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. 2025 Feb 12;18(2):e70101. doi: 10.1111/1751-7915.70101

Multifaceted Antipathogenic Activity of Two Novel Natural Products, Chermesiterpenoid B and Chermesiterpenoid B Seco Acid Methyl Ester, Against Pseudomonas aeruginosa

Dan‐Dan Li 1,2,3, Ying Wang 1, Huiyan Li 1,2, Wen‐Xin Niu 1,2, Jongki Hong 4, Jee H Jung 1,2,✉, Joon‐Hee Lee 1,2,✉
PMCID: PMC11815713  PMID: 39936740

ABSTRACT

Pseudomonas aeruginosa is an opportunistic human pathogen that causes both acute and chronic infections due to its virulence factors, biofilm formation and the ability to suppress the host immune system. Quorum sensing (QS) plays a key role in regulating these pathogenic traits and also downregulates the expression of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) in host cells. In this study, we isolated two novel natural products from the jellyfish‐derived fungus Penicillium chermesinum, chermesiterpenoid B (Che B) seco acid methyl ester (Che B ester) and Che B. Both compounds act as partial agonists of PPAR‐γ and exhibit anti‐QS activity. Che B ester and Che B were found to inhibit biofilm formation, reduce the production of proteases and decrease the infectivity of P. aeruginosa , all without affecting bacterial growth. In host cells, Che B ester and Che B reduced P. aeruginosa ‐induced inflammation by activating PPAR‐γ. This multifaceted function makes these compounds promising candidates for developing new antipathogenic agents against bacterial infections with few side effects.

Keywords: antibiofilm, anti‐QS, immunostimulators, PPAR‐γ, Pseudomonas aeruginosa

PPAR‐γ agonist Che B ester against Pseudomonas aeruginosa by inhibiting QS.

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1. Introduction

Pseudomonas aeruginosa is an ubiquitous Gram (−) opportunistic human pathogen which causes both acute and chronic infections that are often life threatening in immunocompromised people (Thi et al. 2020). Pseudomonas aeruginosa is a leading cause of nosocomial infections (Kamali et al. 2020). The production of virulence factors and biofilm formation are two important factors in the pathogenesis of P. aeruginosa : The former mainly plays a key role in acute infections, and the latter plays a key role in chronic infections (Bedi et al. 2017). Quorum sensing (QS), a cell‐to‐cell communication mechanism, plays a crucial role in promoting biofilm formation and the production of virulence factors (Bedi et al. 2017; De Kievit 2009). Interestingly, bacterial QS also plays a role in disrupting the physiological activity of host cells (Wu and Luo 2021). Therefore, QS is an important target in preventing P. aeruginosa infection, and discovering new anti‐QS compounds is a very promising strategy for suppressing both acute and chronic infections and overall pathogenicity of P. aeruginosa .

Pseudomonas aeruginosa has two main types of QS signals: acyl homoserine lactones (acyl‐HSLs) and 2‐alkyl‐4‐quinolones (AQs) (De Kievit 2009; Hwang et al. 2021; Smith and Iglewski 2003). In the acyl‐HSL‐based QS system, N‐3‐oxododecanoyl‐HSL (3OC12‐HSL) and N‐butyryl‐L‐HSL (C4‐HSL) activate the Las and Rhl regulons. In the AQ‐based QS system, 3,4‐dihydroxy‐2‐heptylquinoline (PQS) and 4‐hydroxy‐2‐heptylquinoline (HHQ) activate the PQS regulon. As the cell density increases, the concentration of QS signals also increases, and when a certain critical concentration is reached, the signal molecules enter the cell and bind to their cognate receptors (LasR and QscR for 3OC12‐HSL; RhlR for C4‐HSL; and PqsR for PQS and HHQ) (Waters and Bassler 2005; Lee et al. 2006; Xiao et al. 2006). The signal‐receptor complexes activate the transcription of the genes involved in the virulence factors production and biofilm formation (Rutherford and Bassler 2012).

In recent years, emerging evidence demonstrated that P. aeruginosa cells (PAO1) and QS molecular 3OC12‐HSL attenuated peroxisome proliferator‐activated receptor γ (PPAR‐γ) expression in bronchial epithelial cells and macrophages of host (Bedi et al. 2016, 2017). PPAR‐γ is a nuclear receptor that belongs to the PPAR family with three isoforms (α, β (formerly δ) and γ) and plays essential roles in the regulation of adipogenesis, lipid metabolism, inflammation and metabolic homeostasis (Tyagi et al. 2011; Ngala et al. 2011; Yu et al. 2008). PPAR‐γ agonists exhibit potent immunostimulatory activity against P. aeruginosa infections by enhancing the clearance of P. aeruginosa from infected mouse lungs and reducing biofilm formation on bronchial epithelial cells (Bedi et al. 2016, 2017). Therefore, increasing PPAR‐γ activity through the administration of PPAR‐γ agonists could enhance the host's ability to combat P. aeruginosa infections. In this respect, P. aeruginosa 's use of QS activity to reduce PPAR‐γ function is a very clever strategy for successful infection by disrupting the host's immune and physiological activities. However, although a significant number of PPAR‐γ agonists have been known so far, it has been reported that PPAR‐γ full agonists, for example, thiazolidinediones (TZDs) that are used to control hyperglycaemia clinically (Boden and Zhang 2006), have various serious side effects, such as weight gain, fluid retention, congestive heart failure and bladder cancer (Nagashree and Krishnamurthy 2019; Ahsan 2019; Piccinni et al. 2011). For this reason, partial agonists are preferred over full agonists.

