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Influence of subinhibitory concentrations of licochalcone A on the secretion of enterotoxins A and B by Staphylococcus aureus

Jiazhang Qiu, Haihua Feng, Hua Xiang, Dacheng Wang, Lijie Xia, Youshuai Jiang, Keji Song, Jing Lu, Lu Yu, Xuming Deng
DOI: http://dx.doi.org/10.1111/j.1574-6968.2010.01973.x 135-141 First published online: 1 June 2010

Abstract

Enterotoxins produced by Staphylococcus aureus are the key pathogenicity factors that can cause a variety of illnesses in humans, including staphylococcal gastroenteritis and food poisoning. It has been proven that licochalcone A is a potentially effective antimicrobial agent against S. aureus. In this study, Western blot assays, tumour necrosis factor release assays, murine T-cell proliferation assays, and real-time reverse transcriptase-PCR were performed to evaluate the effect of subinhibitory concentrations of licochalcone A on the secretion of two major enterotoxins (SEA and SEB) by S. aureus. The results show that licochalcone A significantly decreased, in a dose-dependent manner, the secretion of SEA and SEB by both methicillin-sensitive S. aureus and methicillin-resistant S. aureus. These results may increase the desirability of using licochalcone A as a lead compound for the design of more potent antibacterial agents based on the chalcone template.

Keywords
  • Staphylococcus aureus
  • licochalcone A
  • subinhibitory concentrations
  • enterotoxins

Introduction

Staphylococcus aureus is one of the most important community- and hospital-acquired pathogens, and it continues to cause a wide spectrum of serious diseases, including skin and soft tissue lesions, as well as lethal infections such as osteomyelitis, endocarditis, pneumonia, and septicaemia (Liang et al., 2006). Owing to the development of drug resistance, the morbidity and mortality associated with S. aureus infections remain high in spite of antimicrobial therapy (Kuroda et al., 2007). In addition, S. aureus secretes a number of exotoxins (e.g. haemolysins, enterotoxins, protein A, TSST-1, and coagulase) that contribute to a variety of diseases (Ohlsen et al., 1997). Exotoxins are produced by S. aureus in a growth-phase-dependent manner, primarily during the postexponential phase of growth (Arvidson & Tegmark, 2001). Furthermore, the expression of virulence factors is generally modulated in response to alternations in cell-population density through a process referred to as quorum sensing (Miller & Bassler, 2001).

Staphylococcal enterotoxins (SEs) are the major virulence factors that cause staphylococcal gastroenteritis and are one cause of food poisoning in humans (Tseng & Stewart, 2005; Bania et al., 2006). Moreover, these enterotoxins have the immunomodulatory properties of superantigens, stimulating T-cell activation and the release of T-cell-derived cytokines (Yoh et al., 2000). To date, a number of SEs have been identified, including SEA-E, SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, and SEO (Omoe et al., 2002). Although their exact mechanisms of action have not been fully elucidated, these enterotoxins are believed to stimulate an enteric-vagus nerve reflex, triggering the vomiting centres of the brain (Sears & Kaper, 1996).

Licochalcone A is one of the many flavonoids present in Chinese liquorice root, which has been used for centuries in traditional Chinese medicine. It has been demonstrated that licochalcone A possesses a variety of biological activities, including antimicrobial (Fukai et al., 2002), anti-inflammatory (Kwon et al., 2008), antiprotozoal (Chen et al., 2001), antitumour (Shibata, 2000), and antioxidative (Haraguchi et al., 1998) activities. Strikingly, previous studies have shown that licochalcone A was potent against methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA), with minimum inhibitory concentrations (MICs) ranging from 3 to 16 μg mL−1 depending on the strain (Hatano et al., 2000; Fukai et al., 2002). These results indicate that licochalcone A could be a potentially effective antimicrobial against S. aureus and could be used to treat patients infected with drug-resistant bacteria. Furthermore, in our previous study, we reported that subinhibitory concentrations of licochalcone A significantly decreased α-toxin production in both MSSA and MRSA isolates (Qiu et al., 2009). However, there were no data on enterotoxin secretion by S. aureus exposed to licochalcone A obtained in this study. The present study was aimed to investigate the influence of subinhibitory concentrations of licochalcone A on the production of enterotoxins A and B by S. aureus.

