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. 2025 Jun 30;31:249. doi: 10.1186/s10020-025-01306-2

Discovery of coniferaldehyde as an inhibitor of caseinolytic protease to combat Staphylococcus aureus infections

Shufang Li 1,2,#, Yan Zhang 3,#, Jianfeng Wang 2, Hongfa Lv 2, Hongxia Ma 4, Lingcong Kong 4, Yonglin Zhou 5, Jingmin Gu 2, Wei Li 1,✉, Qiaoling Zhang 2,✉
PMCID: PMC12207802  PMID: 40588743

Abstract

The rising incidence of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant threat to global public health, highlighting the urgent need for novel therapies and treatments in clinical settings. Caseinolytic protease P (ClpP) serves as a key component of bacterial degradation systems, playing a crucial role in maintaining cellular homeostasis and contributing to pathogenicity. Targeting ClpP function inhibition has demonstrated potential in combating antibiotic resistance and offers a promising therapeutic strategy for treating S. aureus infections. In this study, coniferaldehyde (CA) was identified as a ClpP inhibitor through ClpP peptidase inhibition assay. CA reduced the hemolysis activity, protease hydrolysis and bacterial invasion ability via regulating the transcription of main virulence factors. Furthermore, CA treatment led to a decreased resistance of S. aureus to adverse stimuli, including heat, acidic pH, high osmotic environment, hydrogen peroxide and NaClO stress assays. Notably, CA enhanced the efficacy of the bactericidal antibiotic tigecycline against growing S. aureus in time-killing assays. Molecular simulations and mutagenesis analyses revealed that the amino acids M31 and G33 were critical for the interaction between CA and ClpP. Importantly, CA exhibited excellent protective efficacy against S. aureus pneumonia in murine infection models. Our findings confirm that CA is an effective ClpP inhibitor with potential as a therapeutic agent for S. aureus infections.

Graphical Abstract

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Keywords: Staphylococcus aureus, Caseinolytic protease, Coniferaldehyde, Molecular simulations, Virulence

Introduction

Staphylococcus aureus (S. aureus) poses a considerable public health risk due to its significant antibiotic resistance and ability to cause severe infections (Tong et al. 2015). Methicillin-resistant S. aureus (MRSA) is linked to a range of health problems, from minor skin infections to serious conditions, including pneumonia, bloodstream infections, and surgical site infections (Chambers and Deleo 2009). The rising incidence of antibiotic-resistant strains further complicates treatment, resulting in increased morbidity and mortality, particularly among vulnerable groups like the elderly and those with weakened immune systems (Vestergaard et al. 2019). The threat posed by MRSA is further exacerbated by antibiotics abuse in both healthcare settings and the community, with the limited number of effective treatment alternatives (Frieri et al. 2017). This creates a pressing need for novel therapeutic strategies to combat MRSA infections (Zhang et al. 2014; Kahan et al. 2021; Jiang et al. 2023).

ClpP, as a promising target for antibacterial and anticancer therapy, has attracted extensive research interest. Currently, ClpP modulators from different categories of chemical structures are being explored and developed for potential clinical applications (Brotz-Oesterhelt and Vorbach 2021). As a chymotrypsin-like serine protease, it has been thoroughly studied for its involvement in regulating virulence and stress responses in several bacterial species, such as S. aureus (Frees et al. 2003), Listeria monocytogenes (Gaillot et al. 2000), and Streptococcus pneumoniae (Kwon et al. 2004). ClpP is responsible for the degradation of proteins that have been mistranslated, misfolded, or aggregated in the bacterial cell as a result of e.g. heat stress or antibiotic interference with the ribosomal machinery (Aljghami et al. 2022). Knockout of the clpP gene in S. aureus weakened virulence in a murine skin abscess model, and a similar action was obtained after suppression activity of ClpP by β-lactones (Bottcher and Sieber 2008). Therefore, our objective was to identify small-molecule compounds with ClpP inhibitory activity to further enhance our arsenal for combating bacterial infections.

Coniferaldehyde (CA; 4-hydroxy-3-methoxycinnamaldehyde) is a phenolic compound present in the bark, leaves, and twigs of various edible plants, including cinnamomum cassia, salvia plebeia, and phyllanthus emblica (Kim et al. 2010; Yi et al. 2011; Zhang et al. 2016). This compound has been shown to possess anti-inflammatory, antioxidative, antiplatelet, and cytoprotective properties (Akram et al. 2016; Karamac et al. 2017). In this study, we present for the first time that CA is a promising candidate for further exploration and potential pharmaceutical development due to its ability to bind to intracellular ClpP and reduce MRSA virulence both in vitro and in vivo.

