Targeting bacterial virulence factors is now gaining interest as an alternative strategy to develop new types of anti-infective agents. It has been shown that thymol, when used at low concentrations, can inhibit the TSST-1 secretion in Staphylococcus aureus. However, there are no data on the effect of thymol on the production of other exotoxins (e.g., α-hemolysin and enterotoxins) by S. aureus. Secretion of α-hemolysin, SEA and SEB in both methicillin-sensitive and methicillin-resistant S. aureus isolates cultured with graded subinhibitory concentrations of thymol was detected by immunoblot analysis. Hemolysin and tumor necrosis factor (TNF) release assays were performed to elucidate the biological relevance of changes in α-hemolysin, SEA and SEB secretion induced by thymol. In addition, the influence of thymol on the transcription of hla, sea, and seb (the genes encoding α-hemolysin, SEA and SEB, respectively) was analyzed by quantitative RT-PCR. Thymol inhibited transcription of hla, sea and seb in S. aureus, resulting in a reduction of α-hemolysin, SEA and SEB secretion and, thus, a reduction in hemolytic and TNF-inducing activities. Subinhibitory concentrations of thymol decreased the production of α-hemolysin, SEA and SEB in both MSSA and MRSA in a dose-dependent manner. These data suggest that thymol may be useful for the treatment of S. aureus infections when used in combination with β-lactams and glycopeptide antibiotics, which induce expression of α-hemolysin and enterotoxins at subinhibitory concentrations. Furthermore, the structure of thymol may potentially be used as a basic structure for development of drugs aimed against these bacterial virulence factors.

Eugenol, an essential oil component in plants, has been demonstrated to possess activity against both Gram-positive and Gram-negative bacteria. This study examined the influence that subinhibitory concentrations of eugenol may have on the expression of the major exotoxins produced by Staphylococcus aureus . The results from a tumor necrosis factor (TNF) release assay and a hemolysin assay indicated that S. aureus cultured with graded subinhibitory concentrations of eugenol (16 to 128 μg/ml) dose dependently decreased the TNF-inducing and hemolytic activities of culture supernatants. Western blot analysis showed that eugenol significantly reduced the production of staphylococcal enterotoxin A (SEA), SEB, and toxic shock syndrome toxin 1 (the key exotoxins to induce TNF release), as well as the expression of α-hemolysin (the major hemolysin to cause hemolysis). In addition, this suppression was also evaluated at the transcriptional level via real-time reverse transcription (RT)-PCR analysis. The transcriptional analysis indicated that 128 μg/ml of eugenol remarkably repressed the transcription of the S. aureus sea , seb , tst , and hla genes. According to these results, eugenol has the potential to be rationally applied on food products as a novel food antimicrobial agent both to inhibit the growth of bacteria and to suppress the production of exotoxins by S. aureus .

The pathogenicity of staphylococcus aureus is dependent largely upon its ability to secrete a number of virulence factors, therefore, anti-virulence strategy to combat S. aureus-mediated infections is now gaining great interest. It is widely recognized that some plant essential oils could affect the production of staphylococcal exotoxins when used at subinhibitory concentrations. Perilla [Perilla frutescens (L.) Britton], a natural medicine found in eastern Asia, is primarily used as both a medicinal and culinary herb. Its essential oil (perilla oil) has been previously demonstrated to be active against S. aureus. However, there are no data on the influence of perilla oil on the production of S. aureus exotoxins. A broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of perilla oil against S. aureus strains. Hemolysis, tumour necrosis factor (TNF) release, Western blot, and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of perilla oil on exotoxins production in S. aureus. The data presented here show that perilla oil dose-dependently decreased the production of α-toxin, enterotoxins A and B (the major staphylococcal enterotoxins), and toxic shock syndrome toxin 1 (TSST-1) in both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). The production of α-toxin, SEA, SEB, and TSST-1 in S. aureus was decreased by perilla oil. These data suggest that perilla oil may be useful for the treatment of S. aureus infections when used in combination with β-lactam antibiotics, which can increase exotoxins production by S. aureus at subinhibitory concentrations. Furthermore, perilla oil could be rationally applied in food systems as a novel food preservative both to inhibit the growth of S. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.

