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Keywords:

  • agr;
  • alpha-toxin;
  • haemolysis;
  • licochalcone A;
  • Staphylococcus aureus

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Aim:  To evaluate the effect of subinhibitory concentrations of licochalcone A (LicA) on alpha-toxin secretion in Staphylococcus aureus.

Methods and Results:  A haemolysin assay was used to investigate the haemolytic activities in culture supernatants of both methicillin-sensitive and methicillin-resistant Staph. aureus isolates cultured with graded subinhibitory concentrations of LicA. Alpha-toxin secretion was detected by immunoblot analysis. Moreover, quantitative RT-PCR was performed to assess the influence of LicA on the transcription of hla (the gene encoding alpha-toxin) and agr (accessory gene regulator). Growth in the presence of LicA markedly inhibited the mRNA levels of hla and agr in Staph. aureus, resulting in a reduction of alpha-toxin secretion and, thus, haemolytic activities.

Conclusion:  The secretion of alpha-toxin in Staph. aureus is decreased by LicA; this effect may be partially dependent upon inhibition of the Agr two-component system.

Significance and Impact of the Study:  The findings in our study may support the use of LicA as a lead compound in the design of more potent antibacterial agents that are based on the chalcone template.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Staphylococcus aureus is a significant community-acquired and nosocomially acquired pathogen that can cause both local and systemic infections in humans (Li and Cheung 2008). Staphylococcus aureus is also the predominant cause of intramammary infection in dairy cattle, sheep and goats and contributes to considerable economic loss (Wang et al. 2008). Owing to the development of resistance to virtually all antibiotics, the morbidity and mortality associated with Staph. aureus infections remain high in spite of antimicrobial therapy (Kuroda et al. 2007). In addition to antimicrobial resistance, Staph. aureus produces numerous extracellular and cell-associated proteins that are involved in pathogenesis, such as enterotoxins, alpha-toxin, protein A and toxic shock syndrome toxin (Ohlsen et al. 1997).

Previous reports have shown that alpha-toxin (encoded by the hla gene) is one of the major virulence factors secreted by most Staph. aureus strains (Bhakdi and Tranum-Jensen 1991). However, like most staphylococcal exoproteins, alpha-toxin is not expressed persistently but is primarily secreted during the postexponential growth phase (Ohlsen et al. 1997). The alpha-toxin is a 33-kDa pore-forming protein that has cytolytic, haemolytic and dermonecrotic activities. The toxin can lyse a wide range of human cells, including erythrocytes, monocytes, lymphocytes, macrophages and epithelial cells.

Most of the virulence factors produced by Staph. aureus are regulated by a network of interacting regulators. Such regulatory systems include the SarA protein family (Oscarsson et al. 2006) and numerous two-component regulatory systems, such as agr (Peng et al. 1988) and sae (Giraudo et al. 1997). Furthermore, the expression of virulence factors is also influenced by some antibiotics (Ohlsen et al. 1998).

The Agr two-component system is one of the best-investigated global regulatory systems and contributes to the postexponential growth phase regulation of many virulence determinants in Staph. aureus (Ohlsen et al. 1997). Similar to most secreted exoproteins in Staph. aureus, the expression of alpha-toxin (hla) is positively regulated by agr (Recsei et al. 1986). The agr locus consists of two transcriptional units, RNAII and RNAIII, transcribed from the divergent promoters, P2 and P3, respectively. The RNAII transcript encodes four proteins: AgrB, AgrD, AgrC and AgrA (Novick et al. 1995). AgrD produces an octapeptide with quorum sensing properties (autoinducing peptide) with the aid of AgrB. When the threshold concentration is reached, the octapeptide binds to AgrC, resulting in the activation of AgrA, the response regulator (Ji et al. 1995). Presumably, activated AgrA promotes the transcription of RNAIII, the effector molecule of the agr response, which ultimately interacts with target genes to regulate transcription (Novick et al. 1995).

Licochalcone A (LicA), a major phenolic constituent of the licorice species, Glycyrrhiza inflata, has been reported to possess various biological activities, e.g. antiprotozoal (Chen et al. 2001), anti-inflammatory (Kwon et al. 2008), antitumour (Shibata 2000), antioxidative (Haraguchi et al. 1998) and antimicrobial (Tsukiyama et al. 2002) activities. Notably, previous studies have shown that LicA was active against methicillin-sensitive Staph. aureus (MSSA) and methicillin-resistant Staph. 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; Tsukiyama et al. 2002; Liu et al. 2008).

