Jiazhang Qiu

Subinhibitory Concentrations of Thymol Reduce Enterotoxins A and B and α-Hemolysin Production in Staphylococcus aureus Isolates

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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 Reduces the Expression of Virulence-Related Exoproteins in Staphylococcus aureus

ABSTRACT 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.

Subinhibitory Concentrations of Perilla Oil Affect the Expression of Secreted Virulence Factor Genes in Staphylococcus aureus

Background 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. Methodology/Principal Findings 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). Conclusions/Significance 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.

Baicalin protects mice from Staphylococcus aureus pneumonia via inhibition of the cytolytic activity of α-hemolysin.

α-Hemolysin (Hla) is a self-assembling, channel-forming toxin that is secreted by Staphylococcus aureus and is central to the pathogenesis of pulmonary, intraperitoneal, intramammary, and corneal infections in animal models. In this study, we report that baicalin (BAI), a natural compound that lacks anti-S. aureus activity, could inhibit the hemolytic activity of Hla. Using molecular dynamics simulations and mutagenesis assays, we further demonstrate that BAI binds to the binding sites of Y148, P151, and F153 in the Hla. This binding interaction inhibits heptamer formation. Furthermore, when added to S. aureus cultures, BAI prevents Hla-mediated human alveolar epithelial (A549) cell injury. In vivo studies further demonstrated that BAI protects mice from S. aureus pneumonia. These findings indicate that BAI hinders the cell lysis activity of Hla through a novel mechanism of interrupting the formation of heptamer, which may lead to the development of novel therapeutics that aim against S. aureus Hla.

Oroxylin A Inhibits Hemolysis via Hindering the Self-Assembly of α-Hemolysin Heptameric Transmembrane Pore

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.

Fisetin Inhibits Listeria monocytogenes Virulence by Interfering With the Oligomerization of Listeriolysin O

Listeriolysin O (LLO), an essential virulence determinant of Listeria monocytogenes, is a pore-forming toxin whose primary function is to facilitate cytosolic bacterial replication by breaching the phagosomal membranes, which is critical for the pathogen to evade host immune recognition. The critical role of LLO in the virulence of L. monocytogenes renders it an ideal target for designing novel antivirulence therapeutics. We found that fisetin, a natural flavonoid without antimicrobial activity, is a potent antagonist of LLO-mediated hemolysis. Fisetin effectively inhibits L. monocytogenes infection in both tissue culture and animal infection models. Molecular modeling and mutational analysis revealed that fisetin directly engages loop 2 and loop 3 of LLO, leading to the blockage of cholesterol binding and the reduction of its oligomerization, thus inhibiting its hemolytic activity. Our results establish fisetin as an effective antitoxin agent for LLO, which can be further developed into novel therapeutics against infections caused by L. monocytogenes.

Subinhibitory concentrations of licochalcone A decrease alpha-toxin production in both methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolates

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

Chrysin protects mice from Staphylococcus aureus pneumonia.

Aim:  To elucidate the effect of chrysin on α‐haemolysin production by Staphylococcus aureus and protection against pneumonia in a murine model.

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

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.

Glycyrrhetinic acid protects mice from Staphylococcus aureus pneumonia

In the present study, the antimicrobial activity of glycyrrhetinic acid (GA) against Staphylococcus aureus, and its influence on the production of S. aureus alpha-haemolysin (Hla) were investigated, along with the in vivo activity of GA against S. aureus-induced pneumonia. GA could not inhibit the growth of S. aureus, but the secretion of Hla by S. aureus was significantly inhibited by low concentrations of GA in a dose-dependent manner. Furthermore, in vivo data show that GA provides protection against staphylococcal pneumonia in a murine model system.