Materials and methods

Bacterial strains and reagents

The clinical isolate MRSA 2985 was isolated at the First Hospital of Jilin University from a blood sample from an infected patient. The MSSA ATCC 29213 isolate was obtained from the American Type Culture Collection (ATCC). Licochalcone A was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and stock solutions at various concentrations were prepared in dimethyl sulphoxide (DMSO) (Sigma-Aldrich, St. Louis, MO). The MIC in Mueller–Hinton broth (BD Biosciences Inc., Sparks, MD) was evaluated in triplicate using a broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (2005). The licochalcone A MIC values for S. aureus strain ATCC 29213 and MRSA strain 2985 were 4 μg mL−1. Furthermore, the MICs of the strains vs. licochalcone A in Luria-Bertani (LB) broth (BD Biosciences Inc.) were also 4 μg mL−1.

Growth curves

Staphylococcus aureus strain ATCC 29213 was grown to an OD600 nm value of 0.3 in LB, and 100-mL volumes of the culture were placed into five 250-mL Erlenmeyer flasks. Licochalcone A (dissolved in DMSO) was added to four of the cultures to obtain final concentrations of 1/16 × MIC (0.25 μg mL−1), 1/8 × MIC (0.5 μg mL−1), 1/4 × MIC (1 μg mL−1), and 1/2 × MIC (2 μg mL−1). The final DMSO concentration for all the conditions was 1‰ v/v. The control culture included 1‰ DMSO alone. Bacteria were further cultured at 37 °C with constant shaking under aerobic conditions, and cell growth was monitored by reading the OD600 nm values at 30-min intervals.

Western blot assay

Culture supernatants from postexponential growth-phase cultures (OD600 nm of 2.5) grown in LB with graded subinhibitory concentrations of licochalcone A were used for the determination of SEA and SEB concentrations. Western blot analysis was performed under the conditions described by Towbin (1979). Antibodies to SEA and SEB were purchased from Sigma-Aldrich.

Determination of proteolytic activity

The proteolytic activity analysis was performed as described by Edwards-Jones & Foster (2002). In brief, 100 μL of the supernatant from postexponential-phase (OD600 nm of 2.5) cultures was added to 1 mL of azocasein (Sigma-Aldrich) and incubated at 37 °C for 1 h. One millilitre of 5% w/v trichloroacetic acid was used to stop the reaction; undigested azocasein was allowed to precipitate for 30 min. The mixture was then centrifuged at 10 000 g for 10 min, and the A328 nm of the supernatant was read.

Tumour necrosis factor (TNF) release assay and murine T-cell proliferation assay

Preparation of bacterial supernatants

Overnight cultures of ATCC 29213 and MRSA 2985 in RPMI 1640 (Invitrogen, CA) were diluted 30-fold in 500 mL of prewarmed RPMI 1640. The diluted cultures were incubated for 30 min at 37 °C with constant shaking and then divided into aliquots of 100 mL. Graded concentrations of licochalcone A (1/16, 1/8, 1/4, and 1/2 × MIC) were added to the diluted bacterial suspensions before incubation for an additional 4 h. The final DMSO concentration for all the conditions was 1‰ v/v. The control culture included 1‰ DMSO alone. Staphylococcus aureus supernatants without antibiotic treatment served as controls. Proteins secreted into the supernatants were filtered through a 0.2-μm pore-size filter and were immediately analysed as described below.

Preparation of spleen cells

Specific pathogen-free BALB/c mice (male, 6–8 weeks old, weighing 18–22 g) were obtained from the Experimental Animal Center of Jilin University (Changchun, China). Animal experiments were approved by the Experimental Animal Center of Jilin University. All animal experiments were performed in accordance with the guidelines for the care and use of laboratory animals published by the US National Institutes of Health. Spleen cell suspensions were prepared in RPMI-1640, washed, and resuspended in a complete RPMI-1640 medium (RPMI 1640 medium supplemented with 10% foetal bovine serum, 2 mM glutamine, penicillin 100 IU mL−1, streptomycin 100 IU mL−1, 15 mM HEPES, and 50 μM 2-mercaptoethanol). A total of 106 (150 μL) cells were dispensed into wells of a 96-well tissue culture plate.

Enzyme-linked immunosorbent assay (ELISA)

Staphylococcus aureus culture supernatants (50 μL) were added to the tissue culture plate. After incubation for 16 h, the supernatants were collected and centrifuged (1000 g for 5 min), and then the TNF level in the supernatants was determined using the Mouse TNF-α ELISA MAX Set Standard (Biolegend Inc., San Diego, CA) according to the instructions of the manufacturer.

