Development of a dual fluorescent reporter system to identify inhibitors of Staphylococcus aureus virulence factors

ABSTRACT

Staphylococcus aureus is a leading cause of bacterial infections due to its resistance to most antibiotics and the production of diverse virulence factors. Various alternative approaches to treating staphylococcal infections have been proposed, including targeting the virulence factors or mechanisms that regulate their synthesis. The accessory gene regulator (agr) and the S. aureus exoprotein expression (sae) are two major virulence regulators of S. aureus. Here, we describe a dual reporter system capable of simultaneously monitoring the transcriptional activities of agr and sae in S. aureus. The reporter plasmid contained two fluorescent protein cassettes arranged back-to-back, with the mCherry cassette driven by the agrP3 promoter and the GFP cassette driven by the sbi promoter. We examined the fluorescent responses of wild-type, agr, and sae mutant strains using fluorescence microscopy and found that the activities of agr and sae systems could be detected without significant crosstalk in the dual reporter strain. The dual reporter system could be used to monitor the dynamic changes in agr and sae activities during batch growth in S. aureus. Additionally, we evaluated the responsiveness of the reporter strain to virulence inhibitors by treating it with several known inhibitors, including autoinducing peptides (AIPs) containing culture supernatants, indole, and flavone. The results suggested that the dual reporter system was sensitive to both agr and sae inhibitors. This reporter construct enables the characterization of the activity of a substance against two different targets in one screening round, significantly reducing screening time and expense.

IMPORTANCE

Staphylococcus aureus is a formidable pathogen responsible for a wide range of infections, and the emergence of antibiotic-resistant strains has posed significant challenges in treating these infections. In this study, we have established a novel dual reporter system capable of concurrently monitoring the activities of two critical virulence regulators in S. aureus. By incorporating both reporters into a single screening platform, we provide a time- and cost-efficient approach for assessing the activity of compounds against two distinct targets in a single screening round. This innovative dual reporter system presents a promising strategy for the identification of molecules capable of modulating virulence gene expression in S. aureus, potentially expediting the development of antivirulence therapies.

INTRODUCTION

Staphylococcus aureus is a common opportunistic pathogen responsible for severe clinical infections. The emergence and spread of multidrug-resistant S. aureus strains have limited the treatment options available for combating these infections (1). Alternative strategies involve the development of antivirulence drugs that target bacterial virulence factors, or the mechanisms regulating their synthesis (2 – 4). To establish an infection in the host, S. aureus produces a plethora of virulence factors, including toxins, enzymes, adhesins, and other surface proteins, which enable the pathogen to survive in hostile conditions and facilitate its dissemination through tissues (5). The expression of these toxins and enzymes is tightly regulated by multiple regulatory systems in S. aureus.

The accessory gene regulator (agr) is the most extensively studied global regulator of virulence determinant production in S. aureus. The agr locus comprises the P2 and P3 promoters. The P2 promoter drives transcription of agrBDCA, which constitutes the structural component of the agr system, while the P3 promoter drives transcription of RNAIII, a regulatory RNA that modulates the transcription or translation of numerous virulence genes in S. aureus (6). In animal infection models, inhibition of agr-mediated signaling diminishes the pathogenicity of S. aureus, suggesting that agr may represent a potential therapeutic target (7).

The S. aureus exoprotein expression (sae) locus is another major two-component regulatory system that governs the expression of toxins and other exoproteins in the organism. The response regulator and the histidine protein kinase are encoded by the genes saeR and saeS, respectively (8). The SaeRS system regulates the production of over 30 virulence factors in S. aureus (9). Multiple animal infection models have demonstrated the crucial role of the sae system in the complete pathogenesis of S. aureus (10 – 13).

Growing evidence suggests that targeting regulators of virulence factors could provide an effective therapeutic approach to bacterial infections (14). Reporter strains designed to monitor the activity of these regulators offer a powerful tool for identifying antivirulence agents. In S. aureus studies, fluorescent proteins are widely employed as reporter genes due to their ease of detection and quantification (15 – 18), which makes them especially useful in investigations involving the identification of antivirulence agents. A notable example is the discovery of savirin, a small molecule inhibitor of AgrA, which was achieved by screening 24,087 compound libraries using a reporter strain expressing GFP under the control of the agrP3 promoter (19). While a single reporter strain can monitor the activity of a specific transcriptional regulator, S. aureus virulence factors are regulated by multiple regulators, necessitating the use of several reporter strains to monitor each individual regulator’s activity. This approach demands multiple tests for a single compound.

