The anti-biofilm compound 4-ethoxybenzoic acid inhibits Staphylococcus aureus virulence factor production via a putative 4EB-binding pocket in key virulence-associated proteins

There is a need for dual action anti-virulence and anti-biofilm agents that target the opportunistic pathogen Staphylococcus aureus. Previous research determined that 0.8 mg/mL 4-ethoxybenzoic acid (4EB) reduced S. aureus ATCC 6538 biofilm formation by 88% relative to untreated controls with moderate inhibition of planktonic cell growth. Here we report that 4EB impacted S. aureus virulence phenotypes across all growth phases, including alpha-hemolysin (Hla) and serine protease (SplB/C) exoprotein production (60% reduction), staphyloxanthin pigment accumulation (73% reduction) and alpha-hemolysis (>87% reduction) compared to untreated control cells. RT-qPCR analysis demonstrated that 4EB downregulated virulence gene expression, including >100-fold reduction of alpha-hemolysin (hla) and leukocidins (lukDvEv), and a 35-fold decrease of the response regulator SaeR. Phenol-soluble modulin (PSM) transcription by biofilm-grown cells was upregulated by 4EB more than 4-fold for α1-4psm and β1-2psm genes, while δ-toxin (hld) was unaffected. In silico molecular docking analysis revealed that 4EB has a strong binding affinity (ΔG < −6.0 kcal/mol) for 9 virulence-associated transcriptional regulators, including SaeS, IcaR and CodY. Analysis of gene transcription during late exponential phase growth determined that genes controlled by 7 of the 9 identified regulators were significantly impacted by 4EB. The docking analysis identified putative 4EB binding sites that share common features including valine and tyrosine amino acid residues. The combined in vitro and in silico analyses identified interactions with well-known virulence genes but also implicated an effect of 4EB on proteins less commonly associated with S. aureus pathogenesis. These findings suggested potential alternative targets for anti-virulence and anti-biofilm therapeutics.

1 Introduction

Staphylococcus aureus exists as both a commensal bacterium and a human pathogen and is a leading cause of hospital- and community-acquired pneumonia, skin and soft-tissue infections, and biofilm-associated endocarditis (Tong et al., 2015; Kourtis et al., 2019). The non-systemic S. aureus infections typically involve biofilm production, which helps the bacterium evade the host immune system and provides a physical barrier to resist antibiotics (Weiner et al., 2016). Additionally, S. aureus contains a plethora of virulence factors that aid in disease progression. These include, but are not limited to, toxins, exoproteins, and pigments (Paharik et al., 2016; Burlak et al., 2007; Schilcher et al., 2024; Vila et al., 2019). Secreted virulence factors are typically produced post-exponentially, advancing the infection process by lysing host immune cells, dysregulating the immune system, and activating T cells (Kim et al., 2021; Chen et al., 2022). The virulence of S. aureus has earned it a spot among the ESKAPE pathogens, all of which are global health threats (De Oliveira et al., 2020).

Staphylococcus aureus infections have become increasingly problematic because of the emergence of multi-drug resistance. Resistance to beta-lactam antibiotics is virtually omnipresent in S. aureus clinical isolates (Lowy, 2003), and some isolates are resistant to almost all antibiotics that are clinically available, including vancomycin, considered the “last resort” antibiotic (Cheung et al., 2021). The prevalence of resistant strains has accelerated a push to find alternative treatments that are non-bactericidal and generate less selective pressure than antibiotic therapies (Cheung et al., 2021). Several anti-virulence compounds to control S. aureus infections were selected to target the agr quorum sensing system, which regulates production of virulence phenotypes, including biofilm formation and hemolysis (Sully et al., 2014; Mahdally et al., 2021). A challenge with this strategy exists because inhibiting agr quorum sensing in S. aureus leads to increased biofilm formation, a behavior observed in agr-deficient clinical isolates obtained from antibiotic-resistant biofilm infections (He et al., 2023; Otto, 2023). Thus, a need exists for additional S. aureus therapeutics with both anti-virulence and anti-biofilm activity.

In previous research, we identified small molecules from Rhamnus prinoides, an East African shrub used in traditional medicine, that exhibited anti-virulence and anti-biofilm activity (Campbell et al., 2019). Further investigation identified 4-ethoxybenzoic acid (4EB), which reduced biofilm formation up to 88% relative to untreated controls with moderate impact on planktonic growth (Campbell et al., 2020). These findings align with other reports in the literature of small molecule natural products that elicit both anti-biofilm and antimicrobial activity (Campbell et al., 2024; Khodaverdian et al., 2013; Qiu et al., 2010). We hypothesized that their small size allows these molecules to interact with multiple virulence-associated proteins, including transcriptional regulators. Accordingly, we hypothesized that 4EB modifies additional virulence phenotypes beyond biofilm formation. In this work, we investigated the impact of 4EB on S. aureus virulence factor production and used molecular docking to assess the ability of 4EB to interact with S. aureus transcriptional regulators and select virulence-associated proteins.

2 Methods

2.1 In vitro assays

2.1.1 Chemicals

All chemicals used in this work, including 4EB, were obtained from Millipore Sigma (United States) unless otherwise indicated.

2.1.2 Bacterial strain and culturing conditions

Staphylococcus aureus ATCC 6538 was grown in Luria-Bertani (LB) broth (Becton Dickinson, United States) and was used in all experiments. Prior to each experiment, overnight cultures were prepared by inoculating cells (stored in 10% glycerol at −80 °C) into sterile LB broth and incubating with aeration for 14–16 h at 37 °C, 200 rpm. For experiments involving 4EB, the concentration of 4EB was 0.8 mg/mL unless otherwise indicated. 4EB was aseptically weighed and added directly into LB broth prior to inoculation. Inocula for experiments were prepared as follows: an overnight culture was grown, the optical density (OD₆₀₀) was measured, and sample cultures were prepared by inoculating sterile LB broth to an OD₆₀₀ of 0.01, with or without 4EB in the growth medium.