Biofilm formation mediated by sortase A (srtA) is important for bacterial colonisation and resistance to antibiotics. Thus, the inhibitor of SrtA may represent a promising agent for bacterial infection. The structure of Streptococcus pneumoniae D39 srtA has been characterised by crystallisation. Site‐directed mutagenesis was used for the determination of the key residues for the activity of S. pneumoniae D39 srtA. An effective srtA inhibitor, quercetin, and its mechanism was further identified using srtA activity inhibition assay and molecular modelling. In this study, the crystal structure of S. pneumoniae D39 srtA has been solved and shown to contain a unique domain B. Additionally, its transpeptidase activity was evaluated in vitro. Based on the structure, we identified Cys207 as the catalytic residue, with His141 and Arg215 serving as binding sites for the peptide substrate. We found that quercetin can specifically compete with the natural substrate, leading to a significant decrease in the catalytic activity of this enzyme. In cells co‐cultured with this small molecule inhibitor, NanA cannot anchor to the cell wall effectively, and biofilm formation and biomass decrease significantly. Interestingly, when we supplemented cultures with sialic acid, a crucial signal for pneumococcal coloniation and the invasion of the host in the co‐culture system, biofilm loss did not occur. This result indicates that quercetin inhibits biofilm formation by affecting sialic acid production. In conclusion, the inhibition of pneumococcal srtA by the small molecule quercetin offers a novel strategy for pneumococcal preventative therapy.

Alpha-hemolysin (α-HL) is a self-assembling, channel-forming toxin produced by most Staphylococcus aureus strains as a 33.2-kDa soluble monomer. Upon binding to a susceptible cell membrane, the monomer self-assembles to form a 232.4-kDa heptamer that ultimately causes host cell lysis and death. Consequently, α-HL plays a significant role in the pathogenesis of S. aureus infections, such as pneumonia, mastitis, keratitis and arthritis. In this paper, experimental studies show that oroxylin A (ORO), a natural compound without anti-S. aureus activity, can inhibit the hemolytic activity of α-HL. Molecular dynamics simulations, free energy calculations, and mutagenesis assays were performed to understand the formation of the α-HL-ORO complex. This combined approach revealed that the catalytic mechanism of inhibition involves the direct binding of ORO to α-HL, which blocks the conformational transition of the critical “Loop” region of the α-HL protein thereby inhibiting its hemolytic activity. This mechanism was confirmed by experimental data obtained from a deoxycholate-induced oligomerization assay. It was also found that, in a co-culture system with S. aureus and human alveolar epithelial (A549) cells, ORO could protect against α-HL-mediated injury. These findings indicate that ORO hinders the lytic activity of α-HL through a novel mechanism, which should facilitate the design of new and more effective antibacterial agents against S. aureus. The mechanism controlling protein-ligand interactions is one of the most important processes in rational drug design. X-ray crystallography is a traditional tool used to investigate the interaction of ligands and proteins in a complex. However, protein crystallography is inefficient, and the development of crystal technology and research remains unequally distributed. Thus, it seems impractical to explore the structure of the α-hemolysin-ORO monomer complex by crystallography. Therefore, we used molecular dynamics simulations to investigate the receptor-ligand interaction in the α-HL-ORO monomer complex. In this study, we found that oroxylin A (ORO), a natural compound with little anti-S. aureus activity, can inhibit the hemolytic activity of α-HL at low concentrations. Through molecular docking and molecular dynamics simulations, we determined the potential binding mode of the protein-ligand interaction. The data revealed that ORO directly binds to α-HL, an interaction that blacks the conformational transition of the critical “Loop” region in α-HL and thus prevents the formation of the α-HL heptameric transmembrane pore, which ultimately inhibits the hemolytic activity of α-HL. This mechanism was confirmed by experimental data. Furthermore, we demonstrated that ORO could protect against α-HL-mediated injury in human alveolar epithelial (A549) cells.