Materials and methods

Bacterial strains and culture conditions

Luria broth (LB) and LB agar plates were used for growth of Escherichia coli (E. coli), and Trypticase soy broth (TSB) and TSB agar plates were used for S. aureus. Kanamycin was used at 50 µg/mL for plasmid selection. Unless otherwise stated, all of the culture were grown aerobically at 37℃ with shaking, and growth was monitored at 600 nm with a spectrophotometer.

Cloning, protein expression, and purification

The clpP gene fragment of S. aureus NCTC 8325-4 was amplified by PCR and inserted into prokaryotic expressing vector pET-28a (+). The recombinant plasmid was then transformed into E. coli BL21 cells, which were cultured under 37℃ until the OD600 nm reached approximately 0.6. At this point, 0.1 mM IPTG was added to induce the expression of ClpP protein. After 24 h of induction at 16℃, the cells were harvested by centrifugation. The fusion protein was solubilized in the lysate buffer (PBS) using sonication, and the resulting supernatant was collected. The supernatant was then applied to a nickel affinity column packed with Ni Sepharose to purify the ClpP protein. After washed with PBS containing 10 mM imidazole or 20 mM imidazole, the ClpP was eluted with 250 mM imidazole for dialysis in dialysis buffer (150 mM NaCl and 20 mM Tris) overnight. Finally, the concentrations of ClpP proteins was determined by Nano drop.

ClpP peptidase inhibition assay

The peptidase activity derived by ClpP for the different compounds were measured by the use of the fluorogenic substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin (Suc-Leu-Tyr-AMC) with the standard activity buffer (100 mM Hepes, pH 7.0, 100 mM NaCl) (Maurizi et al. 1994). 1 µM ClpP were incubated in ClpP activity buffer with varying concentrations of compounds or DMSO for control experiments for 10 min. Then, 200 µM Suc-Leu-Tyr-AMC were added up to 50 µL. Fluorescence was measured by a microplate reader (excitation 340 nm/emission 450 nm).

CESTA assay

S. aureus NCTC 8325-4 was cultured at 37 °C in TSB and treated with DMSO or CA for 2 h. After resuspended in PBS buffer, and then exposed to heat treatment for 10 min at temperatures ranged from 25 °C to 62.8℃. After incubation, the cells were rapidly cooled, and the effects of heat exposure on protein stability were analyzed by western blot analysis. Briefly, the protein bands were incubated by mouse polyclonal anti-ClpP antibody overnight at 4℃ and then washed with TBST for three times. Following, the HRP-conjugated goat anti-mouse IgG(H + L) antibody (proteintech, China) was diluted at ration of 1:2000 in this assay. Finally, the relative intensities of the targeted proteins were visualized and quantified by Image J software.

Creatine kinase (CK) activity assay

A mixture with 52 µg/mL creatine phosphate kinase (CPK), 20 µM ADP, 20 µM creatine phosphatase (CP) were added to ClpP activity treated with different concentrations of CA or DMSO and incubated at 37℃ for 10 min. Following this, 50 µL Kinase-Glo reagent (Promega) was added and incubated at 37℃ for another 10 min. Luminescence changes were measured using a SpaK 10 m (Tecan) plate reader after transferring the samples to a 96-well clear bottom plate.

ClpP protease activity assay

Each ClpXP reaction contained 3 µM ClpP, 5 µM ClpX, and an ATP-regenerating system (1 mM ATP, 40 mM creatine kinase and 40 mM creatine phosphate). The GFP-tag SsrA was then added to each well and incubated at 37℃. The proteins were then analyzed by SDS-PAGE methods.

Quantitative polymerase chain reaction (qPCR)

The total RNA was extracted from S. aureus NCTC 8325-4 using the TRIzol reagent kit (Mei5 Biotechnology Co.Ltd) and the total RNA was assessed with spectrophotometer. cDNA was synthesized from the total RNA using the StarScript Pro All-in-one RT Mix with gDNA Remover kit (Genstar). Real-time PCR was performed on the resulting cDNA using the Thermo Fisher Real-Time PCR System. The selected genes were analyzed using the primers were referred to previous research (Gao et al. 2018). The relative quantification of genes was determined using the 16SrRNA as the endogenous control.

Hemolysis and proteolysis assay

S. aureus strains (NCTC 8325-4 (WT), ∆clpP, and ∆clpP/pYJ335::clpP (C-clpP) were cultured overnight in TSB medium. Hemolysis was tested on 5% sheep blood agar plates and proteolysis was assessed on LB agar plates containing 1% skimmed milk with different concentrations of CA. Stationary phase cultures of S. aureus (2.5 µL) were inoculated on the plates, and after overnight incubation at 37 °C for 12 h, the diameters of the zones around the bacterial colonies were measured.