2.1.3 Biofilm imaging

Biofilms were cultivated in flows cells suitable for imaging by confocal laser scanning microscopy (CLSM). Details on flow cell construction, biofilm cultivation and imaging can be found in the Supplementary materials. Briefly, biofilms were grown for 24 h in continuous flow conditions with or without 4EB, were stained with acridine orange and were imaged using an LSM510 CLSM (Zeiss) at 100 × magnification. Zeiss Zen Lite software was used to visualize and quantify the biofilms. The data presented are the average pixel intensity measured by the microscope for the collected images.

2.1.4 Exoprotein extraction procedure

Exoproteins were isolated as described (Traber and Novick, 2006) with modifications. Cultures were grown for 15 h (early stationary growth phase) at 37 °C, 200 rpm. At 15 h, the cultures were placed on ice for 1 h, and then the OD₆₀₀ of each 4EB-treated culture was normalized to the untreated culture so that each treated and untreated culture had approximately the same number of cells (OD₆₀₀ = 6.0 to 6.5). Culture supernatants were collected by centrifugation at 4,000 x g for 20 min at 4 °C, followed by filtering through 0.22 μm membrane filters to remove cell debris. Exoproteins were precipitated overnight at 4 °C using 1:1 (v/v) ice-cold 10% trichloroacetic acid added into the filtrate. The following day, samples were centrifuged at 4,000 x g at 4 °C for 20 min, and the protein precipitate was washed with 95% ethanol and air-dried at 4 °C. The protein precipitate was subsequently resuspended in 1x sample buffer (NuPAGE™ LDS Sample Buffer) and stored at −20 °C until needed for exoprotein profiling.

2.1.5 Exoprotein profiling

Exoproteins were profiled using the NuPAGE™ Mini Protein System (Invitrogen, United States). Exoprotein extracts were reduced and incubated at 70 °C for 10 min. The exoproteins were profiled on Bis-Tris Mini Gels (4–12%) using MOPS SDS Running Buffer along with a protein standard. The SDS-PAGE gels were subsequently stained with 1% Coomassie Brilliant Blue and de-stained overnight before imaging (Gauci et al., 2013).

2.1.6 Amino acid sequence determination

Bands of interest were extracted from an SDS-PAGE gel and trypsin-digested. The digested protein samples were sequenced using a nanoLC MS/MS platform (Creative Proteomics; Shirley, NY). The peptide mapping data were used to construct a peptide sequence by combining the initial sequence data within the N-terminal cleavage window and C-terminal cleavage window, removing any sequence overlap. The final amino acid sequence was subsequently aligned using the NCBI BLASTp database (Altschul et al., 1990).

2.1.7 Exoprotein analysis procedure

Each SDS-PAGE gel was analyzed for band intensity determination using ImageJ (Schneider et al., 2012), and the intensity of the image was set to 8-bit (black and white) and inverted. The intensity values and areas were measured for the bands of interest and were used to calculate the percent change from the control.

2.1.8 Hemolysis assay

4EB treatments of 0.2, 0.4, 0.6, and 0.8 mg/mL were used. All samples were incubated at 37 °C, 200 rpm for 15 h. After incubation, the OD₆₀₀ of each 4EB-treated culture was normalized to the untreated culture so that each treated and untreated culture had approximately the same number of cells (OD₆₀₀ = 6.0 to 6.5). 5 mL of each culture was centrifuged at 4,000 x g for 20 min, 4 °C. The supernatant was removed and was sterilized using a 0.22 μm filter. The filtered supernatant was added (200 μL) into tubes containing nuclease-free water with 50 μL of 5% defibrinated sheep’s blood (Lampire, United States), and the samples were incubated at 37 °C for 1 h. Sterile LB broth served as the negative control and a sample containing 1% Triton X-100 (Research Products International) was used as the positive control. After the 1-h incubation, samples were centrifuged at 17,000 x g for 2 min and 150 μL of supernatant was transferred into polystyrene 96-well plates in triplicate. Hemolysis was analyzed at 540 nm using a Victor® 3V plate reader.

2.1.9 Staphyloxanthin assay

Staphyloxanthin (STXN) production was analyzed as described (Vila et al., 2019). Treatments were prepared containing 0.2, 0.4, 0.6, and 0.8 mg/mL 4EB. The samples were incubated at 37 °C, 200 rpm for 24 h. After incubation, the OD₆₀₀ of each 4EB-treated culture was normalized to the untreated culture so that each treated and untreated culture had approximately the same number of cells (OD₆₀₀ = 6.0 to 6.5). The samples were then centrifuged at 4,000 x g, 4 °C for 20 min. The supernatants were removed, and the cell pellets were washed with 1x TBS (tris-buffered saline, pH 7.4–8.0) to remove the residual 4EB. The cell pellets were resuspended in 1 mL of 100% methanol (Fisher Scientific), thoroughly vortexed, and were incubated at 65 °C, 300 rpm for 5 min. The samples were subsequently centrifuged at 17,000 x g for 1 min, and the supernatants containing the pigment were transferred (150 μL) in triplicate to polystyrene 96-well plates. Methanol (100%) served as the negative control. STXN production was analyzed at 465 nm using a Victor® 3V plate reader.

2.1.10 Total RNA extraction

Cultures were incubated at 37 °C, 200 rpm, for 15 h. The sample cultures were subsequently harvested at 3, 9, 15, and 24 h and were maintained on ice for 1 h to preserve the total RNA. The OD₆₀₀ of each 4EB-treated culture was normalized to the untreated culture so that each treated and untreated culture had approximately the same number of cells (OD₆₀₀ = 6.0 to 6.5). The cell samples were centrifuged, the supernatants decanted, and the pellets were maintained on ice for the remainder of the RNA extraction procedure.