Some species of Penicillium have been reported to exhibit anti‐QS activity (Rasmussen et al. 2005). To discover new PPAR‐γ agonists with anti‐QS activity, Penicillium chermesinum M42, a jellyfish‐derived fungus, was selected for investigation. Substances with anti‐QS and PPAR‐γ agonist activities were tracked. Interestingly, both activities were found in the same fraction, suggesting the presence of compounds with dual anti‐QS and PPAR‐γ agonist properties. Fortunately, a new compound was subsequently isolated from this fraction and identified as chermesiterpenoid B ester (Che B ester), a lactone derivative of Che B. Both Che B ester and Che B were characterised as PPAR‐γ partial agonists, with a lower potential for side effects, and demonstrated anti‐QS activity. Additionally, Che B ester and Che B demonstrated immunostimulatory effects against P. aeruginosa and anti‐inflammatory effects on host cells, as well as inhibited the virulence of P. aeruginosa . As is common with anti‐QS compounds, they also showed antibiofilm effects against P. aeruginosa . These findings suggest that Che B ester and Che B are promising lead compounds with multifaceted activity against P. aeruginosa infections.

2. Experimental Procedures

2.1. Organisms, Bacterial Strains, Culture Conditions and Plasmids

The organisms and plasmids used in this study are listed in Table S1. PAO1, a P. aeruginosa wild‐type strain, was used in most experiments. For the Escherichia coli dual‐plasmid reporter system, the E. coli DH5a strain was used. These bacterial strains were mostly grown in Luria–Bertani medium (LB; 10 g/L tryptone, 5 g/L yeast extract and 5 g/L NaCl) at 37°C with vigorous shaking. Growth was measured by optical density at 600 nm (OD600). l‐arabinose (0.4%) was used to induce protein expression and antibiotics were added at the following concentrations: carbenicillin, 150 μg/mL; ampicillin, 100 μg/mL; and gentamicin, 10 μg/mL.

2.2. Isolation of the Compounds With PPAR‐γ Agonist and Anti‐QS Dual Activity

Penicillium chermesinum M42 was grown for 21 days in malt extract medium (ME; 75% seawater, 2% glucose, 2% ME and 0.1% peptone). The whole culture was extracted with ethyl acetate (EtOAc) and then partitioned between aqueous MeOH (90% v/v) and n‐hexane. The aqueous MeOH layer obtained was subjected to step‐gradient medium‐pressure liquid chromatography (ODS‐A, 120 Å, S‐30/50 mesh) and eluted with 20%–100% MeOH/H 2 O to afford 19 fractions. Fraction 13 was subjected to reverse‐phase HPLC (YMC‐Triart C18, 250 × 10 mm, 5 μm) using 52% MeOH/H 2 O (1 mL/min, t R  = 67 min) as eluant to yield Che B ester and Che B. The details about identification of Che B ester and Che B are presented in Table S2, Figures S3, S6–S8. Che B ester is colourless oil, and for 1 H and 13C NMR data, see Table 2; HRESIMS m/z 287.2218 [M + H] + and 309.2045 [M + Na] + (calcd for C 16 H 30 O 4, 287.2217). Che B ester can be converted to Che B after dissolving in chloroform or methanol at room temperature.

2.3. Measurement of Anti‐QS Activity

The E. coli dual‐plasmid reporter system was used to measure the activity of QS regulators (Hwang et al. 2021). In this system, the E. coli reporter strains harbour two compatible plasmids: One is the pJN105‐based plasmids to express the QS regulators and the other is the pQF50 based with the regulator‐specific promoter‐lacZ fusion plasmid to reflect the regulator activity. For reporting the LasR and QscR activities, respectively, pJN105L (LasR expression plasmid) and pSC11 (lasI p‐lacZ fusion plasmid), or pJN105Q (QscR expression plasmid) and pJL101 (PA1897p‐lacZ fusion plasmid) were introduced into E. coli DH5ɑ, respectively (Hwang et al. 2021). Both LasR and QscR respond to 3OC12‐HSL (Hwang et al. 2021). The E. coli reporter strains were cultured at 37°C with shaking up to OD600 = 0.3 in LB broth containing antibiotics. Then, 50 nM (for LasR) or 100 nM (for QscR) 3OC12‐HSL, 0.4% arabinose, and 0.001, 0.01 or 0.1 μM compound Che B ester and Che B were added. After further cultivation for 1.5 h with shaking, the β‐galactosidase activities that reflect the activities of LasR or QscR were measured.