The objective of the present study was to investigate the effect of subinhibitory concentrations of LicA on alpha-toxin expression by MSSA and MRSA and to verify, via quantitative RT-PCR, whether the effect was correlated with the accessory gene regulator.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Bacterial strains and reagents

The MSSA strain ATCC 29213 was obtained from the China Medical Culture Collection Centre. Twenty-five clinical Staph. aureus isolates (16 MSSA and 9 MRSA) and the MRSA strain 2985 were isolated at the First Hospital of Jilin University from blood samples of infected patients. Mueller–Hinton broth II (MHB II) was purchased from BD Biosciences, Inc. (Sparks, MD, USA). LicA was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and stock solutions of various concentrations were made in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO, USA).

Antibiotic MIC

MICs of LicA for Staph. aureus were evaluated in triplicate by a broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI 2005). The MICs were defined as the lowest concentration at which no visible growth was observed.

Growth curves

Staphylococcus aureus strain ATCC 29213 was grown to an opticl density (OD) of 0·3 at 600 nm in MHB II, and 100 ml volumes were aliquoted into five 500-ml Erlenmeyer flasks. LicA (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) or 1/2× MIC (2 μg ml−1). The final DMSO concentration for all conditions was 1% (v/v). The control culture included the addition of 1% DMSO alone. Bacteria were further cultured at 37°C with constant shaking under aerobic conditions, and cell growth was monitored spectrophotometrically (OD600 nm at 30 min intervals). Three millilitre samples of each culture were collected immediately after the addition of LicA (t0), and after 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 and 360 min.

Haemolysin assay

Bacteria were cultured in the presence of graded subinhibitory concentrations of LicA to the postexponential growth phase (OD600 nm of 2·5). Alpha-toxin levels in bacterial culture supernatants were determined based on the method of Rowe and Welch (1994). Bacterial samples (0·5 ml) were centrifuged (5500 g, 4°C, 1 min), the supernatant was removed and 0·1 ml of supernatant was brought up to 1 ml in haemolysin buffer (0·145 mol l−1 NaCl, 0·02 mol l−1 CaCl2) prior to the addition of 25 μl of defibrinated rabbit blood. After incubation for 15 min at 37°C, the samples were centrifuged (5500 g, room temperature, 1 min), and the ODs of the supernatants were measured at 543 nm.

Immunoblot analysis of alpha-toxin

The bacterial culture supernatants described earlier were also used for immunoblot analysis of alpha-toxin production by Staph. aureus. SDS-PAGE was performed as described by Laemmli (1970) with discontinuous 12·5% acrylamide gels. For immunoblot analysis, proteins were transferred to polyvinylidene fluoride membranes (Wako Pure Chemical Industries, Ltd, Osaka, Japan) using a semidry transfer cell (Bio-Rad, Munich, Germany). Following blotting of the membranes, blocking was performed with 5% bovine serum albumin (Wako) in phosphate-buffered saline (PBS) for 2 h. The filters were then incubated for 1 h with a polyclonal anti-alpha-toxin antibody (Sigma-Aldrich) in PBS containing 0·05% Tween-20 followed by 0·5 h of incubation with horseradish peroxidase-conjugated anti-rabbit antiserum (Sigma-Aldrich) diluted to 1 : 4000. The blots were developed using the ECL substrate (GE Healthcare, Buckinghamshire, UK).

Determination of proteolytic activity

A 100-μl volume of supernatant from the 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. Then, 1 ml 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 absorbance of the supernatant was read at 328 nm.

Quantitative RT-PCR

Strain ATCC 29213 was grown in MHB II with graded subinhibitory concentrations of LicA to the postexponential growth phase (t = 300 min). The RNA was prepared as described by Sambanthamoorthy et al. (2006). Briefly, cells were harvested by centrifugation (5000 g for 5 min at 4°C) and resuspended in TES buffer (10 mmol l−1 Tris-Cl, 1 mmol l−1 EDTA, 0.5% SDS) containing 100 μg ml−1 lysostaphin (Sigma-Aldrich). The samples were incubated at 37°C for 10 min and were then applied to a Qiagen RNeasy Maxi column to isolate total bacterial RNA according to the manufacturer’s directions. The optional on-column RNase-free DNase I (Qiagen, Hilden, Germany) treatment was performed 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, USA) and incubating them at 37°C for 20 min. Samples were used immediately or stored at −80°C. The quality, integrity and concentration of the RNAs were determined by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) as described by the manufacturer. The primer pairs used in quantitative RT-PCR are listed in Table 1. RNA was reverse transcribed into cDNA using the Takara RNA PCR kit (AMV) Ver. 3.0 (Takara, Kyoto, Japan) according to the manufacturer’s instructions, and cDNA was stored at −20°C until needed. The PCRs were performed in 25 μl total volume and contained SYBR Premix Ex Taq™ (Takara) as recommended by the manufacturer. The reactions were performed using the 7000 Sequence Detection System (Applied Biosystems, Courtaboeuf, France). Cycling conditions were as follows: one cycle at 95°C for 30 s; 40 cycles at 95°C for 5 s, 55°C for 30 s and 72°C for 40 s; and a 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 normalized against 16S rRNA gene expression. Relative expression levels were determined by the (ΔΔCT) method (Applied Biosystems User Bulletin no. 2 1997).