Briefly, following a modified protocol for the RNeasy Mini Kit (QIAGEN), cells were lysed by bead-beating for 3 min using Lysing Matrix B tubes and Buffer RLT. Cell lysate was transferred into the RNeasy Spin Column, centrifuged, and washed according to the manufacturer’s instructions. Total RNA was eluted from the spin columns with nuclease-free water, and samples were stored at −80 °C. Two RNA extractions were performed for each biological replicate, and all timepoints were conducted in triplicate. RNA was subsequently treated with DNase 1 (Promega RQ1 RNase-free DNase) following the suggested protocol, and the DNase-treated samples were cleaned and concentrated (Monarch® RNA Cleanup Kit; NEB). Following a confirmation amplification of the RNA to confirm the absence of DNA, cDNA was synthesized (ProtoScript® First Strand cDNA Synthesis Kit; NEB) following the manufacturer’s recommended instructions with an RNase inhibitor added (Applied Biosystems). For each RNA sample, three cDNA syntheses were performed (plus the negative control reaction), and the three cDNA synthesis products were combined to create one cDNA pool per total RNA product. The cDNA was stored at −20 °C and was subsequently used for real-time quantitative PCR (RT-qPCR) analysis.

2.1.11 Biofilm RNA extraction

Untreated and 4EB-treated cultures (150 μL) were pipetted into polystyrene 96-well plates, laboratory film-sealed and incubated with aeration at 37 °C, 200 rpm, for 15 h. The 96-well plates were harvested at 15 h and maintained on ice for 1 h to preserve the total RNA. The supernatants were separated from the biofilms and discarded. Using sterile RNase-free scraping tools and ice-cold DEPC-treated water, the biofilms were scraped from the polystyrene plates and transferred into sterile tubes. The harvested biofilm cells were vortexed, and the OD₆₀₀ of each sample was measured. The OD₆₀₀ of each 4EB-treated biofilm sample was normalized to the untreated biofilm sample. All samples were centrifuged at 17,000 x g for 2 min, the supernatants were discarded, and the biofilm pellets were maintained on ice. The remaining total RNA extraction procedure was completed as described above using the RNeasy Mini Kit (QIAGEN).

2.1.12 Reverse transcription-quantitative PCR

For assessing in vitro gene expression, RT-qPCR was performed for the gene transcripts of interest relative to 16S rRNA expression using a dye-based assay. Quantitative PCR was performed using the Applied Biosystems™ 7,500 Fast Real-Time PCR system with SYBR Select Master Mix (2x) (ROX dye). All samples were assayed in triplicate. The primer sequences were as follows: 16S rRNA forward primer (1369) 5′- CGGTGAATACGTT-CYCGG −3′, reverse primer (1492) 5’-GGWTACCTTGTTACGACTT-3′ (Suzuki et al., 2000). All other primers are listed in Table 1. Cycle conditions were as follows: hold at 50 °C for 2 min, hold at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 s and anneal/extend at 56 °C for 30 s. A standard curve and melt curve analysis were obtained in tandem for each assay. The relative expression of the 4EB-treated samples was determined using the Livak 2^-ΔΔCt method (Riedel et al., 2014).

Table 1

Table 1. Primers for PCR and reverse-transcription quantitative PCR (RT-qPCR) analysis.

2.1.13 Resazurin assay

The metabolic activity of S. aureus ATCC 6538 was analyzed using the resazurin assay as described (Muniyasamy and Manjubala, 2024; Chung et al., 2021) with modifications. A 1 mg/mL aqueous resazurin stock solution was prepared in advance as described (Oeschger and Erickson, 2021), and the stock solution was stored at −20 °C. Prior to the start of the assay, the 1 mg/mL stock solution was used to prepare a 0.01% (v/v) working stock using sterile 1x PBS (phosphate buffer saline). Cultures were incubated at 37 °C, 200 rpm, for 3, 6, 15, and 24 h. At each timepoint, 100 μL of the cells were immediately transferred into polystyrene (white, opaque) 96-well plates in triplicate. 60 μL of 0.01% resazurin was pipetted into each well, and the microtiter plates were incubated in the dark at 37 °C, 200 rpm, for 3 h. Sterile LB broth was used as the negative control. The resorufin fluorescence was analyzed at 540/590 nm (excitation/emission) using a SpectraMax® iD5 Multi-Mode Microplate Reader (Molecular Devices). The resazurin assay was repeated for a total of three biological replicates.

2.2 In silico assays

2.2.1 Phenol-soluble modulin identification

Phenol-soluble modulin (PSM) genes were identified within the S. aureus ATCC 6538 genome¹ using the PSM peptide sequences listed in Schwartz et al. (2012). The α1-4 psm and β1-2 psm genomic regions and the δ-toxin (hld) gene were PCR-amplified using primers listed in Table 1. PCR amplification was carried out with DreamTaq Green PCR Mastermix (2X) (Thermo Scientific™) following the manufacturer’s instructions, and the amplicons were Sanger-sequenced (Georgia State University, United States). The psm gene sequences were reverse translated² and these predicted peptide sequences were aligned with the peptide sequences published in Schwartz et al., 2012 for gene confirmation. All putative psm genes were PCR-amplified and sequenced for confirmation (Supplementary Table 1). Due to each αpsm gene being 63–69 nucleotides in length, the α1/α2 and α3/α4psm genes were transcriptionally analyzed in combination.

2.2.2 Molecular docking analysis

One hundred proteins consisting of 75 transcriptional regulators and 25 virulence-associated enzymes were identified within the S. aureus ATCC 6538 genome (Makarova et al., 2017). The crystal structures of proteins were obtained from the Protein Data Bank (Berman et al., 2000) and predicted protein structures were obtained from AlphaFold (Jumper et al., 2021). The protein structure identifications are listed in Table 2.

Table 2

Table 2. Protein structure identifications used in this work.