2.4. β‐Galactosidase Activity Assay

β‐Galactosidase activity was measured using Galacto‐Light Plus kit (Applied Biosystems, USA) as described elsewhere (Hwang et al. 2021). Briefly, the cell culture was measured for OD600, mixed and vortexed with 10 μL chloroform and incubated for 15 min. Then, 10 μL supernatant was taken and mixed with 100‐μL substrate solution in 96‐well plate. After 60 min incubation in dark place, 150 μL Accelerator II was added to generate luminescence, which was measured in multiwell plate reader (Tristar LB941; Berthold). β‐Galactosidase activity was presented as luminescence/OD600.

2.5. Protease Activity and Virulence Assay

Protease activity and virulence of P. aeruginosa were investigated by using Tenebrio molitor (an insect) infection model, as described elsewhere (Hwang et al. 2021). Briefly, P. aeruginosa cells were grown at 37°C with shaking in LB broth with 10 μM Che B ester and Che B up to OD600 = 2. The same volume of DMSO was added to LB as a control. Then, the cell‐free culture supernatant (CS) was prepared by centrifugation at 4°C and filtration through 0.2 μm filter (GVS Abluo syringe filter) of the cultures. For protease activity assay, 5 μL of the CSs was loaded onto the discs placed on the skim milk medium (0.5% skim milk, 0.5% peptone, 0.1% glucose and 1.5% agar). After overnight incubation at 37°C, the size of clear zone around discs was measured.

For the virulence assay, the cell‐free CSs were concentrated 10 times using 10 kDa cut‐off Centricon (Vivaspin; Satorious) and 5 μL of the concentrated CSs was injected into T. molitor larvae. Alternatively, the culture of P. aeruginosa was diluted to 1% (1 × 106 CFU/mL) in insect saline (IS; 130 mM NaCl, 5 mM KCl and 1 mM CaCl2), and 5 μL of the cell diluent was directly injected into T. molitor larvae. Five microlitres of IS was injected into larvae as a control. After injection, the larvae were incubated at 25°C for 24 h to see survival and melanisation. The survival of T. molitor larvae was calculated and the graph for survival rate was made.

2.6. Static Biofilm Formation Assay

Pseudomonas aeruginosa cells were grown in LB overnight and inoculated 1% into fresh M63 minimal medium (KH2PO4, 3 g/L; K2HPO4, 7 g/L; (NH4)2SO4, 2 g/L, 0.12 g/L MgSO4) supplemented with 0.5% (wt/vol) casamino acid (CAA) and 0.2% (wt/vol) citrate as carbon source on 96‐well plate. Che B ester and Che B were added and further incubated for 24 h. After OD600 was measured by multiwell plate reader, planktonic cells were removed and the well surface‐attached biofilm was washed with water, dried and stained by 180 μL crystal violet (0.1% wt/vol) for 10 min. After excess crystal violet was removed and washed with water, the biofilm‐staining crystal violet was dissolved by 200 μL 30% acetic acid and measured by absorbance at 600 nm (A600). The amount of biofilm was presented as A600/OD600.

2.7. Measurement of Intracellular c‐di‐GMP

The intracellular c‐di‐GMP levels were measured by using the cdrA p‐lacZ fusion reporter plasmid (pSKcdrA, Table S1), as described elsewhere (Hwang et al. 2021). pSKcdrA was introduced into P. aeruginosa cells and the pSKcdrA‐harbouring P. aeruginosa cells were grown in LB containing 0.01–10 μM Che B ester or Che B, or 5 μM sodium nitroprusside (SNP) as a positive control for 5 h. Then, the β‐galactosidase activity that reflects the intracellular c‐di‐GMP levels was measured.

2.8. Animal Cell Culture, Drug Treatment and P. aeruginosa Infection

RAW 264.7 (murine macrophages), Ac2F (rat liver cells) and SH‐SY5Y (neuroblastoma cell) cells were obtained from the American‐type culture collection (ATCC, Rockville, MD, USA), cultured at 37°C in a 5% CO2 humidified incubator and maintained in Dulbecco's modified Eagle medium (DMEM)/high glucose (HyClone, Logan, UT, USA) containing 10% heat‐inactivated foetal bovine serum (FBS; Gibco, Grand Island, NE, USA), 100 units/mL penicillin and 100 μg/mL streptomycin. For drug treatment and P. aeruginosa infection, the cells were dispensed into 96‐well plate (1 × 105 cells/mL), treated with Che B ester, Che B or rosiglitazone (10 μM) for 12 h (Ac2F) or 24 h (RAW 264.7 and SH‐SY5Y) and then infected with PAO1 (MOI = 50) for another 8 h.