MTT assay

Cell proliferation was determined by an MTT assay as described previously (Wang et al., 2009). Staphylococcus aureus culture supernatants (50 μL) were added to the tissue culture plate described above. After incubation at 37 °C with 5% CO2 for 72 h, 20 μL of 5 mg mL−1 MTT dissolved in PBS was added to each well, and the plate was incubated at 37 °C for 4 h. The cells were collected by centrifugation for 10 min at 500 g. The pellet was redissolved in 150 μL DMSO at room temperature for 10 min, and the OD570 nm was measured using a microplate reader (Tecan, Austria). The viability and number of splenocytes are represented by the OD570 nm value.

Real-time reverse transcriptase (RT)-PCR

Strain ATCC 29213 was cultured in LB at 37 °C with graded subinhibitory concentrations of licochalcone A to the postexponential growth phase (t=240 min). RNA was isolated as described by Qiu (2009). Briefly, cells were collected by centrifugation (5000 g for 5 min at 4 °C) and resuspended in TES buffer (10 mM Tris-HCl, 1 mM EDTA, 0.5% SDS) including 100 μg mL−1 lysostaphin (Sigma-Aldrich). Following incubation at 37 °C for 10 min, a Qiagen RNeasy Maxi column was used to isolate total bacterial RNA in accordance with the manufacturer's directions. The optional on-column RNAse-free DNAse I (Qiagen, Hilden, Germany) treatment was carried out to remove contaminating DNA. After isolation of RNA, traces of contaminating DNA were further eliminated by treating RNA samples with RNAse-free DNAse I (Ambion, Austin, TX) at 37 °C for 20 min. RNA concentrations were determined from the OD260 nm, and the RNA was run on an RNAse-free 2% agarose gel to test for generalized degradation. The primer pairs used in real-time RT-PCR are shown in Table 1. RNA was reverse transcribed into cDNA using the Takara RNA PCR kit (AMV) Ver. 3.0 (Takara, Kyoto, Japan), in accordance with the manufacturer's directions; cDNA was stored at −20 °C until needed. The PCR reactions were performed in a 25-μL final volume and contained SYBR Premix Ex Taq (Takara), as recommended by the manufacturer. The reactions were carried out using the 7000 Sequence Detection System (Applied Biosystems, Courtaboeuf, France). Cycling parameters were as follows: 95 °C for 30 s; 45 cycles at 95 °C for 5 s, 54 °C for 30 s, and 72 °C for 20 s, and one dissociation step of 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s. All samples were analysed in triplicate, and the 16S rRNA gene was used as an internal control housekeeping gene to normalize the levels of expression between samples. The real-time RT-PCR data were analysed using the Embedded Image method described in Applied Biosystems, User Bulletin no. 2.

View this table:
Table 1

Primers used in real-time RT-PCR

PrimerSequenceLocation within gene
16S rRNA gene-forward5′-GCTGCCCTTTGTATTGTC-3′287–305
16S rRNA gene-reverse5′-AGATGTTGGGTTAAGTCCC-3′446–465
sea-forward5′-ATGGTGCTTATTATGGTTATC-3′335–356
sea-reverse5′-CGTTTCCAAAGGTACTGTATT-3′477–498
seb-forward5′-TGTTCGGGTATTTGAAGATGG-3′480–501
seb-reverse5′-CGTTTCATAAGGCGAGTTGTT-3′612–633
agrA-forward5′-TGATAATCCTTATGAGGTGCTT-3′111–133
agrA-reverse5′-CACTGTGACTCGTAACGAAAA-3′253–274

Statistical analysis

Experimental data were analysed using spss 12.0 statistical software. Data were expressed as the mean±SD. Statistical differences were examined using an independent Student's t-test. A P-value of <0.05 was considered statistically significant.

Results

Influence of subinhibitory concentrations of licochalcone A on S. aureus growth

The growth curve of S. aureus ATCC 29213 is shown in Fig. 1. We found that 1/16 × MIC, 1/8 × MIC, and 1/4 × MIC of licochalcone A had no obvious effects on the growth of S. aureus. Although S. aureus grew in the presence of 1/2 × MIC of licochalcone A, the growth velocity was much slower, and after 30 min, the OD value was only 51.5% of that of the control culture. However, after 360 min of licochalcone A treatment, there was no significant difference in the OD value among all the cultures.