Molecular docking between 4EB and the proteins of interest was quantified using AutoDock Vina v1.1.2 (Trott and Olson, 2010) and Open Babel v3.0.1 software (O'Boyle et al., 2011). Water molecules and any additional ligands were removed using AutoDockTools (Huey et al., 2012). For AutoDock Vina, the exhaustiveness was set to 20 for thoroughness of search and accuracy, and all other default parameters were used. For each protein, the form of the grid box was defined as a hexahedron that covered the entire protein domain, allowing for 4EB to bind in several conformations. All 100 molecular docking experiments were replicated three times, with eight of the best-fit ligand poses being generated during each experimental replication. The ligand poses with the most negative ΔG values (strongest binding affinity) were averaged to determine the mean ΔG values of the three docking experiments. Structural images of the protein-ligand binding sites were generated using BIOVIA (Visualizer BDS, 2024) for 2D imaging and PyMOL v3.0.2 (DeLano, 2002) for 3D imaging. The experimental process of molecular docking was carried out using a scripting workflow.

2.2.3 Phylogenetic analysis

Phylogenetic analysis was performed for the 100 proteins of interest. Protein PDB files (Table 2) were aligned using MUSCLE Multiple Sequence Alignment (Edgar, 2004). The alignment was transformed into a phylogenetic tree and was edited and annotated using TreeViewer software (Bianchini and Sánchez-Baracaldo, 2024). The CLUSTAL multiple sequence alignment by MUSCLE (FASTA format) was added onto the phylogenetic tree for alignment visualization.

For conserved domain identification, proteins with the most favorable binding to 4EB were analyzed using the NCBI Conserved Domain Database (Yang et al., 2020). Known protein DNA-binding sites and known active sites were located using Protein Data Bank (Berman et al., 2000), AlphaFold (Jumper et al., 2021), and the NCBI Conserved Domain Database.

2.2.4 Statistical analysis

Data were analyzed for statistical significance using Student’s t-test. All experiments were conducted with a minimum of three independent biological replicates. p < 0.05 was considered significant.

3 Results

3.1 Effect of 4EB on Staphylococcus aureus growth and metabolic activity

At a concentration of 0.8 mg/mL, 4EB attenuated planktonic growth by 47 ± 7% after 24 h of growth (MIC₅₀; Kowalska-Krochmal and Dudek-Wicher, 2021; Supplementary Figure 1) and reduced biofilm formation by 88% (Campbell et al., 2020). The corresponding metabolic activity was analyzed using resazurin, a viability and activity indicator. During treatment of planktonically grown cells with 0.8 mg/mL 4EB, S. aureus metabolism was reduced 0–18% over 24 h compared to an untreated control (Supplementary Figure 2).

3.2 4EB impacts Staphylococcus aureus 3D biofilm structure

Biofilms treated with 0.8 mg/mL 4EB prior to inoculation formed characteristic tower structures after 24 h but lacked the extensive surface area coverage seen in untreated control biofilms (Figures 1A,B). The maximum height of the 4EB-treated biofilms was significantly less than the untreated controls (control: 131 ± 5 μm, 4EB-treated: 88 ± 3 μm; p < 0.03). Tower structures in the 4EB-treated biofilms appeared “patchy” relative to the more uniform and bulbous structures seen in the controls (Figures 1C,D). The mean fluorescent intensity for each biofilm is indicative of the amount of biomass that is present. A comparison of 4EB-treated biofilms versus untreated control biofilms determined a 47 percent greater average fluorescence intensity in the control biofilms (1.77 ± 0.09 × 104 RFU) compared to the 4EB-treated biofilms (1.20 ± 0.03 × 104 RFU). These data indicated that 4EB treatment reduced biofilm biomass accumulation (p < 0.04). Together these quantitative measurements demonstrated that 4EB treatment impacted the three-dimensional biofilm structure.

Figure 1

Figure 1. 3D biofilm structures after 24 h of growth. Representative orthogonal views of the untreated control (A) and 0.8 mg/mL 4EB (B). Corresponding 3D views for the representative images with a 35% threshold value to remove noise for the untreated control (C) and 0.8 mg/mL 4EB (D). Cells were stained with 100 ppm acridine orange before imaging on an LSM510 (Zeiss). Images were visualized using Zeiss Zen Lite software.

3.3 4EB inhibits Staphylococcus aureus alpha-hemolysin and serine protease production

We evaluated extracellular protein production in response to 4EB treatment. Treated samples and untreated controls were normalized to the same cell concentration prior to protein extraction (OD₆₀₀ = 6.0 to 6.5). Concentrations of 4EB ranging from 0.05 to 1.0 mg/mL were selected and the exoproteins were profiled by SDS-PAGE (Figure 2A). Exoprotein production decreased with increasing 4EB concentration. Two prominent protein bands, indicated by the blue and pink arrows, had the greatest decrease in expression. These bands were extracted and sequenced. The protein band at ~35 kDa was identified to be alpha-hemolysin (Hla, 86.7% identity), and the protein band at ~20 kDa was identified to be serine protease C (SplC, 90.6% identity) (Supplementary Figures 3, 4). Exoprotein production was measured for cells grown for 15 h with 0.8 mg/mL for five trials (Figure 2B). The average Hla and SplC production were significantly reduced relative to untreated controls by 64 ± 16% (p < 0.001) and 56 ± 9% (p < 0.001), respectively, following 4EB treatment.

Figure 2

Figure 2. Staphylococcus aureus Hla and SplC exoprotein production during 4EB treatment. (A) Exoprotein production during treatment with various concentrations of 4EB at 15 h of growth using normalized cell amounts. (B) The intensity (%) of exoprotein production was quantified for five separate experiments (trials 1–5; shown) during treatment with 0.8 mg/mL 4EB. (C) Relative expression of hla and splC genes during treatment with 0.8 mg/mL 4EB. The results represent the mean ± SEM of the triplicate RT-qPCR determinations of each cDNA sample obtained from three replicate planktonic cultures. Statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Reverse-transcription quantitative PCR (RT-qPCR) analysis was performed to determine the effect of 4EB on hla and splC expression. Transcript levels were quantified at 3, 9, 15 and 24 h of growth with 0.8 mg/mL 4EB. Transcription of hla and splC was most significantly downregulated (~100 fold or more) at 9 h and 15 h (early- and mid-stationary phases of growth) compared to the untreated control (Figure 2C).