A recent study generated a dual reporter construct for observing the simultaneous expression of two secreted staphylococcal enzymes, nuclease and coagulase, during S. aureus biofilm development (20). Inspired by this work, we envision that a dual reporter strategy, if applied to concurrently monitor major virulence regulators in S. aureus, which play critical roles in the regulatory network of S. aureus virulence factor expression, could provide a more efficient method for screening antivirulence factor compounds compared to single-reporter approaches. Building upon this concept, in the current study, we established a dual fluorescent protein reporter strain to simultaneously monitor the transcriptional activities of the agr and sae signaling systems in S. aureus. The reporter strain was validated by assessing its responsiveness to known inhibitors of agr or sae. Our results demonstrate that this dual reporter system can be effectively employed for the efficient screening of compounds targeting either the agr or sae systems as potential antivirulence agents for treating S. aureus infections.

RESULTS AND DISCUSSION

Construction of mCherry-GFP dual reporter plasmid

To establish a reporter strain capable of simultaneously monitoring the transcriptional activities of both agr and sae systems in S. aureus, we constructed an mCherry-GFP dual expression cassette reporter plasmid, designated as pAgrP3-mCherry/Sbi-GFP (Fig. 1). We used an Escherichia coli-S. aureus shuttle plasmid, pLI50, as the starting vector. For the constructed dual reporter plasmid, ampicillin served as the antibiotic resistance marker for E. coli, while chloramphenicol was used for S. aureus. To minimize interference between the transcription processes of the mCherry and GFP expression cassettes, they were arranged in a back-to-back orientation. The mCherry expression cassette was driven by the agrP3 promoter, known for its high output and wide dynamic range (21), while the GFP cassette was driven by the sbi promoter, which is exclusively regulated by the sae system and exhibits strong transcriptional activity (9, 22), making it an ideal reporter for the sae transcriptional activity. The combination of mCherry and GFP for the dual reporter system was selected due to their nonoverlapping spectral characteristics and the absence of interference between their fluorescent signals.

Fig 1.

Schematic representation of the dual reporter vector (pAgrP3-mCherry/Sbi-GFP). The plasmid features two expression cassettes arranged in a back-to-back configuration, with the mCherry cassette and GFP cassette driven by the agrP3 and sbi promoters, respectively. Additionally, the plasmid contains two origins of replication (Ori), pBR322 for E. coli maintenance and pUB110 for S. aureus maintenance. The ampicillin and chloramphenicol resistance genes facilitate selection in E. coli and S. aureus, correspondingly.

Fluorescence signal of S. aureus wild-type, agrA mutant, and saeS mutant strains transformed with dual reporter vector

The dual reporter plasmid, pAgrP3-mCherry/Sbi-gfp, was introduced into the wild-type, agrA, and saeS mutant strains of S. aureus. Fluorescence images of bacterial cells were taken after a 12-h cultivation period of the transformed strains (Fig. 2). In the wild-type S. aureus strain, both mCherry and GFP signals were evident, indicating the preservation of agrP3 and sbi promoter activities within the dual reporter vector. In contrast, only the GFP signal was observable in the agrA mutant, while the saeS mutant exclusively exhibited the mCherry signal. We also used a microplate reader to measure the fluorescence signals emitted by the dual reporter vector in the wild-type S. aureus, agrA mutant, and its complemented strain, as well as in the saeS mutant and its complemented strain (Fig. S1C). The results were similar to those obtained through fluorescence microscopy. Specifically, inactivation of AgrA or SaeS resulted in the loss of mCherry or GFP signal, respectively. However, reintroduction of the agrA or saeS genes led to fluorescence signal restoration, reaching levels comparable to that of the wild-type strain. These findings demonstrate that the dual reporter strain enables effective detection of agr and sae system activities, with minimal crosstalk interference.

Fig 2.

