001), and Cultivation Fund Project of the National Natural Science Foundation in Beijing Children’s Hospital, Capital Medical University (grant number GPQN202001).

Compliance with ethical standards

Conflict of interest The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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