2.9. Cell Viability Assay

Cell viabilities were evaluated using water‐soluble tetrazolium (WAT) regent (EZ‐CyTox; Daeil Lab Service Co. Ltd., Seoul, Korea), which was added to each well (10 μL) and incubated at 37°C for 1 h. Absorbance was read using the Microplate Absorbance Reader at a wavelength of 450 nm.

2.10. Luciferase Reporter Assay

Ac2F cells were cotransfected with PPRE‐X3‐TK‐luc (a peroxisome proliferator response element [PPRE]‐driven luciferase reporter) and one of pPPAR‐α, pPPAR‐β and pPPAR‐γ (expression vectors of PPAR‐α, PPAR‐β and PPAR‐γ, respectively). The transfected cells were treated with Che B ester, Che B, WY‐14643, GW501516 or rosiglitazone as described above. After treatment, the cells were lysed and assayed using the ONE‐GloTM Luciferase assay system (Promega, Madison, WI, USA). Luciferase activities were measured using a multiwell plate reader.

2.11. Docking Simulation

As we described in previous data (Li et al. 2022).

2.12. Measurement of NO Production

After drug treatment, Raw264.7 cells were coincubated with 0.1 μg/mL of lipopolysaccharide (LPS) for 24 h. NO concentrations in medium were determined by Griess assay. The cell culture medium (80 μL) was mixed with Griess reagent (80 μL) and incubated at 37°C for 15 min in dark place. Absorbance was then measured at 520 nm (A520). NO concentration was determined using 0–100 μM sodium nitrite standards. Dexamethasone (20 μM) was used as the positive control.

2.13. Western Blot

After drug treatment, the Ac2F and Raw264.7 cells were collected and lysed in cell/tissue lysis buffer. The protein concentration was determined by BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). All protein was separated by sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) and transferred into PVDF membranes. Then, the membranes were incubated with anti‐PPAR‐γ or anti‐COX‐2 antibody at 4°C overnight, and incubated with secondary antibody for 1 h at room temperature. The bands were visualised by ECL kit and the ChemiDoc Touch Imaging System (Bio‐Rad Laboratories, Hercules, CA, USA).

2.14. Immunofluorescence Assay

After drug treatment, the Raw264.7 cells were fixed by 10% formalin solution, and incubated in 0.3% Triton X‐100 for 15 min and 10% BSA for 30 min. Then, anti‐PPAR‐γ and Alexa 488 secondary antibodies were successively hybridised at 4°C overnight and for 30 min at room temperature, respectively. After treatment with RNase (10 μg/mL) and propidium iodide (10 μg/mL) for 20 min, fluorescence was analysed by ZEISS LSM 800 confocal microscope.

2.15. Statistical Analysis

Standard error of the mean (SEM) is indicated in the data in the graphs. The significant difference between groups was analysed using Student's t‐test by GraphPad Prism 5 (San Diego, CA, USA) software. p < 0.05 was considered significant.

3. Results and Discussion

3.1. Che B and Che B Ester Obtained From P. chermesinum Are Novel PPAR‐γ Partial Agonists

A fungus was isolated from the marine jellyfish Aurelia aurita which was collected from the southern coast of South Korea in 2017, and identified as P. chermesinum M42 by internal transcribed spacer (ITS) sequences (GenBank Accession No. KJ767051.1) (Figure S1). This fungus was cultured in large quantities, disrupted and fractionated, and the fractions were tested for activity that increases PPAR‐γ activity (Figure S2). In addition, since it has been previously reported that some Penicillium species exhibit anti‐QS activity (Rasmussen et al. 2005), we also tested the fractions for anti‐QS activity. We found some fractions with the PPAR‐γ agonist or anti‐QS activities, and interestingly many of them have both activities (Figure S2). From the fractions with both PPAR‐γ agonist and anti‐QS activities, two compounds were isolated to have dual activities, and their molecular structures were identified as Che B (Hu et al. 2020), a lactone derivative, and Che B seco acid methyl ester (Che B ester) by 1H NMR, 13C NMR, DEPT135, 1H–1H COSY, HMBC, HRESIMS spectra and ECD calculation (Figure 1, Figures S3, S6–S8 and Table S2).

FIGURE 1.

FIGURE 1

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The molecular structures of chermesiterpenoid B (Che B) and Che B seco acid methyl ester (Che B ester).