Table 1.   Primers used for quantitative RT-PCR in the study
PrimerN315 ORF*Sequence
  1. *ORF, open reading frame.

16S rRNA senseSArRNA015′-CGTGCTACAATGGACAATACAAA-3′
16S rRNA antisenseSArRNA015′-ATCTACGATTACTAGCGATTCCA-3′
hla senseSA10075′-TGAATCCTGTCGCTAATG-3′
hla antisenseSA10075′-TATCATCCGACCTTTCACT-3′
agrA senseSA18445′-TGAAATTCGTAAGCATGACCC-3′
agrA antisenseSA18445′-CATCGCTGCAACTTTGTAGAC-3′

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Growth of Staphylococcus aureus in the presence of subinhibitory concentrations of LicA

The MICs of LicA against 27 Staph. aureus strains ranged from 2 to 8 μg ml−1. The MIC values of Staph. aureus strain ATCC 29213 and MRSA strain 2985 vs LicA were 4 μg ml−1. These results were consistent with previous studies (Fukai et al. 2002; Tsukiyama et al. 2002; Liu et al. 2008), indicating that LicA is a potentially effective antimicrobial agent against Staph. aureus. The growth curves of Staph. aureus ATCC 29213 are shown in Fig. 1, wherein we found that 1/16×, 1/8× and 1/4× MIC of LicA had no obvious effects on the growth of Staph. aureus. However, while Staph. aureus grew in the presence of a 1/2× MIC of LicA, the growth velocity was attenuated remarkably, after 30 min, the OD value was c. 50% of the untreated culture.

image

Figure 1.  Growth curve for Staphylococcus aureus strain ATCC 29213 in the presence or absence of licochalcone A (LicA). (□) untreated Staph. aureus; (bsl00001) Staph. aureus plus 0·25 μg ml−1 LicA; (bsl00066) Staph. aureus plus 0·5 μg ml−1 LicA; (○) Staph. aureus plus 1 μg ml−1 LicA and (+) Staph. aureus plus 2 μg ml−1 LicA.

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LicA attenuated haemolysis of Staphylococcus aureus by reducing the level of alpha-toxin secretion

Both strain ATCC 29213 and MRSA strain 2985 were cultured with graded subinhibitory concentrations of LicA, and the bacterial culture supernatants were used for a haemolysin assay. A 95% reduction was found with ATCC 29213, and for MRSA strain 2985, reduction was nearly 93% when cultured with 1/2× MIC of LicA (Fig. 2). As expected, a dose-dependent attenuation of haemolysis was observed in both strains. In addition, LicA itself did not cause haemolysis of rabbit erythrocytes at 1× or 2× MIC concentrations, and there was little effect on the haemolytic activity of culture supernatants when pre-incubated with a 2× MIC of LicA (data not shown).

image

Figure 2.  Haemolysis of rabbit erythrocytes by culture supernatants of strain ATCC 29213 (a) and methicillin-resistant Staphylococcus aureus strain 2985 (b) in the absence or presence of graded concentrations of licochalcone A (LicA). Values represent the mean ± SD for three independent experiments. Student’s paired t-test was used to compare each culture with LicA to the untreated culture (*P < 0·05; **P < 0·01).

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The supernatants were also used for immunoblot analysis to detect the secretion of alpha-toxin. As shown in Fig. 3, LicA reduced, in a dose-dependent manner, the secretion of alpha-toxin. Growth in the presence of 1/16× MIC of LicA led to a recognizable reduction in alpha-toxin secretion; at 1/2× MIC, no immunoreactive protein could be detected in strains ATCC 29213 and MRSA 2985.

image

Figure 3.  Immunoblot analysis of alpha-toxin production by strain ATCC 29213 (a) and methicillin-resistant Staphylococcus aureus strain 2985 (b) after growth with different subinhibitory concentrations of licochalcone A.

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The proteolytic activity was determined to confirm whether the reduction of alpha-toxin secretion in Staph. aureus was because of the increase in protease secretion induced by LicA. There was no significant effect on protease secretion by ATCC 29213 or MRSA 2985 cultured with 1/2× MIC of LicA (data not shown).