Fluorescence microscopy images of mCherry and GFP in S. aureus wild-type, agrA mutant, and saeS mutant strains carrying the dual reporter plasmid. The engineered strains were cultured for 12 h, and fluorescence images of the cells were acquired using red and green channels, respectively.

Dynamics of agr and sae system activities during batch growth in a dual reporter strain

To confirm that the established dual reporter system is suitable for monitoring the dynamic changes in the activity of the agr and sae systems in response to varying environments, we conducted a time-course analysis of the transcriptional activity of the agrP3 and sbi promoters during the batch growth of S. aureus strains carrying the dual reporter plasmid. Overnight cultures of wild-type S. aureus harboring the dual reporter vector were inoculated into fresh TSB medium at a 1:100 dilution, and the cultures were incubated at 37°C with shaking at 220 rpm for 16 h. Samples were collected at indicated time points, and mCherry and GFP signals were measured using a microplate reader and normalized to the cell density at 600 nm (Fig. 3). During the early to midexponential growth phases (from 1 to 4 h), the agrP3 promoter activity was relatively low, but it progressively increased and eventually peaked in the post-exponential growth phase (at 12 h). In contrast, the highest transcriptional activity of the sbi promoter was observed at the termination of the exponential growth phase (at 6 h). These observations are consistent with previous reports (23, 24), thus confirming the efficacy of our dual reporter system in real-time monitoring of agr and sae activities concurrently.

Fig 3.

Dynamic changes in the transcriptional activities of the agr and sae systems during batch growth. S. aureus strains carrying the dual reporter plasmid were cultured at 37°C, and samples were collected at appropriate time points. At each time point, the red and green fluorescence signals were quantified using a microplate reader and normalized to the cell density at 600 nm. Error bars represent the standard deviation.

Dual reporter strain responses to agr system agonists and antagonists

To assess the responsiveness of our developed dual reporter system to agr and sae inhibitors, we investigated the impact of chemicals known to exhibit inhibitory activity on the agr or sae signaling pathways, on the levels of mCherry and GFP expressions in the dual reporter strain.

Crosstalk among different agr specificity classes in S. aureus strains is one mechanism of virulence regulation by agr (25). S. aureus isolates are classified into four agr specificity classes based on an autoinducing peptide (AIP) (amino acid sequence and length) and the corresponding receptor, AgrC (19). AIP from one class can stimulate agr in strains from the same class while inhibiting agr from another class (25). In S. aureus, agr-I and agr-II are the two most distantly related agr specificity groups, with their respective AIPs sharing only the central cysteine residue (26). In this study, S. aureus ATCC 6538, which belongs to the agr-I specificity class, was selected as the host bacterium for the dual reporter plasmid. Therefore, the culture supernatant of the ATCC 6538 strain, which secretes AIP-I (27), was selected as an agonist of the agrP3 promoter in the dual reporter strain. The culture supernatant of Mu 50 strain, which secretes AIP-II (6), was used as an inhibitor, and TSB culture medium was used as a blank control (Fig. 4). Compared to the control group using TSB medium, the addition of AIP-I containing culture supernatant increased the transcriptional activity of agrP3 by 1.6-fold, while the addition of AIP-II containing culture supernatant reduced the transcriptional activity by 5.9-fold. In contrast, culture supernatants containing either AIP-I or AIP-II had no significant impact on the sae transcriptional activity in the reporter strain. The results indicate that the dual reporter strain is sensitive to both agonists and antagonists of the agr system and can be used for screening compounds that inhibit agr.

Fig 4.

Assessment of the dual reporter strain response to culture supernatants containing AIP-I or AIP-II. Culture supernatants from S. aureus strains ATCC 6538 and Mu 50 were utilized as sources of AIP-I and AIP-II, respectively. Upon reaching an OD ₆₀₀ of 1.0 at 37°C, the dual reporter strain was incubated for 3 h with culture supernatants containing either AIP-I or AIP-II. The transcriptional activities of the agr and sae systems were evaluated using the OD₆₀₀-normalized mCherry and GFP signals, respectively. Error bars represent the standard deviation. Statistical significance was determined using an unpaired, two-tailed Student’s t-test. *P < 0.05.