We first investigated the toxicity of these two compounds by performing cell viability assay with three different cell lines (Ac2F, Raw264.7 and SH‐SY5Y). Our results showed no significant cytotoxicity up to 40–60 μM (Figure 2A–C). When we tested the ability to activate PPAR isotypes, both Che B ester and Che B significantly activated only PPAR‐γ (Figure 2D–F). WY‐14643, GW501516 and rosiglitazone were employed as the positive agonists of PPAR‐α, PPAR‐β and PPAR‐γ. Since the degree of PPAR‐γ activation of Che B ester and Che B was smaller compared to rosiglitazone, a known full agonist, we measured the degree of PPAR‐γ activation according to the concentration of the two substances more accurately. As shown in Figure 2G, both Che B ester and Che B activated PPAR‐γ to a lesser extent at higher concentrations compared to rosiglitazone, which was similar to that of amorfrutin 1–4, a PPAR‐γ partial agonist (Weidner et al. 2012). So, we concluded that both Che B ester and Che B are novel PPAR‐γ partial agonists. We emphasise that just because it is not a full agonist, it does not mean that it is not the material we wanted. In the case of PPAR‐γ full agonist, side effects have been reported, and it has been suggested that a partial agonist may be better. For example, TZDs, the PPAR‐γ full agonists, have been used to control hyperglycaemia clinically (Boden and Zhang 2006), but they exhibited serious side effects in clinical. On the contrary, amorfrutins, the PPAR‐γ partial agonists, have been reported to have low side effects (Weidner et al. 2012).

FIGURE 2.

FIGURE 2

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Cytotoxicity and PPAR‐γ‐activating effect of Che B ester and Che B. (A–C) Cytotoxicity of Che B ester and Che B on Ac2F, Raw264.7 and SH‐SY5Y cells was measured by using cell viability assay. (D–F) Activation effects of Che B ester and Che B on PPAR‐α, PPAR‐β and PPAR‐γ were measured. WY‐14643, GW501516 and rosiglitazone (Rosi), the known agonists for PPAR‐α, PPAR‐β and PPAR‐γ, respectively, were used as positive control. (G) Comparison of the PPAR‐γ‐activating effectiveness between Rosi and Che B ester/Che B according to concentration. (H, I) Docking simulation between the ligand binding domain (LBD) of human PPAR‐γ (crystal data; 2PRG) and Che B ester or Che B. The docking scores of Che B ester and Che B were −6.7 and −7.0 kcal/mol, respectively. Green dashed line, H‐bond; pink dashed line, hydrophobic interaction; green ribbon, H12. This experiment was performed at least three times. *р <  0.05; ***р <  0.001.

The molecular docking simulation showed that both Che B ester and Che B bind into the ligand binding domain (LBD) of PPAR‐γ: Che B ester showed H‐bond–mediated interaction with His449 (H11), Gln286 (H3), Cys285 (H3) and Arg288 (H3), occupying the region close to the H3 (Figure 2H), and Che B showed H‐bonds with His323 and Ser289, and hydrophobic interaction with Tyr473, Leu469, His323, Cys285, Phe363, Met364, His449 and Tyr327, occupying the region near H3 and H12 (Figure 2I). Our previous study showed that rosiglitazone, the PPAR‐γ full agonist forms H‐bonds with Tyr473, His323, Ser289 and His449 and stabilises H12 (Figure S4), and amorfrutin 1, the PPAR‐γ partial agonist, interacts with β‐sheet and H3 by H‐bond and hydrophobic interaction (Figure S4). Based on these simulations, it has been proposed that the H‐bond with Tyr473 stabilises the conformation of H12 and is a feature found only in the full agonist (Li et al. 2022). Consistent with this suggestion, Che B and Che B esters, which showed partial agonist activity in our results, did not form H‐bonds with Tyr473 in the simulation (Figure 2H,I). This simulation result also supports our conclusion that Che B ester and Che B are partial agonists of PPAR‐γ.

3.2. Che B Ester and Che B Inhibited the Virulence of P. aeruginosa by Inhibiting QS

The crude fractions including Che B ester and Che B had both PPAR‐γ agonist and anti‐QS activities (Figure S2). Therefore, we investigated the anti‐QS activity of Che B ester and Che B. For this, the activity of LasR, the major QS regulator that first triggers QS response by perceiving 3OC12‐HSL in P. aeruginosa, was measured using the E. coli dual‐plasmid reporter strain harbouring pSC11 (lasI p ‐lacZ) and pJN105L (Hwang et al. 2021). Our results showed that both Che B and Che B ester could inhibit LasR significantly (data not shown). When a concentration‐dependent inhibitory effect was investigated, Che B ester was able to inhibit the activity of LacZ expressed from the lasR promoter by about 40% even at a very low concentration of 1 nM (Figure 3A). To confirm this result, we investigated how much Che B ester and Che B inhibit the activity of QscR, another 3OC12‐HSL receptor protein of P. aeruginosa (Lee et al. 2006). Che B ester inhibited QscR activity by about 29% at 10 nM, and Che B inhibited it by about 26% at 100 nM (Figure 3B). In both cases, the inhibitory effect did not increase even if the concentration was increased as in LasR. In both LasR and QscR, the inhibition ability of Che B ester was stronger than that of Che B, and both Che B ester and Che B inhibited LasR better than QscR, although the affinity of QscR to 3OC12‐HSL is weaker than that of LasR (Lee et al. 2006; Oinuma and Greenberg 2011).

