Xiaodi Niu

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.

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.

Morin hydrate attenuates Staphylococcus aureus virulence by inhibiting the self-assembly of α-hemolysin.

To investigate the mechanism by which morin hydrate inhibits the haemolytic activity of α‐hemolysin (Hla), a channel‐forming toxin that is important for the pathogenesis of disease in experimental animals, and its therapeutic effect against Staphylococcus aureus pneumonia in a mouse model.

Allicin Reduces the Production of α-Toxin by Staphylococcus aureus

Staphylococcus aureus causes a broad range of life-threatening diseases in humans. The pathogenicity of this micro-organism is largely dependent upon its virulence factors. One of the most extensively studied virulence factors is the extracellular protein α-toxin. In this study, we show that allicin, an organosulfur compound, was active against S. aureus with MICs ranged from 32 to 64 μg/mL. Haemolysis, Western blot and real-time RT-PCR assays were used to evaluate the effects of allicin on S. aureus α-toxin production and on the levels of gene expression, respectively. The results of our study indicated that sub-inhibitory concentrations of allicin decreased the production of α-toxin in both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in a dose-dependent manner. Furthermore, the transcriptional levels of agr (accessory gene regulator) in S. aureus were inhibited by allicin. Therefore, allicin may be useful in the treatment of α-toxin-producing S. aureus infections.

Capsaicin Protects Mice from Community-Associated Methicillin-Resistant Staphylococcus aureus Pneumonia

Background α-toxin is one of the major virulence factors secreted by most Staphylococcus aureus strains, which played a central role in the pathogenesis of S. aureus pneumonia. The aim of this study was to investigate the impact of capsaicin on the production of α-toxin by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain USA 300 and to further assess its performance in the treatment of CA-MRSA pneumonia in a mouse model. Methodology/Principal Findings The in vitro effects of capsaicin on α-toxin production by S. aureus USA 300 were determined using hemolysis, western blot, and real-time RT-PCR assays. The influence of capsaicin on the α-toxin-mediated injury of human alveolar epithelial cells was determined using viability and cytotoxicity assays. Mice were infected intranasally with S. aureus USA300; the in vivo protective effects of capsaicin against S. aureus pneumonia were assessed by monitoring the mortality, histopathological changes and cytokine levels. Low concentrations of capsaicin substantially decreased the production of α-toxin by S. aureus USA 300 without affecting the bacterial viability. The addition of capsaicin prevented α-toxin-mediated human alveolar cell (A549) injury in co-culture with S. aureus. Furthermore, the in vivo experiments indicated that capsaicin protected mice from CA-MRSA pneumonia caused by strain USA 300. Conclusions/Significance Capsaicin inhibits the production of α-toxin by CA-MRSA strain USA 300 in vitro and protects mice from CA-MRSA pneumonia in vivo. However, the results need further confirmation with other CA-MRSA lineages. This study supports the views of anti-virulence as a new antibacterial approach for chemotherapy.

β-sitosterol interacts with pneumolysin to prevent Streptococcus pneumoniae infection.

Pneumolysin is one of the major virulence factors elaborated by Streptococcus pneumoniae; this toxin is a member of the cholesterol-dependent cytolysins. Engagement of cholesterol induces the formation of a multi-subunit complex by pneumolysin that lyses host cells by forming pores on the membrane. Because pneumolysin released by bacteria which have been killed by conventional antibiotics is still active, agents capable of directly attacking the toxin are considered advantageous against antimicrobials in the treatment of S. pneumoniae infections. Here we found that the phytosterol, β-sitosterol, effectively protects against cell lysis caused by pneumolysin. This compound interacts with the toxin at Thr459 and Leu460, two sites important for being recognized by its natural ligand, cholesterol. Similar to cholesterol, β-sitosterol induces pneumolysin oligomerization. This compound also protects cells from damage by other cholesterol-dependent toxins. Finally, this compound protects mice against S. pneumoniae infection. Thus, β-sitosterol is a candidate for the development of anti-virulence agents against pathogens that rely on cholesterol-dependent toxins for successful infections.

Inhibition of α-toxin production by subinhibitory concentrations of naringenin controls Staphylococcus aureus pneumonia

Staphylococcal pneumonia provoked by methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening infection in which α-toxin is an essential virulence factor. In this study, we investigate the influence of naringenin on α-toxin production and further assess its therapeutic performance in the treatment of staphylococcal pneumonia. Remarkably, the expression of α-toxin was significantly inhibited when the organism was treated with 16 μg/ml of naringenin. When studied in a mouse model of S. aureus pneumonia, naringenin could attenuate the symptoms of lung injury and inflammation in infected mice. These results suggest that naringenin is a promising agent for treatment of S. aureus infection.

Molecular Modeling Reveals the Novel Inhibition Mechanism and Binding Mode of Three Natural Compounds to Staphylococcal α-Hemolysin

α-Hemolysin (α-HL) is a self-assembling, channel-forming toxin that is produced as a soluble monomer by Staphylococcus aureus strains. Until now, α-HL has been a significant virulence target for the treatment of S. aureus infection. In our previous report, we demonstrated that some natural compounds could bind to α-HL. Due to the binding of those compounds, the conformational transition of α-HL from the monomer to the oligomer was blocked, which resulted in inhibition of the hemolytic activity of α-HL. However, these results have not indicated how the binding of the α-HL inhibitors influence the conformational transition of the whole protein during the oligomerization process. In this study, we found that three natural compounds, Oroxylin A 7-O-glucuronide (OLG), Oroxin A (ORA), and Oroxin B (ORB), when inhibiting the hemolytic activity of α-HL, could bind to the “stem” region of α-HL. This was completed using conventional Molecular Dynamics (MD) simulations. By interacting with the novel binding sites of α-HL, the ligands could form strong interactions with both sides of the binding cavity. The results of the principal component analysis (PCA) indicated that because of the inhibitors that bind to the “stem” region of α-HL, the conformational transition of α-HL from the monomer to the oligomer was restricted. This caused the inhibition of the hemolytic activity of α-HL. This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay. This study can facilitate the design of new antibacterial drugs against S. aureus.