LicA diminished the transcription of hla and agrA in Staphylococcus aureus

Quantitative RT-PCR was performed to detect the transcriptional level of alpha-toxin gene (hla). Because the hla gene is positively regulated by the Agr two-component system (Recsei et al. 1986), the transcription of agrA was also investigated. As shown in Fig. 4, a remarkable reduction of hla and agrA transcription was observed in the strains analysed. When cultured with 1/2× MIC of LicA, the transcription levels of hla and agrA were decreased by 4·2- and 3·4-fold, respectively. Both genes were affected by LicA at the transcriptional level in a dose-dependent manner.

image

Figure 4.  Relative expression levels of hla and agrA in Staphylococcus aureus with different subinhibitory concentrations of licochalcone A (LicA). Values represent the mean ± SD for three independent experiments. Student’s paired t-test was used to compare each culture with LicA to the untreated culture (*P < 0·05).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Licorice root, derived from the plant Glycyrrhiza glabra or Glycyrrhiza radix, has been used for centuries in traditional Chinese and Indian medicine for the treatment of pulmonary diseases, rheumatic and other pain, gastric ulcers and inflammatory processes because of its wide-ranging therapeutic properties (Shibata 2000; Baltina 2003). Presently, licorice root is widely used in the tobacco, food and pharmaceutical industries (Tsukiyama et al. 2002). The research on licorice constituents has revealed the pharmacological importance of phenolic compounds (Shibata 1994; Nomura and Fukai 1998; Liu et al. 2008). LicA, the most abundant phenolic component of licorice, was originally isolated by Saitoh (Saitoh and Shibata 1975). Subsequent studies resulted in reports of a variety of biological effects of LicA (Shibata 2000; Kwon et al. 2008; Liu et al. 2008).

A number of virulence factors secreted by Staph. aureus play an important role in pathogenesis. Therefore, the clinical performance of antibiotics used for the treatment of Staph. aureus infections not only depends on the respective bacteriostatic or bactericidal effects, but also on the ability to prevent virulence factor release by dying or stressed bacteria (Bernardo et al. 2004). It has long been known that the expression of alpha-toxin by Staph. aureus was affected by subinhibitory concentrations of many antibiotics (Kernodle et al. 1995; Ohlsen et al. 1998; Worlitzsch et al. 2001). Some β-lactam and fluoroquinolone antibiotics enhance the production of alpha-toxin by stimulating hla expression (Ohlsen et al. 1998), suggesting that the symptoms of Staph. aureus infections may be aggravated when patients are treated with these antibiotics. In contrast, clindamycin, erythromycin and several aminoglycosides significantly diminish alpha-toxin secretion by inhibiting the expression of the gene coding for the protein (Ohlsen et al. 1998; Herbert et al. 2001). Furthermore, (−)-Epicatechin gallate (ECg) disrupts secretion of alpha-toxin by Staph. aureus, which indicates that ECg may be useful for the treatment of Staph. aureus infections when used in combination with β-lactam antibiotics (Shah et al. 2008). In this study, LicA has been shown by haemolysin assays, immunoblot analysis and quantitative RT-PCR to repress alpha-toxin secretion by Staph. aureus.

Previous studies have indicated that subinhibitory concentrations of antibiotics may interfere with translation of one or more regulatory gene products in Staph. aureus and thereby may affect transcription of the exoprotein-encoding genes (Herbert et al. 2001; Kuroda et al. 2007). For example, subinhibitory clindamycin concentrations differentially inhibit the transcription of exoprotein genes in Staph. aureus and act partly through sar (Herbert et al. 2001); subinhibitory concentrations of β-lactams induce haemolytic activity in Staph. aureus through the SaeRS two-component system (Kuroda et al. 2007). Accordingly, in this report, we performed quantitative RT-PCR to investigate the influence of LicA on the agr locus of Staph. aureus. Our results showed that LicA noticeably inhibited agrA transcription. However, the mechanisms by which Staph. 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 and Foster 1998). Therefore, we presume that the effect of LicA on alpha-toxin production may partially depend on inhibition of Agr two-component system.

It is uncommon that compounds isolated from medical plants have such powerful antimicrobial activities on both MSSA and MRSA, as does LicA. In addition to this, LicA can decrease alpha-toxin secretion. Therefore, it is promising that LicA could play an important role in the treatment of Staph. aureus infections. More detailed studies should be performed to reveal the bactericidal mechanism(s) of LicA on Staph. aureus. Significantly, LicA 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 Staph. aureus strains.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This work was supported by the National Nature Science Foundation of China (nos. 30871889, 30972212).

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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