FIGURE 3.

FIGURE 3

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QS inhibitory effects of Che B ester and Che B. Inhibition of the Pseudomonas aeruginosa QS by Che B ester and Che B was measured using the Escherichia coli dual‐plasmid reporter. (A) The E. coli reporter strain harbouring pSC11 and pJN105L was treated with 50 nM 3OC12 and 0.4% arabinose to activate LasR, and treated with various concentrations of Che B ester and Che B. Then, β‐galactosidase activity was measured. (B) The E. coli reporter harbouring pJL101 and pJN105Q was treated with 100 nM 3OC12 0.4% arabinose to activate QscR, treated with Che B ester and Che B and β‐galactosidase activity was measured. (C) The protease activity was measured using skim milk plate assay and the size of clear zones was presented graphically on the right. The P. aeruginosa harbouring pSC11 (D) and pJL101 (E) as a reporter, and then β‐galactosidase activity was measured after treatment with 100 nM Che B ester and Che B. *р < 0.05; **р < 0.01; ***р < 0.001. This experiment was performed at least three times.

In order to confirm the anti‐QS activities of Che B ester and Che B in P. aeruginosa , the reporter plasmids pSC11 and pJL101 were introduced into P. aeruginosa . When the reporter‐harbouring cells were treated with 100 nM Che B ester and Che B, both significantly inhibited the LasR activity (Figure 3D). Interestingly, somewhat different result was obtained for QscR compared to E. coli : Che B did not affect QscR activity, while Che B ester slightly increased QscR activity (Figure 3E). Despite this difference, it does not mean that Che B ester does not act as antipathogenics, but rather means that Che B ester can inhibit the virulence of P. aeruginosa in additional way because it has been reported that QscR acts as an antagonist of QS regulators such as LasR and RhlR (Chugani et al. 2001; Ding et al. 2018; Ledgham et al. 2003). Therefore, increasing the activity of QscR in P. aeruginosa means that it can have an additional suppressing effect on LasR. The exact reason for the different result in E. coli and P. aeruginosa is unclear, but such differences may occur in heterologous hosts because there may be differences in efflux, membrane permeability and/or chemical modification between E. coli and P. aeruginosa , and even if a substance binds to a target protein and changes its conformation, it may not always inhibit the activity, but may have a similar effect with the original ligand, or may even increase the activity depending on the host's intracellular condition.

Since both Che B ester and Che B were able to inhibit LasR activity of P. aeruginosa , we checked whether they could actually inhibit the virulence of P. aeruginosa . For this purpose, we measured the total activity of extracellular protease, a representative virulence factor produced by P. aeruginosa through the QS mechanism. The skim milk assay showed that both Che B ester and Che B significantly decreased total activity of extracellular proteases (Figure 3C). As in the QS inhibition, Che B ester inhibited protease activity more strongly than Che B. Finally, we investigated whether Che B ester and Che B can actually inhibit P. aeruginosa infection using Tenebrio molitor (an insect) infection model (Park et al. 2014; Yeom et al. 2013). P. aeruginosa cells were treated with Che B ester or Che B, and either concentrated CS (Figure 4A) or diluted P. aeruginosa cells (Figure 4B) were injected into T. molitor larvae by syringe. Since most virulence factors are already secreted into the CS during the cultivation, using the CS allows us to measure the amount of preproduced virulence factors. Conversely, when injecting live cells, the preproduced virulence factors are diluted to negligible levels before injection, thus allowing us to observe the bacteria's ability to produce virulence factors within the host. Through this separate investigation of virulence, we found that both Che B and Che B ester reduced the toxicity of CS (Figure 4A), and Che B ester also reduced the virulence of live cells (Figure 4B). Therefore, we confirmed that both compounds significantly attenuated P. aeruginosa .

FIGURE 4.

FIGURE 4

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Attenuation of Pseudomonas aeruginosa by treating with Che B ester and Che B. (A) The concentrated culture supernatants of P. aeruginosa cells were injected into Tenebrio molitor larvae, and the survival was counted on 2nd day. (B) Five microlitres of the insect saline (IS)‐diluted live P. aeruginosa cells were injected into T. molitor larvae and survival was counted on 2nd day. LB or IS was injected as a control, and DMSO was treated as a solvent control for Che B ester and Che B. *р < 0.05; **р < 0.01. This experiment was performed at least three times.