For detecting neutralizing activity of CA against Hla, cultures of S. aureus NCTC 8325-4 or USA300 were grown for 8 h. The supernatant was harvested and mixed with 5% sheep red blood cells at a 1:1 ratio and different concentrations of CA. After incubation, the released hemoglobin in the supernatant was measured at 570 nm.

Cytotoxicity study

The LDH assay was performed to evaluated the cytotoxicity of CA against Raw 264.7 or A549 cells. Briefly, the cells were seed into 96-well plates for 12 h and then treated with different concentrations of CA ranged from 4 µg/mL to 128 µg/mL at 37 °C for 6 h. After that, collected the culture supernatant at 1000 g, 10 min for LDH release detection with the optical densities at 492 nm and calculated cell viability based on its specification. The cells treated with DMSO or Triton X 100 were visualized with negative control or positive control respectively.

Invasion assay

Overnight cultures of S. aureus strains (NCTC 8325-4, ΔclpP, and ΔclpP/pYJ335::clpP) with or without CA treatment were diluted and added to A549 cells at a multiplicity of infection (MOI) of 50. After 2 h of incubation at 37℃, the cells were washed to remove non-adherent bacteria, followed by the addition of fresh medium containing 50 µg/mL gentamicin to kill any remaining extracellular bacteria. The cells were then lysed in 1% saponin. and samples plated on TSB agar plates to determine the recovered colony-forming units (CFU).

Cellular infections

To investigate the protective effects of CA against S. aureus infection on A549 cells, both LDH release assays and live/dead staining assays were conducted. A549 cells are infected with an overnight culture of S. aureus NCTC 8325-4, ΔclpP or USA300 at a multiplicity of infection of 100, with or without PCA treatment and centrifuged at 1000 g for 10 min. After 6 h of infection, the supernatants were mixed with LDH assay reagents to quantify the LDH release.

Additionally, the infected cells were subjected to live/dead staining using the Live/Dead Viability kits (Beyotime). Calcein-AM stains live cells green, while propidium iodide (PI) penetrates only dead or damaged cells, staining them red. After the staining procedure, the cells are examined under a fluorescence microscope.

Stress assays

S. aureus strains (NCTC 8325-4, USA 300 and ∆clpP) was cultured at 37℃ and incubated with DMSO or CA for 2 h, and then the bacterial cultures were subjected to different environmental stress conditions.

For the oxidative stress assay, bacteria were treated with 0.5% NaClO or 1% H₂O₂ for 30 min after which the surviving cells were plated, and the survival rate was calculated relative to the DMSO control. To assess sensitivity to heat shock, cultures were incubated at 50℃ for 30 min, and then allowed to recover for 12 h before calculating the survival rate. In the acidic stress assay, bacteria were grown in medium containing 1 g/L tryptone, 5 g/L NaCl, and pH adjusted to 5.5 or 3.5. Following incubation, serial dilutions were plated onto TSB medium, and the bacterial concentration was determined to calculate the survival rate. Lastly, to evaluate the effect of the ClpP inhibitor under hypertonic conditions, cultures were grown in TSB medium with or without 10% NaCl for 6 h, with the survival rate calculated accordingly.

Time killing assay

S. aureus (NCTC 8325-4 and ΔclpP) strains were diluted in TSB and cultured to OD600nm of 0.6 at 37℃. The cultures were then diluted to 5 × 105 CFU/mL in TSB and treated with tigecycline (0.2 µg/mL), CA (64 µg/mL) or their combination. Following this, all groups are incubated at 37℃, with samples taken at predetermined time intervals (0, 2, 4, 6, 8, and 12 h). At each time point, aliquots of the culture are removed and diluted appropriately before plating onto agar plates to determine viable counts by calculating CFU.

Combined disk test

The combined disk test assay was performed to visualize the synergistic effect between CA and tigecycline. The overnight cultures were diluted with TSB to the OD600 nm=0.1. And then, the strains were poured into the plate with 32 µg/mL or 64 µg/mL CA. The disks containing the 0.2 µg/mL tigecycline were placed the center of the plate. After incubation for 24 h at 37℃, the inhibition zone was recorded.

Live/dead bacteria staining

S. aureus strains (NCTC 8325-4 and ΔclpP) were diluted in TSB medium and then incubated with tigecycline (0.2 µg/mL), CA (64 µg/mL) or their combination at 37℃ for 6 h. After centrifuge, the bacteria were resuspended to 0.5 at OD600 nm with PBS. The bacterial suspension was mixed with the dyes using the LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen) according to the manufacturer’s instructions. After incubation, the samples are observed under the fluorescence microscope.