Upon observing that Hla exoprotein production was significantly reduced by 4EB treatment, hemolysis activity was measured (at 15 h when Hla production levels were significantly inhibited; Figure 2B). Alpha-hemolysis was reduced by up to 87 ± 14% (p < 0.001) during treatment with 0.8 mg/mL 4EB (Figures 3A,B).

Figure 3

Figure 3. Hemolysis during 4EB treatment. S. aureus alpha-hemolysis was measured at 15 h of growth, and the percentage of hemolysis relative to the untreated control was measured during treatment with 0.8 mg/mL 4EB (A) Percentages of hemolysis are relative to the untreated control (B) Hemolysis supernatants; the negative control was sterile LB broth, and the positive control was 1% Triton X-100. Statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

3.4 4EB affects additional Staphylococcus aureus virulence factors

We investigated whether additional virulence factors were affected by 4EB treatment. We visually observed that staphyloxanthin (STXN) pigmentation decreased when the cells were treated with 4EB. To test the impact of 4EB on STXN production, methanol extractions were performed followed by spectrophotometric analysis. During treatment with 4EB, STXN was decreased by up to 73 ± 7% (p < 0.001) following treatment with 0.8 mg/mL 4EB (Figures 4A,B).

Figure 4

Figure 4. STXN production during treatment with 4EB. (A,B) STXN pigmentation production was measured during treatment with various concentrations of 4EB. Statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

In S. aureus, three categories of toxins are produced that eliminate neutrophils and leukocytes: alpha-toxin (alpha-hemolysin), leukocidins, and phenol-soluble modulins (PSMs). We observed that 4EB decreased alpha-hemolysin production and alpha-hemolysis. Therefore, we also measured the effect of 4EB on leukocidin and PSM gene transcription. Strain ATCC 6538 does not contain lukA or lukB leukocidin genes, so only transcript levels for lukEv and lukDv leukocidins were quantified. Following 0.8 mg/mL 4EB treatment, lukEv and lukDv were both significantly downregulated at all timepoints and were reduced by 500-fold and 40-fold at both the 9 and 15 h times, respectively (Figure 5A). Two enterotoxins encoded by entA and entD are present within the ATCC 6538 genome, and their transcript levels were quantified. Transcription of entA was downregulated by 17-fold at 3 h. However, levels of transcripts for entD were variable with 4EB treatment, although they were downregulated 22-fold at 15 h. These results indicated that 4EB treatment significantly downregulated the expression of leukocidins and enterotoxins.

Figure 5

Figure 5. Relative expression levels for genes associated with S. aureus toxins during treatment of 0.8 mg/mL 4EB. The fold change shown is relative to the transcript levels in untreated control. (A) Leukocidins (lukDv and lukEv) and enterotoxins (entA and entD) were analyzed. (B) The phenol-soluble modulins (αpsm, βpsm, and δ-toxin) genes were analyzed. The results represent the mean ± SEM of the triplicate RT-qPCR determinations of each cDNA sample obtained from three replicate planktonic cultures. Statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

PSM toxin expression in response to 4EB treatment was investigated by quantifying gene expression levels. There are three classes of PSMs: αPSMs (α1, α2, α3, α4psm), βPSMs (β1, β2psm), and δ-toxin (hld). Using published PSM amino acid sequences (Schwartz et al., 2012), all psm genes were identified within the strain ATCC 6538 genome. The psm genes and hld are only expressed during the post-exponential growth phase (Wang et al., 2023); therefore, transcript levels for psm genes were quantified at the 15 h timepoint only. PSMs have been shown to have variable levels of expression during biofilm formation, when compared to planktonic growth (Wang et al., 2023), and so psm expression levels were quantified for both planktonic and biofilm modes of growth (Figure 5B). All psm genes were similarly expressed in both the planktonic and biofilm cultures treated with 4EB (Figure 5B). All psm genes were significantly upregulated following 4EB treatment, except for hld (δ-toxin), which was unaffected by 4EB treatment.

To identify possible mechanisms of 4EB activity, the levels of transcripts for the following genes were measured: accessory gene regulator A (agrA), sae response regulator (saeR), V8 serine protease (sspA), dehydrosqualene synthase (crtM; STXN synthesis), RNAIII, and intracellular adhesion gene A (icaA) (Figure 6). All the genes tested were downregulated in response to 4EB treatment except for crtM, which was significantly upregulated at 9 and 15 h. Apart from agrA, transcript levels for all other genes changed from one- to two-log units over the sampling period.

Figure 6

Figure 6. Relative expression levels for S. aureus virulence-associated genes. Transcript levels for regulatory-, biofilm-, and virulence-associated genes were quantified after 0.8 mg/mL 4EB treatment. The results represent the mean ± SEM of the triplicate RT-qPCR determinations of each cDNA sample obtained from three replicate planktonic cultures. Statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

We observed a change in the expression of select biofilm and virulence-associated genes by 3 h after treatment with 0.8 mg/mL 4EB (Figures 5, 6). To understand the relationship between 4EB concentration and its influence on early-stage gene expression, we investigated the expression of select biofilm and virulence-associated genes at two sub-MIC concentrations: 0.4 mg/mL and 0.8 mg/mL, and at two early-stage timepoints, 3 h and 9 h of growth. At 3 h, there was a significant difference in the level of gene expression for all of the evaluated genes, with the absolute expression level (i.e., either upregulation or downregulation) at 0.8 mg/mL greater than at 0.4 mg/mL (Supplementary Figure 5A). At 9 h, there was no significant difference in gene expression for any tested gene at 0.4 mg/mL versus 0.8 mg/mL (Supplementary Figure 5B).