3.3. Che B Ester and Che B Inhibited the Biofilm Formation of P. aeruginosa by Reducing Intracellular c‐di‐GMP Levels

In addition to QS, biofilm formation is also an important feature of P. aeruginosa infection. Biofilms provide pathogens with strong resistance to host immunity and antibiotic treatment (Rasamiravaka et al. 2015; Yan and Wu 2019). To determine the effect of Che B ester and Che B on the biofilm formation, we treated P. aeruginosa with Che B ester and Che B in a concentration range of 0.01–10 μM and measured biofilm formation. Both Che B ester and Che B began to significantly inhibit biofilm formation at 0.01 μM and maximally inhibited biofilm formation at 1 μM (Figure 5A). No further inhibition was observed even when the concentration was further increased (Figure 5A). The extent to which Che B ester and Che B inhibited biofilm was about 30%, which was about half the effect compared to when SNP, a well‐known biofilm inhibitor (Fida et al. 2018; Barraud et al. 2009), was treated at a concentration of 10 μM (Figure 5A).

FIGURE 5.

FIGURE 5

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Inhibition of biofilm formation by Che B ester and Che B. (A) Static biofilm assay was performed to measure biofilm formation with the Che B ester and Che B treatment at 0.01–10 μM. For comparison, 10 μM SNP was treated in the same manner. (B) The pSKcdrA‐harbouring Pseudomonas aeruginosa cells were treated with Che B ester and Che B for 5 h, and β‐galactosidase activity that reflects the intracellular c‐di‐GMP levels was measured. Data were presented relative to the sample without treatment (which corresponds to 100%). (C) Growth of P. aeruginosa was monitored with the Che B ester and Che B treatment (10 μM) by OD 600. This experiment was performed at least three times. *р < 0.05; **р < 0.01; *** p < 0.001.

Bis‐(3′‐5′)‐cyclic diguanosine monophosphate (c‐di‐GMP) is an important messenger molecule that regulates biofilm formation (Hengge 2009; Cotter and Stibitz 2007). When its intracellular level increases, it induces biofilm formation, and when its intracellular level decreases, it induces planktonic life. Since the cdrA promoter activity parallels intracellular c‐di‐GMP levels (Kim et al. 2020), we measured the expression of the cdrA promoter that reflects the intracellular level of c‐di‐GMP, and the cdrA promoter expression was decreased by Che B ester and Che B in the same pattern as the inhibition of biofilm formation (Figure 5B). This means that Che B ester and Che B inhibit biofilm formation of P. aeruginosa by reducing the level of intracellular c‐di‐GMP. The extent to which Che B ester and Che B reduced the activity of c‐di‐GMP promoter was also about half that of 10 μM SNP (Figure 5B). Taken together, Che B ester and Che B are partial agonists that activate PPAR‐γ in the host and are substances that simultaneously inhibit virulence and biofilm formation of pathogens. We would like to note that Che B ester and Che B perform these actions without affecting the growth of P. aeruginosa itself. Figure 5C shows that treatment of Che B ester and Che B at a high concentration of 10 μM has no effect on the growth of P. aeruginosa . In general, as antibiotics are used, resistant bacteria are selected due to their growth‐inhibiting effect on bacteria, which is the main cause of the development of antibiotic‐resistant bacteria. Since Che B ester and Che B have no effect on bacterial growth, they do not impart such selective pressure and are therefore expected not to generate resistance. Substances that specifically inhibit the virulence of pathogens without inhibiting growth are called antipathogenics, or antivirulence agents (Dickey et al. 2017). Che B ester and Che B can be said to have the characteristics of antivirulence agents.

3.4. Che B Ester and Che B Inhibited the Interaction of P. aeruginosa With Macrophages

It has been reported that the following conflicting effects exist between PPAR‐γ activity and P. aeruginosa infection: P. aeruginosa infection downregulated the expression of PPAR‐γ, and conversely, increasing PPAR‐γ activity has an effect of suppressing P. aeruginosa infection (Bedi et al. 2016, 2017). When Ac2F and Raw264.7 macrophages were infected with P. aeruginosa , PPAR‐γ expression decreased as expected (Figure 6A and Figure S5A). However, when cells were treated with Che B ester, the PPAR‐γ expression was restored again (Figure 6A). Che B failed to restore the PPAR‐γ expression significantly (Figure 6A). Similarly, direct immunofluorescence detection of PPAR‐γ in P. aeruginosa ‐infected cells also confirmed that Che B ester increased the expression of PPAR‐γ, but Che B did not (Figure 6B). These results demonstrated that Che B ester is a PPAR‐γ agonist and potential immunostimulator against P. aeruginosa infection.

FIGURE 6.

FIGURE 6

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Decrease in PPAR‐γ expression by Pseudomonas aeruginosa infection and its recovery through Che B ester treatment. (A) The expression of PPAR‐γ in P. aeruginosa ‐infected RAW264.7 cells was measured by Western analysis. Che B ester, Che B and rosiglitazone (Rosi) were treated at 10 μM. The band intensity for PPAR‐γ was normalised by β‐ACTIN, an internal control, and presented as bar graph below. (B) Anti‐PPAR‐γ antibody was added into Raw264.7 cells to detect PPAR‐γ and visualised by green fluorescence. Propidium iodide was used to stain nucleus (red). Intensity of green fluorescence was quantified and presented graphically on the right. *р < 0.05; ***р < 0.001; # p < 0.05. This experiment was performed at least three times.