Figure 1

Growth curve for Staphylococcus aureus strain ATCC 29213 in the presence or absence of licochalcone A. ◻, untreated S. aureus; ▪, S. aureus plus 0.25 μg mL−1 licochalcone A; ▲, S. aureus plus 0.5 μg mL−1 licochalcone A; ○, S. aureus plus 1 μg mL−1 licochalcone A; and +, S. aureus plus 2 μg mL−1 licochalcone A.

Effects of licochalcone A on SEA and SEB secretion by S. aureus

The secretion of two major enterotoxins (SEA and SEB) by S. aureus, when exposed to subinhibitory concentrations of licochalcone A, was analysed in the study; both MSSA ATCC 29213 and MRSA strain 2985 were investigated. As shown in Fig. 2, the addition of licochalcone A reduced the secretion of SEA and SEB in a dose-dependent manner. Growth in the presence of 1/16 × MIC licochalcone A led to a measurable reduction in SEA and SEB secretion; at 1/2 × MIC, no immunoreactive protein could be detected in cultures of ATCC 29213 and MRSA 2985.

Figure 2

Western blot analysis of SEA and SEB production by strain ATCC 29213 (a) and MRSA strain 2985 (b) after growth with graded subinhibitory concentrations of licochalcone A. Supernatants were subjected to SDS-PAGE. After transfer to polyvinylidene fluoride membranes, proteins were stained with the indicated antibodies against SEA and SEB. A horseradish peroxidase-conjugated goat anti-rabbit antiserum was used as the secondary antibody, and the blots were developed using the ECL substrate (GE Healthcare, UK).

The proteolytic activity of the cultures was determined to confirm whether the reduction of SEA and SEB secretion by S. aureus was due to an increase in protease secretion induced by licochalcone A. There was no significant effect on protease secretion by ATCC 29213 or MRSA 2985 cultured with 1/2 × MIC of licochalcone A (data not shown).

Licochalcone A reduces TNF-α release and proliferation of T cell caused by S. aureus supernatants

It is well known that among the proteins released, enterotoxins are the most important exotoxins secreted by S. aureus that could act as superantigens, stimulating T cells to release proinflammatory cytokines and stimulating T-cell proliferation (Balaban & Rasooly, 2000). Therefore, in this study, a TNF release assay and a murine T-cell proliferation assay were performed to clarify the biological relevance of the reduction in SEA and SEB secretion caused by licochalcone A. As expected, the culture supernatants of S. aureus grown in the presence of graded subinhibitory concentrations of licochalcone A elicited much lower TNF-α production by spleen cells (Fig. 3) and stimulated a significantly lower level of T-cell proliferation (Fig. 4). In addition, licochalcone A itself did not induce TNF release or stimulate T-cell activation at 1 × MIC or 2 × MIC concentrations. Apparently, licochalcone A reduced the TNF-inducing and T-cell-activating activities in a dose-dependent manner.

Figure 3

TNF release from splenocytes stimulated with the supernatants of Staphylococcus aureus grown in the presence of graded subinhibitory concentrations of licochalcone A in RPMI 1640. Values represent the mean±SD for three independent experiments. *P <0.05, **P <0.01.

Figure 4

Induction of murine splenocyte proliferation by Staphylococcus aureus supernatants. Staphylococcus aureus strains were cultured with graded subinhibitory concentrations of licochalcone A in RPMI 1640. Values represent the mean±SD for three independent experiments. *P <0.05, **P <0.01.

Licochalcone A diminishes the transcription of sea, seb, and agrA in S. aureus

Real-time RT-PCR was performed to evaluate the transcriptional level of sea, seb, and agrA after treatment with subinhibitory concentrations of licochalcone A. As shown in Fig. 5, licochalcone A markedly decreased the transcription of sea, seb, and agrA in S. aureus strains ATCC 29213. When cultured with 1/2 × MIC of licochalcone A, the transcriptional levels of sea, seb, and agrA in strain ATCC 29213 were decreased by 6.2-, 7.6-, and 4.2-fold, respectively. The investigated genes were affected by licochalcone A at the transcriptional level in a dose-dependent manner.

Figure 5

Relative gene expression of sea, seb, and agrA in Staphylococcus aureus strain ATCC 29213 after growth with subinhibitory concentrations of licochalcone A. Values represent the mean and SE of three independent experiments. *P <0.05, **P <0.01.