3.5 Molecular docking analysis of 4EB interactions with transcriptional regulators

We tested the hypothesis that 4EB interacts with transcriptional regulatory proteins using in silico molecular docking. One hundred proteins within the ATCC 6538 genome were selected using the Protein Data Bank (Berman et al., 2000) and AlphaFold (Jumper et al., 2021); 75 proteins were transcriptional regulators, and 25 proteins were other virulence-associated enzymes (Supplementary Table 2). We docked the 100 proteins with 4EB using AutoDock Vina v1.1.2 (Trott and Olson, 2010). 27 proteins had binding affinities (ΔG values) between −5.6 and −5.9 kcal/mol, and 14 proteins had ΔG values more negative than −6.0 kcal/mol, potentially indicating a specific association (Table 3). All ΔG values for each protein-4EB interaction were chosen for the pose with the most favorable binding complex.

Table 3

Table 3. In silico docking results for proteins of interest with 4EB.

The validity of the 4EB-protein associations for the nine identified transcriptional regulators with ΔG values more negative than −6.0 kcal/mol (Table 3) were evaluated in vitro using RT-qPCR analysis. For each regulator, the expression of genes known to be under their control (Supplementary Table 3) was measured after 9 h of growth (late exponential/early stationary phase). For seven of the nine transcription regulators, 4EB caused a significant change in their transcription relative to the untreated control (Figure 7). The extent of the change varied from 2-fold to more than 80-fold at the measured timepoint.

Figure 7

Figure 7. Relative expression levels for select S. aureus genes identified by in silico molecular docking to bind with high affinity to 4EB. Transcript levels for downstream regulons of the proteins of interest (red, top of figure) were quantified after 0.8 mg/mL 4EB treatment at 9 h. The results represent the mean ± SEM of the triplicate RT-qPCR determinations of each cDNA sample obtained from three replicate planktonic cultures. Statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

The 4EB-amino acid residue interactions for each of the 27 proteins were analyzed using the optimal binding confirmation of each complex (Supplementary Figure 6). The alkoxy moiety of 4EB formed alkyl bonds with hydrophobic amino acids during 84% of the interactions, with valine binding most frequently (Figure 8). The phenyl ring of 4EB associated with hydrophobic amino acids forming a pi-alkyl bond 83% of the time, with valine binding most frequently. The phenyl ring also formed pi-pi stacked bonds involving only tyrosine and phenylalanine residues. The carboxy moiety of 4EB formed conventional hydrogen bonds with several amino acids, most frequently arginine and glycine. 4EB-residue interactions for each of the 14 proteins with 4EB-binding complexes of ΔG < −6.0 kcal/mol are listed in Figure 8.

Figure 8

Figure 8. Amino acid residues that were found within the 4EB-binding pockets. Residues in pink represent binding with the alkoxy side chain of 4EB. Residues in orange represent binding with the phenyl ring of 4EB. Residues in blue represent binding with the carboxylic acid side chain of 4EB. Each of the proteins in the table associated with 4EB with a binding affinity (ΔG) less than −6.0 kcal/mol.

To understand the effect of the 4EB-residue interactions within each protein, we identified known DNA-binding sites, ligand binding sites, and active sites of the proteins listed within Figure 8 (ΔG < −6.0 kcal/mol) using the UniProt database (UniProt Consortium, 2015). All residues involved with binding 4EB (Figure 8) were cross-referenced to known binding sites (Supplementary Table 4). Proteins MalR, TreR, IcaR, CodY, and DegA are DNA-binding transcriptional regulators with the helix-turn-helix (HTH) DNA-binding motif (Table 3). The in silico analysis demonstrated that 4EB did not interact within the HTH region of these proteins (Figure 8; Supplementary Table 4). Proteins WalK, GraS, and SaeS are sensor kinase proteins, the essential phosphorylation component of two-component regulatory systems (TCSs). The in silico data determined that 4EB interacts with the histidine kinase domain of WalK, GraS, and SaeS (Figure 8; Supplementary Table 4). 4EB did not interact with the transmembrane domains or ligand binding sites of the sensor protein kinases (Supplementary Table 4). The sensor protein kinase SaeS was selected as a representative example for visualizing 4EB binding within the histidine kinase domain (Figure 10A). The transcriptional regulator CodY was selected as a representative example for visualizing 4EB’s interaction outside of the DNA-binding HTH-motif (ΔG = −6.03 kcal/mol) (Figure 10B).

Figure 9

Figure 9. Graphical representation of 4EB binding affinities. In silico molecular docking analysis results revealed that 4EB binds favorably to hydrophobic residues within the proteins of interest. The amino acid residues that interacted with the 4EB molecule are listed, and residues that bind the most frequently are indicated by an asterisk (*).

Figure 10

Figure 10. In silico molecular docking of (A) SaeS and (B) CodY with 4EB. 3D images are representations of the putative 4EB-binding pockets.

Based on the shared features among the 27 proteins with which 4EB associated most strongly, we hypothesized that these proteins potentially derived from a common ancestral structure or shared a conserved domain. To test this hypothesis, we aligned the 100 proteins of interest using a MUSCLE Multiple Sequence Alignment tool (Edgar, 2004) to visualize the protein phylogeny (Supplementary Figure 7). Some proteins, such as GraR, SarA, RsbU, and Nuc clustered within the phylogenetic tree. However, most of the proteins with the most favorable binding to 4EB were not clustered. The conserved domains for the 27 proteins were analyzed and were compared using the NCBI Conserved Domain Database (Yang et al., 2020). Neither common ancestry nor conserved domains were shared among all the 27 proteins with favorable ΔG values (Supplementary Figures 8A,B).

4 Discussion

Anti-virulence strategies are essential for controlling pathogens in an era of multidrug resistance. In previous work, we reported the anti-biofilm effects of several subinhibitory concentrations of 4EB, with 0.8 mg/mL 4EB reducing biofilm formation by 88% (Campbell et al., 2020). The present work confirmed that 0.8 mg/mL 4EB was the MIC₅₀ (concentration attenuating 50% of planktonic growth relative to untreated controls) and that it inhibited no more than 18% of metabolic activity across all growth stages. The reason for the discrepancy in metabolic inhibition compared to growth inhibition is not currently understood, although 4EB may interfere with normal cell division processes including those dependent upon the WalK/WalR functions (cell wall metabolism (Dubrac et al., 2007)), as suggested by the in silico results showing favorable binding of 4EB within the histidine kinase conserved domain of WalK. Similarly, the quorum sensing inhibiting molecule Azan-7 in combination with clindamycin was found to significantly decrease biofilm formation by approximately 60% while minimally inhibiting overall metabolism (Bernabè et al., 2021), an observation that the authors suggested may be due to inhibition of the icaABCDR operon.