Dual reporter strain responses to sae system inhibitors

Similarly, we tested the responsiveness of the dual-reporter strain to sae inhibitors. The sae system plays a crucial role in regulating the production of various virulence factors in S. aureus, including toxins, exoenzymes, and immunomodulatory proteins, which contribute to the pathogenesis of S. aureus. Inactivation of the sae system has been shown to reduce the pathogenicity of S. aureus (11, 12, 28, 29). Previous studies have demonstrated that indole and flavone can inhibit sae transcription, leading to a reduction in the virulence of S. aureus (30, 31). To validate the efficacy of the dual reporter platform in screening for sae inhibitors, we selected indole and flavone as candidate compounds for this study. S. aureus strains carrying the dual reporter plasmid were cultured in TSB medium supplemented with varying concentrations of indole or flavone at 37°C in the presence of 5 µg/mL of chloramphenicol. We first evaluated the effects of indole and flavone on the growth of S. aureus (Fig. 5A). Batch culture analysis revealed a slight initial delay in the growth of S. aureus in the presence of 50 mM indole or 50 µg/mL flavone (the highest concentrations of these compounds used in this study); however, by 14 h (for the indole-treated group) or 16 h (for the flavone-treated group), the cell density recovered to levels similar to those of the untreated group. Therefore, to minimize the potential influence of bacterial growth on the measurement of fluorescence signals in the reporter strain, we chose the 16 h time point, when the bacterial growth of both drug-treated and untreated groups had become similar, to assess the fluorescence intensity of mCherry and GFP, and subsequently normalized these values to the cell density at 600 nm.

Fig 5.

Validation of the dual reporter system using sae inhibitors indole and flavone. (A) Growth curves of S. aureus ATCC 6538 strains at 37°C in TSB medium alone, with 50 mM indole, and with 50 µg/mL flavone. (B and C) Evaluation of the dual reporter strain in the presence of varying concentrations of indole (B) and flavone (C). For both (B) and (C), S. aureus strains harboring the dual reporter plasmid were cultured in TSB medium for 16 h in the presence of different concentrations of indole or flavone, and the transcriptional activities of the agr and sae systems were assessed using OD₆₀₀-normalized mCherry and GFP signals, respectively. (D) Quantitative real-time PCR analysis of changes in the expression of agrA and saeR genes in S. aureus treated with 50 mM indole or 50 µg/mL flavone. Error bars in all panels indicate the standard deviation, and statistical significance was determined using an unpaired, two-tailed Student’s t-test (*P < 0.05).

As illustrated in Fig. 5B, our study demonstrates that indole concentrations of 0.10, 0.25, and 0.50 mmol/L led to a reduction in sae system transcriptional activity by 1.1-, 1.4-, and 2.2-fold, respectively, in the reporter strain compared to the untreated group, while exerting no significant impact on agrP3 activity. These findings concur with a prior investigation that utilized quantitative PCR (qPCR) techniques to assess saeS transcript levels in response to indole (31). Exposure of the dual reporter strain to flavone at concentrations of 10, 25, and 50 µg/mL resulted in reductions in the transcriptional activities of the sae system by 1.5-, 3.2-, and 6.1-fold, respectively (Fig. 5C), aligning with the observations reported by Lee (30). Intriguingly, our data indicate that the aforementioned flavone concentrations also reduced agrP3 transcriptional activities by factors of 1.9, 2.5, and 4.3, in contrast to the conclusions drawn by Lee (30). Lee’s study employed qPCR methods and revealed an upregulation of the agrA gene expression in S. aureus following flavone treatment. This inconsistency highlights the need for further validation of our dual reporter system findings through additional experiments.

To corroborate the findings of our dual reporter system, we conducted qPCR analysis to investigate the effects of indole and flavone on the expressions of agrA and saeR genes, key components of the agr and sae systems, respectively (Fig. 5D). Treatment with indole significantly downregulated saeR expression while showing no significant effect on agrA expression. In contrast, flavone treatment significantly downregulated both saeR and agrA expressions. These qPCR results are consistent with our observations from the dual reporter strain, supporting the reliability of our established dual reporter system. Notably, similar to the dual reporter assay, our qPCR results investigating the effects of flavone on agrA expression also contradicted the findings reported by Lee (30). This discrepancy may arise from differences in reference gene selection between the studies. Lee employed the 16S rRNA gene as the reference gene, which has been subject to debate regarding its suitability as an internal control gene for S. aureus (32, 33). In our study, we selected rpoB and ftsZ genes as reference genes based on a geNorm pilot experiment conducted in our previous research, which demonstrated that these two genes are the most stably expressed housekeeping genes in S. aureus when exposed to flavone (22). Additionally, the inhibitory effect of flavone on agrA expression was also confirmed by RNA-seq analysis in the same prior study (22).