Scanning electron microscopy (SEM)

Overnight cultures of S. aureus (NCTC 8325-4 and ΔclpP) strains were diluted in fresh TSB medium and incubated at 37℃ with tigecycline (0.2 µg/mL), CA (64 µg/mL) or a combination of both for 3 h. The bacterial cells were then collected, washed with PBS, and fixed in 2.5% glutaraldehyde overnight at 4℃. After fixation, the samples underwent dehydration using increasing concentrations of ethanol (30%, 50%, 70%, 90%, and absolute 100%), followed by critical-point drying to prevent osmotic damage during the imaging process. Dried samples were gold-coated and analyzed under the scanning electron microscope.

Galleria mellonella infection model

The Galleria mellonella infection model was performed to explore the protective effect of CA against S. aureus USA300. The 200 mg-230 mg Galleria mellonella was infected by S. aureus USA300 at a dose of 1 × 107 CFU per larva via the leg injection. After infected for 2 h, the Galleria mellonella were treated with 25 mg/kg or 50 mg/kg CA once. The survival rate of G. mellonella was monitored every 6 h for 30 h.

Circular dichroism (CD) spectra analysis

The CD spectra of ClpP with or without CA were analyzed over a wavelength range of 190 nm to 250 nm using a CD spectropolarimeter, with the temperature controlled to maintain consistency across measurements. The secondary structure of the protein was analyzed using the BeStSel web server.

Molecular dynamics (MD) simulations

The PDB code for the initial 3D structure of ClpP was 7wid, and the standard docking procedures of CA and ClpP were performed using AutoDock Vina, following docking, selected poses were subjected to molecular dynamics (MD) simulations using GROMACS software. The complex was solvated in a water box, and appropriate Na+ were added to neutralize the system. The MD simulations were performed at physiological temperature and pressure, while monitoring the root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and hydrogen bonds to analyze the stability and interactions within the complex. The binding free energy between the protein and the ligand was calculated using the molecular mechanics Poisson Boltzmann surface area (MM-PBSA) method.

Mutagenesis

The primers were used to amplify the entire plasmid pET28a-ClpP for generation of site-specific mutations. The mutated products were extracted and transformed into DH5α competent bacterial cells. And the mutated sequences of the clpP gene were verified and confirmed by DNA sequencing. Variant ClpP proteins were expressed in E. coli BL21(DE3) and purification with Ni-NTA affinity chromatography methods, which was detailed describe in “2.2 Cloning, protein expression, and purification” section. Moreover, the mutant proteins were also purified for further enzymatic cleavage assays using Suc-LY-AMC as the substrate.

Fluorescence quenching analysis

The binding constants (Ka) between CA and binding sites on ClpP, M31A, I30A, L25A, and G33A were determined using a fluorescence-quenching method at excitation wavelength of 290 nm and emission wavelength of 500 nm following previously described methods (Xu et al. 2022).

Pneumonia model experiment

Female C57BL/6J mice (6–8 weeks old) were obtained from Liaoning Changsheng Biotechnology Co., Ltd. Mouse experiments were approved by the Animal Care and Use Committee of Jilin University (SY202412062). S. aureus strain NCTC 8325-4 and ∆clpP were cultured to the mid-log growth phase in TSB, washed with PBS, resuspended in sterile saline at 1 × 107 CFU/100 µL for the sublethal pneumonia model.

The mice were infected with S. aureus by nasal drip method. (50 µL/mouse) and randomized into three groups (solvent control, 15 mg/kg CA, and 30 mg/kg CA). Another group received the same volume of S. aureus ∆clpP and this served as a control. One hour after infection, mice were treated with subcutaneous injection of the designated concentrations of compound CA (15 mg/kg or 30 mg/kg) or control solvent every 12 h. For the sublethal pneumonia model, mice from each group were euthanized at 48 h post infection, and the lungs were fixed with 4% formaldehyde for the histological assessment. Then, the bacterial counts were determined from bronchoalveolar lavage fluid (BALF) and lung homogenates on TSB agar plate medium. In addition, cytokines (TNF-α, IL-6 and IL-1β) were assessed using ELISA kits (BioLegend).

Statistical analyses

All experiments in this study were performed in triplicates unless specified otherwise. Students t test in GraphPad Prism Software 8.0 was used for all statistical analyses. And all data are showed as mean ± SD. Asterisks indicate statistically significant differences by t-test (*, p < 0.05, **, p < 0.01, ns, not significant).