A differential effect of a high or a low concentration of 4EB on biofilm and virulence gene expression (Zhang et al., 2024) was evident at 3 h but not at 9 h. This pattern suggests that treatment with 0.8 mg/mL caused 4EB to penetrate the cell and reach its intracellular targets faster than treatment with 0.4 mg/mL, leading to a stronger impact on gene expression by 0.8 mg/mL 4EB. In contrast, by 9 h, the concentration-dependent difference was no longer evident, potentially because the intracellular targets of 4EB were fully saturated, regardless of the initial concentrations that were present. This finding may be relevant to preventing biofilm formation, where the early stages of surface attachment impact the progress to mature biofilms (Moormeier et al., 2014) and which in turn impact the success of S. aureus infections (Tran et al., 2023). These data are also consistent with earlier findings that treating with 0.8 mg/mL 4EB reduced biofilm formation by S. aureus to a greater extent than 0.4 mg/mL 4EB or lower concentrations (Campbell et al., 2020). Notably, in recent work on the impact of 4EB on S. aureus biofilm development, 0.8 mg/mL 4EB was found to significantly alter S. aureus biofilm structure and reduce overall biofilm formation at 6 h following inoculation, which corresponds to the multiplication phase of S. aureus biofilm development (Marchesani et al., 2025).

Due to the substantial prevention of biofilm formation and the moderate growth inhibition, 0.8 mg/mL 4EB was selected for further experimentation, with the intent of using the knowledge gleaned to identify more potent anti-virulence compounds in future work. In vitro experiments demonstrated for the first time that 4EB significantly downregulated diverse S. aureus virulence factors across several growth stages. In silico analysis predicted that 4EB binds to common features within several regulatory and non-regulatory proteins, notably interacting with valine and tyrosine residues in a putative 4EB binding pocket. The in silico findings were supported by in vitro gene expression measurements that validated the ability of 4EB to alter transcription of key virulence-related genes, indicating its potential as an anti-virulence agent.

4EB significantly reduced the production of Hla, an exoprotein that is essential for disease progression during host-pathogen interactions (Burlak et al., 2007), with a corresponding reduction in hemolysis. Transcriptional regulators AgrAC and SaeRS have been reported to impact Hla production (Schilcher et al., 2024; Liu et al., 2016; Gudeta et al., 2019; Pendleton et al., 2022). During 4EB treatment, the transcription of agrA was not significantly impacted at 15 h, but a significant downregulation of saeR was observed, consistent with the measured decrease in extracellular Hla. Hla has been shown to be essential for biofilm development in S. aureus (Caiazza and O'Toole, 2003). Additionally, molecular docking indicated that the sensor histidine kinase SaeS complexed effectively with 4EB within the conserved histidine kinase domain, suggesting that the observed downregulation of saeS, saeR, sspA, hla and splC and the significant reduction in exoprotein production all resulted from a reduction in SaeS activity. These findings are consistent with previous research that investigated the essential phosphorylation of SaeR by SaeS; if this phosphorylation step is inhibited, then the downstream transcription of saeR, saeS, hla, and exoprotein-related genes in the sae-regulon is nearly abolished (Mainiero et al., 2010; Liu et al., 2016).

The STXN carotenoid pigment has been extensively studied for its ability to provide resistance to reactive oxygen species (Vila et al., 2019; Liu et al., 2005; Antonic et al., 2013). The pigment is synthesized through a triterpenoid biosynthesis pathway that is encoded by the crtMNOPQ operon (Antonic et al., 2013). We observed that STXN production was decreased by 4EB treatment; however, we were initially perplexed by the finding that crtM was upregulated by 4EB. In silico analysis indicated that the decrease in STXN production was potentially due to 4EB complexing with and inhibiting the enzymatic function of the CrtN and CrtP proteins. Previous research has identified several STXN-inhibiting compounds, including compounds 5 m, a benzofuran analog (Wang et al., 2016), NP16, a sulfonamide derivative (Gao et al., 2017), and 1,4-benzodioxan derivatives (Ni et al., 2018) that are all predicted to inhibit CrtN activity. The interaction sites of these molecules with CrtN are currently unknown to the best of our knowledge. No CrtP inhibitors have been reported to date; the putative interaction between 4EB and CrtP warrants further investigation.

Three categories of pore-forming toxins are commonly present within S. aureus: alpha-toxin, leukocidins, and PSMs. We determined that 4EB treatment downregulated alpha-toxin and leukocidins and conversely upregulated α1-4psm and β1-2psm genes. During treatment with 4EB, lukEvDv transcription was downregulated approximately 500-fold during the log phase of growth. Elsewhere, transcription of the leukocidin genes lukAB was almost abolished in an S. aureus USA300 saePQRS-knockout mutant (Schilcher et al., 2024), indicating that the sae-operon is a direct transcriptional regulator of these virulence factors. Our results show that saeR and saeS transcription was substantially downregulated with 4EB, and lukEv and lukDv were both downregulated at levels in concordance with hla. These data suggest that lukEvDv are regulated by SaeRS in strain ATCC 6538. Enterotoxin (entA and entD) transcription patterns in response to 4EB were variable, consistent with other reports (Borst and Betley, 1994; Bayles and Iandolo, 1989). These findings highlight the complex regulatory network influenced by 4EB treatment.