Advantages of dual reporter system in screening virulence inhibitors

Antivirulence therapy offers several advantages over conventional antibiotic treatment, including a wider range of targets, preservation of commensal bacteria, and reduced selective pressure for resistance (1). However, due to the diversity and functional redundancy of S. aureus virulence factors, targeting a single factor may not provide sufficient protection to the host. Therefore, targeting virulence regulators such as agr and sae, which control a broader range of virulence factors, may be advantageous. High-throughput screening technologies are effective techniques for identifying virulence regulator inhibitors. In this study, we demonstrated that our developed dual-reporter system is sensitive to both agr and sae inhibitors and can serve as a valuable tool for screening virulence inhibitors. Furthermore, the integration of two reporters controlled independently by the agr and sae systems in a single expression vector significantly improves the efficiency of screening for virulence regulator inhibitors compared to a similar single-reporter construct.

Conclusions

In this study, we successfully developed a dual reporter system for S. aureus, enabling simultaneous and robust monitoring of the transcriptional activities of both agr and sae regulatory systems without significant crosstalk. The reporter plasmid featured two fluorescent protein cassettes, mCherry and GFP, driven by the agrP3 and sbi promoters, respectively. We demonstrated the effectiveness of the dual reporter system by monitoring dynamic changes in agr and sae activities during batch growth of S. aureus. Additionally, the system proved sensitive to both agr and sae inhibitors, as evidenced by its responsiveness to known inhibitors, such as AIPs containing culture supernatants, indole, and flavone.

The integration of two reporters into a single screening platform offers a time- and cost-effective approach for characterizing the activity of a substance against two different targets in one screening round. However, further research is necessary to apply this system to the screening of synthetic and natural compound libraries, which will be a crucial area of future work. By utilizing the established dual-reporter system to identify promising inhibitors, it may facilitate the discovery of effective antivirulence therapeutic agents against S. aureus infections.

MATERIALS AND METHODS

Bacterial strains, plasmids, and reagents

The strains and plasmids used in this study are listed in Table S1, while the primers are listed in Table S2. E. coli strains DH5α and BL21 were cultivated on Luria-Bertani agar or in Luria-Bertani broth. S. aureus strains ATCC 6538 and Mu 50 were grown on tryptic soy agar (TSA; Difco) or in tryptic soy broth (TSB; Difco). Plasmids were maintained in E. coli DH5α. To circumvent the SauUSI restriction barrier (34), we propagated DNA plasmids in E. coli BL21, a DNA cytosine methyltransferase-deficient strain, prior to transformation into S. aureus. Plasmid DNA extracted from BL21 was directly electroporated into the desired S. aureus strains (17 kV/cm, 5 ms pulse, 1 mm cuvette). When necessary, antibiotics were added to the growth media at the following concentrations: ampicillin, 100 µg/mL; kanamycin, 50 µg/mL; chloramphenicol, 5 µg/mL; and erythromycin, 10 µg/mL. New England Biolabs supplied the following products: NEBuilder HiFi DNA Assembly for cloning, Q5 High-Fidelity DNA Polymerase for PCR, restriction enzymes, T4 DNA ligase, and T4 polynucleotide kinase. Flavone and indole were obtained from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO). All other chemical reagents employed in this study were of analytical grade.