Results

Identification of CA as an ClpP inhibitor

To identify the novel inhibitors of ClpP, we evaluated the ClpP-catalyzed hydrolysis of the SLY-AMC peptide (schematic diagram as shown in Fig. 1A) using an in-house library of natural origin compounds (Fig. 1B). Notably, the compound CA exhibited the most significant inhibition, with an IC50 of 18.40 µg/mL (Fig. 1C) and had no effect on the bacteria growth (Fig. 1D). To explore the direct interaction between CA and ClpP, we performed thermal stability analysis using CESTA. The results indicated that ClpP treated with increasing concentrations of CA remained more stable at 58.2 °C, in contrast to the control, suggesting a strong affinity between CA and ClpP (Fig. 1E and F). ClpP can degrade SsrA in the help of ClpX in an ATP-dependent process derived by creatine kinase (CK) (Gottesman et al. 1998; Kim et al. 2000). Furthermore, we assessed the impact of CA on the degradation of SsrA peptide sequence by ATPases activate ClpP. As shown in Fig. 1G and H, CA did not interfere the ATP regeneration system derived by creatine kinase but inhibited the degradation activity of ClpP against SsrA in Fig. 1H. And the relative degradation of SsrA peptide was shown in the Fig. 1I. These findings demonstrated that CA effectively inhibits the proteolytic activity of the ClpP without hindering ATP production, which indicated that CA is an effective ClpP inhibitor.

Fig. 1.

Fig. 1

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CA was discovered as an ClpP inhibitor in S. aureus.A A schematic diagram of natural compound inhibitor screening process, showing the ability of the ClpP enzyme to cleave the Suc-LY-AMC substrate. B Effectiveness of different compounds in inhibiting ClpP. Compounds showing more than 70% inhibition were identified as potential inhibitors. C CA inhibited the ClpP hydrolytic ability against SUC-LY-AMC oligopeptide. D The growth curves of S. aureus treated with or without CA. E and F Western blot lines (E) and optical density analysis (F) of CETSA assay showing the heat stability of ClpP with or without CA. G - I CA did not affect the creatine kinase activity in the SsrA dagration reaction (G), but inhibited the ClpP’s ability to hydrolyze SsrA in the presence of ClpX at the concentrations of 32 µg/mL or 64 µg/mL (H). And the relative degradation of SsrA peptide was also analyzed (I)

Attenuation of S. aureus virulence by CA in vitro

The successful inhibition of ClpP by CA raised the question whether the compound could also impair its natural function, leading to a reduced production of virulence factors in ∆clpP mutant S. aureus strain. As shown in Fig. 2A, the major virulence factor genes of hla, pvl, psm and agrA were significantly downregulated by CA in S. aureus.

Fig. 2.

Fig. 2

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CA reduced the virulence of S. aureus in vitro. A The virulence related gene expression level in S. aureus was determined by real time PCR with the treatment of CA (32 µg/mL or 64 µg/mL). B-E CA inhibited the hemolytic activity of culture supernatant and proteolytic ability of S. aureus in 1% skim milk agar plates (B and C) Moreover, the ratio of halo diameter to colony diameter (D/d) reflected the hemolysis and extracellular proteolysis ability of strains was shown in D and E. F and G CA showing no cytotoxicity in Raw264.7 and A549 cells at less than 128 µg/ml. H and I Reduced the bacterial intracellular colonization number (H) and protect the cells from toxicity induced by S. aureus infection (I). J: Visualizing the protective of CA at 32 µg/mL or 64 µg/mL against S. aureus infection using the Live/dead cell staining kit

The pathogenicity of S. aureus requires a large number of cell surface-associated and secreted proteins. The deficiency of clpP leads to dysregulation of the expression of toxicity-related proteins, highlighting the role of clpP in the pathogenic mechanism of S. aureus (Frees et al. 2005). Among the secreted proteins include extracellular proteases and α-hemolysin (Frees et al. 2012). The collected S. aureus NCTC8325-4 culture supernatants were cultured with erythrocytes to assess the level of secreted Hla. A dose-dependent inhibition effect of CA on hemolysis against erythrocytes was observed (Fig. 2B). Agar plate-based assays with 5% sheep blood further confirmed inhibition of hemolysis in S. aureus for CA (Fig. 2C). In addition, corresponding assays on 1% skim milk agar plates revealed that CA inhibited extracellular proteolysis (Figs. 2C). The ratio of halo diameter (D) to colony diameter (d) (D/d) reflected the hemolysis and extracellular proteolysis ability of strains. Our D/d value indicated that CA possessed the significantly inhibition effect of hemolysis and proteolysis at above the 128 µg/mL (Figs. 2D and E).