COX‐2 is a key enzyme to cause inflammation. Che B ester and Che B were able to inhibit the induction of COX‐2 in P. aeruginosa infection (Figure 7A), suggesting that Che B ester and Che B can suppress inflammation in P. aeruginosa infections. To verify this, inflammation was induced with LPS in RAW 264.7 cells. Normally, nitric oxide (NO) production is highly induced as shown in Figure S5B. However, when RAW 264.7 cells were pretreated with Che B ester and Che B, NO production induced by LPS was significantly inhibited (Figure 7B). In any case, Che B ester and Che B are PPAR‐γ agonists and appear to have both immune‐stimulating and inflammation‐suppressing functions.

FIGURE 7.

FIGURE 7

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Inhibitory effect of Che B ester and Che B on inflammatory factor expression. (A) The expression of COX‐2 in RAW264.7 cells was measured with pretreatment of compounds and Pseudomonas aeruginosa infection. Rosi, rosiglitazone (B) RAW264.7 cells were treated with Che B ester and Che B for 1 h, and then 100 ng/mL LPS were treated for 24 h. The NO production was measured by Griess reagent. Dexamethasone (Dex) was used as positive control. **р < 0.01; ***р < 0.001; # p < 0.05; ## p < 0.01. This experiment was performed at least three times.

Substances with an immunostimulatory effect can clearly play a role in protecting against P. aeruginosa infection, and substances that suppress inflammation can also play a role in protecting the host from infection (Codagnone et al. 2018; Lin and Kazmierczak 2017). Che B ester and Che B can perform both of these actions simultaneously. In addition, it can suppress QS, virulence and biofilm formation of pathogens. Therefore, these natural compounds are very promising materials that can be developed as new antipathogenic agents.

4. Conclusion

In conclusion, we isolated two novel natural compounds, Che B ester and Che B, from a jellyfish‐derived fungus P. chermesinum M42. They had PPAR‐γ partial agonist activity on host and anti‐QS effects on P. aeruginosa . Che B ester and Che B were found to inhibit biofilm formation, reduce the production of virulence factors and decrease the infectivity of P. aeruginosa , all without affecting bacterial growth. This indicates that these compounds specifically target the pathogenic mechanisms of the bacteria rather than killing them, which could help minimise the development of resistance. Che B ester and Che B exhibited antibiofilm activities by reducing intracellular c‐di‐GMP levels of P. aeruginosa . In host cells, Che B ester and Che B enhanced the expression of PPAR‐γ and inhibited P. aeruginosa ‐induced inflammation. Therefore, we propose that Che B ester and Che B are antipathogenic substances with multifaceted effects that activate immunity and suppress inflammation in the host, and at the same time suppress pathogenicity and biofilm formation in pathogen.

Author Contributions

Dan‐Dan Li: conceptualization, funding acquisition, methodology, writing – original draft. Ying Wang: conceptualization, methodology, writing – original draft, visualization. Huiyan Li: methodology, visualization. Wen‐Xin Niu: methodology, visualization. Jongki Hong: methodology. Jee H. Jung: writing – review and editing, funding acquisition. Joon‐Hee Lee: funding acquisition, writing – review and editing, writing – original draft, conceptualization, project administration.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1.

MBT2-18-e70101-s001.doc (3.2MB, doc)

Acknowledgements

This research was supported by the National Research Foundation of Korea [201901920001], [NRF‐2019R1A2C1010087] and [RS‐2024‐00353202], and the Scientific Research Foundation of Tianjin University of Traditional Chinese Medicine for Recruiting Teachers [XJS2024102]. We thank Xiao‐Wei Luo for ECD calculation, and Jongki Hong for MS analysis. We thank Professor Hae Young Chung (College of Pharmacy, Pusan National University, Busan 46241, Republic of Korea) for generously donating the plasmids pcDNA3, PPAR‐α and β/δ. We thank Dr. Christopher K. Glass (University of California, San Diego) who donated PPRE luciferase reporter plasmid. We thank Dr. Chatterjee (University of Cambridge, Addenbrooke's Hospital) for donating PPAR‐γ luciferase reporter plasmid.

Funding: This work was supported by the National Research Foundation of Korea (201901920001, NRF‐2019R1A2C1010087 and RS‐2024‐00353202) and the Scientific Research Foundation of Tianjin University of Traditional Chinese Medicine for Recruiting Teachers (XJS2024102).

Dan‐Dan Li and Ying Wang contributed equally to this work.

Contributor Information

Jee H. Jung, Email: jhjung@pusan.ac.kr.

Joon‐Hee Lee, Email: joonhee@pusan.ac.kr.

Data Availability Statement

The authors have nothing to report.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1.

MBT2-18-e70101-s001.doc (3.2MB, doc)

Data Availability Statement

The authors have nothing to report.

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