Discussion

The continued emergence of multiple-antibiotic-resistant S. aureus isolates originating from community and nosocomial sources necessitates the development of new and improved antimicrobial agents for the prevention and treatment of these life-threatening infections (Hall et al., 2003). To date, many studies have focused on naturally occurring compounds (Smith-Palmer et al., 2004). Our previous research has shown that the MICs of licochalcone A against 27 S. aureus strains ranged from 2 to 8 μg mL−1(Qiu et al., 2009). It is uncommon for compounds isolated from medical plants to have such powerful antimicrobial activities on both MSSA and MRSA. Consequently, licochalcone A may potentially be used as a lead compound for the design of more potent antibacterial agents (based on the chalcone template) to be used to fight drug-resistant S. aureus strains.

On the other hand, an alternative strategy that is now gaining interest to treat with S. aureus infections is the targeting of bacterial virulence factors (e.g. haemolysins, enterotoxins, adhesins) (Song et al., 2009). A number of virulence factors secreted by S. aureus play a significant role in pathogenesis. Therefore, the clinical performance of antibiotics used for the treatment of S. aureus infections not only depends on the respective bacteriostatic or bactericidal effects but also on the ability to prevent the release of virulence factors by dying or stressed bacteria (Bernardo et al., 2004). Previous studies have indicated that enterotoxins secreted by S. aureus are affected by many antibiotics, especially at suboptimal concentrations. Protein synthesis inhibitors such as linezolid can reduce the expression of S. aureus virulence factors including enterotoxins A and B at subgrowth-inhibitory concentrations (Bernardo et al., 2004). In contrast, β-lactam antibiotics induce or increase enterotoxin production, suggesting that the symptoms of S. aureus infections, especially MRSA infections, may be aggravated when patients are treated with these antibiotics (Stevens et al., 2007). Furthermore, it has been shown that some plant compounds (e.g. oleuropein) and plant essential oils (e.g. oils of bay, cinnamon, and clove) can also influence the production of enterotoxins when used at subinhibitory concentrations (Tranter et al., 1993; Smith-Palmer et al., 2004). The antibiotic-induced regulation of virulence factors may result in either aggravation or attenuation of the disease. Therefore, the up- or downregulation of toxin secretion is significant for diseases caused by S. aureus, and the ability of antibiotics to affect these properties may be an important criterion in selecting an antibiotic for therapy (Blickwede et al., 2005). In this study, licochalcone A was shown by Western blot assay, TNF release assay, murine T-cell proliferation assay, and real-time RT-PCR to repress SEA and SEB secretion by S. aureus in a dose-dependent manner.

The expression of most virulence factors by S. aureus is regulated by a network of interacting regulators, such as agr, sar, and sae (Goerke et al., 2001). Previous research has indicated that subinhibitory concentrations of antibiotics may interfere with the translation of one or more regulatory gene products in S. aureus and may thereby affect transcription of the exoprotein-encoding genes. For example, subinhibitory concentrations of clindamycin differentially inhibit the transcription of exoprotein genes in S. aureus and act partly through sar (Herbert et al., 2001). Additionally, subinhibitory concentrations of β-lactams induce haemolytic activity in S. aureus through the SaeRS two-component system (Kuroda et al., 2007). In the study, real-time RT-PCR was performed to investigate the influence of licochalcone A on the agr locus of S. aureus. Our results showed that licochalcone A significantly inhibited agrA transcription. However, the mechanisms by which S. aureus controls virulence gene expression are fairly intricate and involve an interactive, hierarchical regulatory cascade among the products of the sar, agr, and other components (Chan & Foster, 1998). Accordingly, we may infer that the reduction of SEA and SEB in S. aureus in the presence of licochalcone A may, in part, originate from the inhibition of the Agr two-component system.

In conclusion, considering the potent antimicrobial activities of licochalcone A on S. aureus, the influence of licochalcone A on α-toxin secretion, as well as the findings in the present study that licochalcone A significantly reduces the production of key pathogenicity factors by S. aureus, namely the enterotoxins A and B, licochalcone A may potentially be used in the food or the pharmaceutical industries.

Acknowledgement

The study was supported by a grant from the 973 programme of China (2006CB504402).

Footnotes

  • Editor: Stefan Schwarz

References

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