Construction of a dual reporter plasmid

In this study, the Gibson assembly method (35) was employed to construct all reporter vectors using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, USA). Initially, an intermediate plasmid, pSbi-GFP, which contains the gfp reporter driven by the sbi promoter, was created. The sbi promoter and terminator were amplified from ATCC 6538 genomic DNA, while the gene for GFP was amplified from plasmid pGFPmut3.1 (36). The E. coli-S. aureus shuttle plasmid pLI50 (37) was digested with EcoRI and HindIII restriction endonucleases. The digested plasmid and PCR products were run on an agarose gel and then purified using TIANquick Midi Purification Kits (TIANGEN BIOTECH Co., Ltd., Beijing, China). The linearized pLI50 plasmid backbone was assembled with the DNA fragments of the sbi promoter, GFP gene, and sbi terminator, generating the intermediate plasmid pSbi-GFP.

Subsequently, the dual reporter plasmid was constructed by fusing the sbi promoter-gfp expression cassette into the agrP3-mCherry expression cassette in a back-to-back transcriptional orientation. The sbi promoter-gfp expression cassette was amplified from the intermediate plasmid pSbi-GFP, the agrP3 promoter fragment from ATCC 6538 genomic DNA, and the mCherry gene from the plasmid pRN11 (18). The three DNA fragments mentioned above were integrated into the pLI50 plasmid backbone digested with BamHI and EcoRI, generating the dual reporter plasmid pAgrP3-mCherry/Sbi-GFP. This plasmid contains the agrP3 promoter for driving mCherry expression and the sbi promoter for driving GFP expression. Positive clones were confirmed by colony PCR using Maxima Hot Start Green PCR Master Mix (Thermo Fisher Scientific) according to the manufacturer’s protocol. Plasmids were isolated from positive colonies using a Mini Plasmid Kit (TIANGEN BIOTECH Co., LTD., Beijing, China) and then sequenced for further verification. The confirmed dual reporter was transformed into the target strains of S. aureus (ATCC 6538 wild-type strain, saeS mutant, and agrA mutant) for subsequent analysis.

Creating and complementing agrA or saeS mutants

Inactivating mutations for SaeS or AgrA in S. aureus were engineered using the pnCasSA-BEC base editing plasmid, a tool specifically designed for highly efficient C-to-T base transitions in S. aureus strains (38). Specifically, in the agrA gene, a mutation replaced the Glu179 codon (CAA) with a stop codon (TAA, Fig. S1A), thereby inactivating the AgrA protein, as previously confirmed (38). Similarly, for the saeS gene, the Glu100 codon was substituted with a stop codon (Fig. S1B), resulting in a truncated protein representing only 28% of the full-length SaeS protein.

To carry out these genetic alterations, spacer oligonucleotide pairs specific to agrA and saeS were designed using the CRISPR-CBEI toolkit (39), then phosphorylated, annealed, and inserted into the BsaI sites of the pnCasSA-BEC plasmid via the Golden Gate assembly. The modified pnCasSA-BEC plasmid, now containing the agrA or saeS spacer, was introduced into the S. aureus ATCC 6538 strain through electroporation (17 kV/cm, 5 ms pulse in 1 mm cuvette). Thereafter, colonies were selected at 30°C on TSA plates with 5 µg/mL chloramphenicol. Confirmation of the agrA or saeS mutation was accomplished by sequencing PCR-amplified genomic regions containing the desired mutations. Once validated, the base editing plasmid was eliminated from the cells by incubating them at 42°C without chloramphenicol for 12 hr.

To complement the agrA or saeS mutants, we constructed plasmids pCN47-agrA and pCN47-saeS using the E. coli-S. aureus shuttle plasmid pCN47 (40). For both pCN47-agrA and pCN47-saeS, the respective promoters (agrP3 for agrA and saeP1 for saeS) and coding genes (agrA and saeS) were PCR amplified from ATCC 6538 genomic DNA. The gene fragments and their corresponding promoter fragments were then assembled into the predigested pCN47 vector using BamHI/EcoRI. The resulting plasmids, pCN47-agrA and pCN47-saeS, were introduced into the corresponding mutant strains, generating the complemented strains agrA mutant/pCN47-agrA and saeS mutant/pCN47-saeS.

Fluorescence microscopy

To visualize fluorescent bacteria, we utilized a differential interference contrast microscope (TE2000U, Nikon) equipped with a high numerical aperture objective (100×, N.A. = 1.40). The mCherry signal, regulated by the agrP3 promoter, was captured in the red channel, while the GFP signal, governed by the sbi promoter, was recorded in the green channel.