The surface-associated proteins possessed by S. aureus to bind to host fibrinogen, fibronectin, collagen, and von willebrand factor, thus enabling the bacteria to colonize and establish a focus of infection. Thus, the effect of CA on invasion of S. aureus to epithelial cells was further evaluated. The results indicated that CA exhibited no significant cytotoxicity to Raw 264.7 and A549 cells within the effective concentration range (Fig. 2F and G), but the invasion rate of S. aureus is significantly reduced following treatment with CA (Fig. 2H), and further damage to the cell membrane was also inhibited by CA according to the live/dead assay (green: live, red: dead) and the lactate dehydrogenase (LDH) assay (Fig. 2I and J). Overall, CA can affect virulence of S. aureus by affecting the specific virulence factors transcription.

Reduction of environmental tolerance by CA of S. aureus

Based on previous research, the ClpP is involved in not only the regulation of protein expression and secretion but also the degradation of misfolded proteins under stress conditions, including temperature shifts, pH changes, and exposure to reactive oxygen species, generated by host phagocytes (Gottesman 2003; Michel et al. 2006). Therefore, we further investigated whether the suppressed ClpP activity by CA is correlation to the reduced stress tolerance of S. aureus. The results indicated that the inhibition of ClpP by CA rendered the cells sensitive to hydrogen peroxide and NaClO (Fig. 3A-D), and the growth of S. aureus treated with CA was impaired in high temperatures, high salt, and low pH conditions experiments (Fig. 3E-H). Thus, we demonstrated that the role of CA in reducing the tolerance of S. aureus towards stress conditions.

Fig. 3.

Fig. 3

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CA weaken the environmental tolerance of S. aureus in vitro. A-F The survival of indicated strains treated with CA above 64 µg/mL was spotted on the LB agar plate in 10-fold serial dilution after H2O2 (A), NaClO (C) or heat (E) stress. Moreover, the corresponding colony count results was showing the B, D and F. G and H The survival rate of strains at different culture condition (pH 3.5, pH 5.5 and 10% NaCl) was determined by plate count

Enhancement of sensitivity to Tigecycline by CA of S. aureus

It has been reported that truncation mutation of clpP confers susceptibility to protein synthesis inhibitor antibiotics (Shoji et al. 2011). Thus, we further determined whether the combination of CA and tigecycline has an effective bactericidal effect in vitro. The combination of CA with tetracycline can reduce the number of tested bacteria to the limit of detection within 12 h compared with monotherapies (Fig. 4A). Similarly, the combination of CA and tigecycline increased the diameter of the inhibition zones compared to the non-combination group (Fig. 4B, C). Consistent with these results, in the samples treated with monotherapy or without treatment, most bacteria survived with a normal cellular morphology, however, combination therapy resulted in increased cell permeability and death, which was evidenced by significantly more red-stained bacteria observed with the Live/Dead BacLight bacterial viability kit and significantly damaged bacteria examined under SEM (Fig. 4D, E). Taken together our findings indicate that CA can effectively restore the bactericidal effect of tigecycline by inhibiting ClpP.

Fig. 4.

Fig. 4

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CA enhanced the bactericidal efficiency of tigecycline against S. aureus in vitro. A Time-killing curves evaluation of CA (64 µg/mL), tigecycline (0.2 µg/mL) or combinations on S. aureus strains every 2 h for 12 h. B and C Eye view of combining drug susceptibility test between CA and tigecycline (B). Corresponding inhibition zone diameter was also recorded (C). D and E The cell permeability and death treated with CA (64 µg/mL), tigecycline (0.2 µg/mL) or combinations were analyzed by Live/Dead bacteria staining and Scanning electron microscopy

CA exhibited an anti-infection ability against S. aureus USA300

In order to explore the anti-infection ability of CA against clinically isolated S.aureus USA300, we conducted several important phenotype experiments including hemolysis test, cellular infections, stress assay, time killing assay in vitro and Galler mellonella infection model in vivo. As shown in Fig. 5A, CA could inhibit the hemolytic activity of S. aureus USA300 culture supernatant at above 32 µg/mL. 32 µg/mL or 64 µg/mL CA also exhibited a protective effect in cellular infections, showing the decreased LDH release in Fig. 5B and injured A549 cells (red fluorescence marked cells) in Fig. 5C. Moreover, CA enhanced the susceptibility of S. aureus USA300 against heat, NaClO and tigecycline stress in Fig. 5D and E. Importantly, 25 mg/kg or 50 mg/kg CA improved the Galler mellonella survival rate in Fig. 5F, compared with S. aureus USA300 infected group. And the survival status was shown in Fig. 5G at 30 h. Above all, these results indicated that CA exhibited an anti-infection ability against S. aureus USA300.

Fig. 5.