The PSMs are small amphipathic proteins that have multiple biological activities, including host cell cytolysis and biofilm structuring (Periasamy et al., 2012; Wang et al., 2007). We determined that the α1-4 and β1-2psm genes were significantly upregulated during 4EB treatment, but hld (δ-toxin) transcription was not affected by 4EB. The agr operon has been identified as the transcriptional regulator of the psm genes and hld (Vuong et al., 2000; Cassat et al., 2006; Queck et al., 2008). Transcript levels for hld and agrA were not significantly affected during 4EB treatment; in contrast, the α1-4 and β1-2psm genes were significantly upregulated. These findings suggest that there is an additional transcriptional regulator, or possibly multiple transcriptional regulators, regulating α1-4 and β1-2psm genes that is independent of agrA transcriptional control. MgrA has been identified as a negative regulator of psm expression in strain NCTC8325 (Jiang et al., 2018); however, molecular docking indicated that 4EB does not form a favorable binding complex with MgrA. This suggests that MgrA inhibition may not be responsible for the upregulated expression of α1-4 and β1-2psm genes that were measured in this work. Previous studies have hypothesized that more transcriptional regulators of αpsm and βpsm operons may exist (Queck et al., 2008), although no additional regulators have been reported to date. Future work will investigate the divergent psm gene expression pattern observed in this study. Notably, this work identified the location of the α1-4 and β1-2psm coding regions within the ATCC 6538 genome (Supplementary Figure 8). Changes in PSM activity caused by 4EB may have contributed to the observed differences in biofilm structure reported here. The impact of the observed increase in psm transcription on S. aureus virulence was not evaluated in this work.

Molecular docking analysis identified nine transcriptional regulatory proteins that strongly complexed with 4EB (binding affinities of ΔG < −6.0 kcal/mol). Three of these were TCS proteins: WalK, SaeS, and GraS (Bleul et al., 2022). The walKR TCS is the only TCS that is essential for S. aureus growth (Villanueva et al., 2018; Monk et al., 2019), and it regulates cell-wall thickening in response to antibiotics as a form of antibiotic resistance (Howden et al., 2011). GraS is involved in cationic antimicrobial peptide (CAMP) resistance through the regulation of the virulence-associated vraFG operon (Dhankhar et al., 2020). GraR has been explored as a target of anti-virulence molecules (Dhankhar et al., 2020), but an inhibitory molecule targeting GraS has not yet been identified. Limited information exists regarding biofilm formation and the transcriptional regulators MalR, TreR, and YqfL, all of which formed favorable binding complexes with 4EB. Additionally, several non-regulatory virulence proteins were analyzed in silico. Notably, the Nuc (secreted nuclease, nuc1) protein has an important role in the S. aureus biofilm development process (Moormeier et al., 2014), and Nuc was determined to form a highly favorable binding complex with 4EB (ΔG = −6.28 kcal/mol). Of the nine regulatory proteins that were identified as 4EB targets in silico, in vitro measurements demonstrated that transcription of seven downstream regulons was significantly impacted by 4EB.

Molecular docking, phenotypic analysis, and in vitro validation suggested that for at least two proteins, 4EB acts as an agonist, increasing binding. Both IcaR and CodY transcriptional regulators formed favorable binding complexes with 4EB in silico, and the downstream regulons of IcaR (icaA) and CodY (ilvD and oppB) were significantly upregulated by 4EB. Two CodY promoter sites have been identified for ilvD and one CodY promoter site for oppB (Majerczyk et al., 2010). The intracellular adhesion icaABCD operon is required for biofilm formation in S. aureus (Cramton et al., 1999; Gerke et al., 1998; Heilmann et al., 1996), and the operon is regulated by the transcriptional repressor IcaR. When IcaR represses transcription of the icaABCD operon, the result is decreased biofilm formation (Yu et al., 2012; Yehia et al., 2022). 4EB inhibited biofilm formation (Campbell et al., 2020), and downregulated icaA transcript levels, suggesting that IcaR is activated by 4EB. CodY regulates the transcription of more than 200 genes, including many virulence factors (Majerczyk et al., 2008). Typically, codY knockout mutants have increased virulence factor production including biofilm formation and hemolysis (Majerczyk et al., 2008; Bulock et al., 2022). Molecular docking indicated that 4EB binds strongly with CodY, and in consideration of the measured phenotypes reported here in combination with the upregulation of ilvD and oppB expression in vitro, it is plausible that 4EB activates CodY.

Molecular docking analysis results revealed several notable features of 4EB’s interaction with S. aureus ATCC 6538 regulatory and virulence-associated proteins. First, there was a wide range of binding affinities, implying that 4EB associates with a subset of the evaluated proteins in a specific way. Second, there were common residues and protein structural features among the proteins that had the strongest association with 4EB. This result suggested that there is a putative 4EB binding site within these proteins. Among the proteins to which 4EB bound most strongly, it did not associate with residues that interact directly with DNA, indicating that the impact of 4EB is allosteric. Currently, the specific physical mechanism by which 4EB alters transcription is unknown. Third, the ability of 4EB to complex with a diverse set of transcriptional regulators is potentially a function of its small molecular size. This finding suggests a strategy by which a combination of two or more small molecules can be used simultaneously to inhibit S. aureus virulence. Notably, many plant-based essential oils with antimicrobial activity are combinations of multiple small molecules (Jafarı-sales and Pashazadeh, 2020; Gao et al., 2020; Han et al., 2021; Sreepian et al., 2022). Overall, a combination of in vitro and in silico analyses demonstrated that 4EB is a multi-mechanistic, anti-virulence and anti-biofilm compound. These findings can assist with understanding the types of molecular features that contribute to effective anti-virulence activity.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author contributions

CT: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. AA-G: Methodology, Software, Writing – original draft. AM: Investigation, Methodology, Writing – original draft. CK: Investigation, Writing – original draft. TP: Investigation, Writing – original draft. LC: Investigation, Writing – original draft. HY: Writing – original draft, Investigation. ZL: Writing – original draft, Investigation. AS: Investigation, Writing – original draft. K-JC: Writing – review & editing, Methodology. EG: Conceptualization, Methodology, Supervision, Writing – original draft, Writing – review & editing.

Funding

The author(s) declared that financial support was not received for this work and/or its publication.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that Generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2025.1704290/full#supplementary-material

Footnotes

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