Measurement of agr and sae transcriptional activities in reporter strain

For the assessment of agr and sae transcriptional activities, strains harboring the reporter vector were initially grown overnight in TSB supplemented with antibiotics. These cultures were subsequently diluted 100-fold into 5 mL of fresh TSB media and incubated at 37°C with shaking at 220 rpm in 50 mL shake flasks. At appropriate time points, 0.2 mL aliquots were sampled from each culture. Cell density (OD₆₀₀) as well as mCherry (excitation at 485 nm, emission at 535 nm) and GFP (excitation at 485 nm, emission at 535 nm) fluorescence signals were measured using an Infinite 200 PRO Microplate Reader (TECAN, Switzerland). An S. aureus culture carrying an empty pLI50 vector was used as a control to measure the autofluorescence of the cells (cell blank). To calculate the agr and sae transcriptional activities, we normalized the mCherry and GFP fluorescence values of each sample to their respective OD₆₀₀ values. The autofluorescence, which was also normalized by OD₆₀₀, was then subtracted. The resulting normalized and corrected mCherry fluorescence values represented the agr transcriptional activity, while the GFP fluorescence values represented the sae transcriptional activity for each sample.

Treatment of the reporter strain with culture supernatants containing AIPs

Culture supernatants containing AIP-I and AIP-II were obtained from strains ATCC 6538 and Mu 50, respectively. To prepare the supernatants, we inoculated 1 mL overnight cultures of ATCC 6538 or Mu 50 into 50 mL of fresh TSB media and incubated them at 37°C for 12 h in a 250 mL shake flask. The cultures were adjusted to the same cell density by normalizing their OD₆₀₀ nm values using TSB medium. Subsequently, the cultures were centrifuged at 9,000 rpm for 10 min at 4°C, and the resulting supernatants were filtered through a sterile 0.2 µm filter. To assess the response of the dual reporter to AIP-I or AIP-II-containing supernatants, we cultured S. aureus carrying the dual reporter plasmid in TSB medium at 37°C until an optical density of 1.0 was reached. The supernatants containing AIP-I or AIP-II, as well as TSB medium as a negative control, were added to the culture at a ratio of 1:4, and the culture was incubated for 3 h. After dilution with PBS at a ratio of 1:4, fluorescence and OD₆₀₀ were measured to quantify the activities of the agrP3 and sbi promoters, respectively.

Treatment of reporter strain with indole or flavone

The S. aureus ATCC 6538 strain carrying the dual reporter was cultured overnight and then inoculated into fresh TSB medium supplemented with 5 µg/mL chloramphenicol and various concentrations of indole or flavone. The culture was then incubated at 37°C with shaking at 220 rpm for 16 h. After diluting the culture with PBS at a ratio of 1:4, the activities of the agrP3 and sbi promoters were quantified using fluorescence and OD₆₀₀ measurements.

Real-time qPCR

Real-time qPCR assays were conducted on untreated S. aureus controls, as well as samples treated with 0.5 mM indole or 50 µg/mL flavone. Total RNA was purified using the RNAprep pure Cell/Bacteria Kit (TIANGEN BIOTECH Co., Ltd., Beijing, China). Complementary DNA (cDNA) was synthesized from the isolated total RNA utilizing HiScript III RT SuperMIX (Vazyme, Hangzhou, China). qPCRs were performed with the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Hangzhou, China) in 20 µL reaction volumes. Amplification efficiency and threshold cycle (Cq) values were derived via LinRegPCR (41). Normalization of target gene expression was accomplished using the geometric mean of the Cq values for two reference genes, rpoB and ftsZ (22). Relative quantification of mRNA was conducted via the efficiency-corrected ΔΔCq method (42). Each real-time qPCR assay was executed in triplicate to ensure experimental reliability.

Statistical analysis

The experimental data analysis was conducted using GraphPad Prism software (version 6.00, La Jolla, CA, USA). Statistical significance was determined by a P-value of less than 0.05. Error bars in line and bar graphs represent the standard deviation.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.00978-23.

Fig. S1, Tables S1 to S2. aem.00978-23-s0001.docx.

Bacterial strains, plasmids, and primers used in study; construction and complementation of agrA and saeA mutant strains.

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