Fig. 5

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CA exhibited an anti-infection ability against S. aureus USA300 (A) CA inhibited the culture supernatant hemolytic activity of S. aureus USA300 at above 32 µg/mL. B The LDH release determination in cellular infections model induced by S. aureus USA300. C Live/dead cells staining assay for visualizing the cells injury in cellular infections model. The green or red marked cells were shown live or dead cells respectively. D and E CA weaken the environmental tolerance of S. aureus USA300 against heat, NaClO (D) and tigecycline stress (E). F and G Galleria mellonella infection model was performed to evaluated the protective effect of CA against S. aureus USA300. The survival rate and state were shown in F and G respectively

Identification of the mechanism of the interaction between CA and ClpP

The stability of ClpP treated with CA was investigated by circular dichroism spectra. The results showed that changes in secondary structures occurred when CA were added to ClpP, the α-helix content decreased and β-sheet content increased (Fig. 6A, B).

Fig. 6.

Fig. 6

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Identification of the mechanism of the interaction between CA and ClpP. A and B Secondary structure changes of ClpP treated with 32 µg/mL CA. C-H CA and ClpP docking complex was created (C), and RMSD (Root Mean Square Deviation) (D), Rg (Radius of Gyration) (E), Hydrogen bond (F), FES (Free energy surface) (G) and RBFE (Relative binding free energy) (H) date were recorded. I and J The binding constants (Ka) of the pivotal mutants between CA was calculated by fluorescence quenching assay. Moreover, ClpP and mutant peptidase activity test were also test with 16–32 µg/mL CA

Molecular docking was employed to further explore the binding interaction between CA and ClpP. A visual analysis of the ClpP-CA docking complex was conducted using PyMol2 and Discovery Studio Visualizer software to generate a 3D interaction map (Fig. 6C). To validate the docking results, molecular dynamics simulations were performed. Analysis of RMSD, Rg, hydrogen bonds, free energy surfaces (FES), and relative binding free energy (RBFE) indicated a compact protein structure and reduced solvent-accessible surface area after 70 ns (Fig. 6D-H). We concluded that, the protein tightly enveloped the drug small molecule in the presence of a certain number of hydrogen bonding interaction forces at steady state.

Mutation experiments and fluorescence quenching analyses were performed to illustrate CA’s detailed action on ClpP. The binding constants (Ka) of the mutants were significantly lower than that of ClpP (Fig. 6I). Additionally, CA’s inhibitory effect on ClpP’s peptidase activity was diminished in the M31A and G33A mutants (Fig. 6J). These observations indicated that the residues M31 and G33 are required for the engagement of CA with ClpP.

Anti-infective effects of CA against S. aureus in vivo

To investigate the protective efficacy of CA against S. aureus, murine models of MRSA pneumonia infection were established. Mice were intratracheally instilled with live S. aureus, and lung tissues were collected after 2 days to enumerate the bacterial loads (schematic diagram as shown in Fig. 7A). As shown in the Fig. 7B, both the CA-treated group (30 mg/kg) and the ∆clpP group exhibited a significant reduction in the colony-forming units of S. aureus in the lungs compared to the vehicle group, suggesting that CA effectively reduced bacterial colonization.

Fig. 7.

Fig. 7

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CA relieved mouse pneumonia injury induced by S. aureus in vivo. A Schematic diagram of the mouse pneumonia model infected with S. aureus. Mice were infected with S. aureus 1 × 107 CFU/mouse and treated with CA (15 mg/kg or 30 mg/kg). Lung tissue was collected for bacterial load (B), cytokines expression level evaluation (C-E) and histopathological examination (F and G). Green arrow: healthy alveolar space; yellow arrow: pulmonary hemorrhage; blue arrow: bronchiole; red arrow: inflammatory cells; black arrow: neutrophils

To further explore the therapeutic benefits of CA in treating acute S. aureus pneumonia, we investigated lung inflammation and cytokine production in mice. The levels of inflammatory cytokines, including TNF-α, IL-6, and IL-1β, were markedly lower in the bronchoalveolar lavage fluid (BALF) of both CA-treated (30 mg/kg) and ∆clpP animals (Fig. 7C-E). Meanwhile, mice treated with solvent developed more obvious pneumorrhagia than mice treated with 30 mg/kg CA at 48 h postinfection (Fig. 7F). Histopathological examination through hematoxylin and eosin (HE) staining revealed less inflammatory cell infiltration and preserved lung architecture in both the CA-treated and ∆clpP groups, as opposed to the untreated group, which showed extensive infiltration, edema, and alveolar damage (Fig. 7G). Taken together, these results indicated that CA treatment relieved inflammation and cytokine levels, which show an excellent protective effect.

Discussion

S. aureus obtained nearly all available antibiotics, highlighting the need for new therapeutic targets and innovative strategies to address S. aureus-related infections. ClpP and ClpX are essential for regulating the expression of important virulence factors and protein transcription in this bacterium (Mei et al. 1997). To coordinate the release of these viluence, S. aureus strains employ a complex global regulatory network (Gimza et al. 2019). The ClpXP protease system promotes the transcription of mgrA and agr, which increases the levels of RNAIII. This, in turn, suppresses the expression of the repressor of toxins (Rot) and promotes the production of multiple toxins, including the pore-forming hemolysin alpha-toxin (Jelsbak et al. 2010; Aljghami et al. 2022). Through this intricate regulatory mechanism, ClpP and ClpX are integral to the pathogenicity of S. aureus, ensuring that virulence factors are expressed in a coordinated manner to optimize infection. Furthermore, the loss of clpP results in a complete transcriptional depression of genes regulated by the ctsR and hrcA heat shock regulon, as well as a partial depression of genes associated with the oxidative stress response, metal homeostasis, and SOS DNA repair, which are controlled by regulatory proteins such as PerR, Fur, MntR, and LexA (Michel et al. 2006). This alteration in gene expression underscores the significant role of ClpP in maintaining the balance of stress responses and homeostasis in S. aureus.

The exploration of inhibitors targeting ClpP has garnered significant attention due to their critical role in bacterial virulence and pathogenesis (Gaillot et al. 2000; Michalik et al. 2012; Zhao et al. 2016). However, the quest for effective caseinolytic protease inhibitors is fraught with challenges. β-Lactones has been displayed ClpP inhibition in a variety of pathogens such as P. alciparum or M. tuberculosis (Rathore et al. 2010; Compton et al. 2013). Despite promising results in vitro, the clinical development of cyclic esters has been hampered by reduced plasma stability due to hydrolysis and their generally low selectivity (Hackl et al. 2015). Bortezomib, a known inhibitor of the 26 S proteasome, has also been shown to inhibit ClpP1P2 (Moreira et al. 2015). However, its high cost, short half-life, and poor pharmacokinetics limited its effectiveness in treating M. tuberculosis. In this study, we demonstrate that the natural compound CA reduces the virulence of S. aureus by inhibiting ClpP. One significant advantage of CA is its status as a legally approved food flavoring (Panossian et al. 2001; Choi et al. 2017; Dong et al. 2020). Analysis of CA levels in mice following oral administration (0.2 mmol/kg) indicated rapid absorption, peaking at around 1 h. Importantly, CA has a favorable safety profile (Oral LD50: mouse, 300 mg/kg; rabbit, 3200 mg/kg; rat, 980 mg/kg), which is well within the effective range tested here (Mei et al. 1997). Thus, CA has the potential to be a promising candidate for further clinical evaluation as a therapeutic agent against S. aureus pneumonia.

Conclusion

We found CA could bind endogenous ClpP in S. aureus cells and exhibited significant efficacy in attenuating S. aureus virulence. Moreover, the persistence of S. aureus treated with CA under conditions of tigecycline exposure, as well as heat, oxidative stress, and osmotic pressure were decreased. Importantly, CA attenuated mice pneumonic injury induced by S. aureus in vivo. This work holds great potential to inspire promising strategies for treating MRSA infections and contributes to the development of potent synthetic antibiotic scaffolds with minimal cytotoxicity to the host.

Acknowledgements

We are grateful to Dr. Hanne Ingmer (The Royal Veterinary and Agricultural University, Denmark) for the gift of S. aureus NCTC 8325-4 and 8325-4/ΔclpP and Dr. Caiguang Yang (Shanghai Institute of Organic Chemistry, China) for the gift of the 8325-4/ΔclpP/pYJ335::clpP.

Authors’ contributions

Shufang Li, Wei Li, Jianfeng Wang and Qiaoling Zhang contributed to the concepts and design of the study. Shufang Li, Yan Zhang, Hongxia Ma, Yonglin Zhou, Lingcong Kong, Hongfa Lv participated in the acquisition, analysis, and interpretation of the data. Shufang Li and Jianfeng Wang drafted the manuscript. Jingmin Gu helped in handling review comments. All authors reviewed the manuscript.

Funding

This work was funded by National Natural Science Foundation of China (32222083 and U23A20242), the Fundamental Research Funds for the Central Universities under Grant 2023-JCXK-01, the Science and Technology Research Project of the Education Department of Jilin Province (JJKH20221092KJ) and the Norman Bethune Program of Jilin University (2023B29).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Experiments involving animals were conducted according to the experimental practices and standards approved by the Animal Welfare and Research Ethics Committee at Jilin University (SY202412062).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

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

Shufang Li and Yan Zhang contributed equally to this work.

Contributor Information

Wei Li, Email: weili8308@jlu.edu.cn.

Qiaoling Zhang, Email: zql@jlu.edu.cn.

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Data Availability Statement

No datasets were generated or analysed during the current study.

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