The Target Confirmation Of Baicalin Against Staphylococcus Aureus α-hemolysin

Staphylococcus aureus is a gram-positive zoonotic pathogen, causing a broadrange of diseases, ranging from relative minor skin and soft tissue infections tolife-threatening pneumonia, endocarditis, meningitis, septicemia and toxic shocksyndrome. In the veterinary clinic, S. aurues is the major pathogen that causedmastitis of cows and sheep, resulting in great economic losses and public health issues.The antibiotic resistance of S. aurues is pretty serious. Actually, it has been resistant toall clinically used antibiotics. Particularly, the dissemination and wide spread ofmethicillin-resistant S. aurues (MRSA) isolates have become a major threat to thehuman and animal health.90%of MRSA isolates have multi-drug resistant featurethat are simultaneously resistant to many antimicrobial agents, such as β-lactams,quinolones, macrolides and tetracyclines. The glycopeptides antibiotic vancomycin ispreviously considered to be the preferred drug for the treatment of MRSA infections.However, the emergence of vancomycin-insensitive S. aurues (GISA) andvancomycin-resistant S. aurues strains (VRSA) has made it difficult to controlstaphylococcal infections. Consequently, there is an urgent need to develop moreeffective antimicrobial agents or alternative therapeutic strategies.With increasing understanding of bacterial pathogenesis and intercellularcommunication has revealed many potential strategies to develop novel drugs for thetreatment of S. aurues-mediated diseases. S. aurues produces a number of virulencefactors, including cell surface proteins and exotoxins, which participated inpathogenesis. Presently, targeting virulence factors has become a promising strategyfor the treatment of S. aurues illnesses. α-hemolysin (Hla) is one of the majorexotoxins of S. aurues that is produced in the post-exponential growth phase. It issecreted as a33.2kDa water-soluble extracellular protein which contains293aminoacids. A wide range of mammalian cells, including erythrocytes, monocytes,lymphocytes, macrophages and epithelial cells, are influenced by Hla. Hla is a self-assembling, channel-forming toxin. Upon binding to the membrane of asusceptible cell, the membrane-bound monomer oligomerizes to form a232.4-kDamembrane-inserted heptamer. The heptameric pore leads to leakage of ions, water,and low molecular weight molecules out of and into the cell, and ultimately causingcell lysis and death. The roles of Hla in S. aurues infections have beenwell-investigated. Strains lacking Hla displayed less virulent in animal models ofdiseases, such as mastitis, peritonitis, keratitis and pneumonia. Based on the cell lyticmechanism and the key roles of Hla in S. aurues infections, Hla could be an importanttarget for the development of anti-S. aurues drugs.Natural compounds are important resources for drug discovery, while thedevelopment of anti-virulence drug relies on newly discovered synthetic or naturalsmall organic compounds that possess anti-virulence characteristics. In the study, weapplied hemolysis assay, western-blot and real-time RT-PCR to investigate theinfluence of22natural compounds on the production of Hla. The results indicated thatisoalantolactone, licochanlcone A, farrerol, capsaicin, thymol, eugenol, luteolin,chrysin and menthol could inhibit the transcription of hla gene (encoding Hla),decrease the production of Hla, and thereby inhibiting the hemolytic activity. Theexpression of Hla is controlled by a network of regulators, among which the Agrtwo-component system is the most important and well-characterized one. The data ofreal-time RT-PCR showed that the transcription of agrA was significantly inhibited bythese compounds, suggesting that the decreased production of Hla may partiallydepend on the inhibition of Agr regulator, although other regulators may also beinfluenced. Baicalin is the major component of Scutellaria baicalensis Georgi (HuangQin), which belongs to the flavonoids. It has various biological effects, includinganti-inflammatory, anti-viral, anti-tumor, anti-allergy and anti-pyretic characteristics.Treatment with baicalin could also inhibit the hemolytic activities of S. aurues culturesupernatants; however, the western-blot results showed that the expression of Hlawere not affected, suggesting that baicalin may directly inhibit Hla. Subsequently, wedemonstrated the possibility by hemolysis assay using purified Hla.Further, we used the theoretical chemistry methods to investigate the mode ofaction that baicalin inactivates Hla. Molecular docking and molecular dynamics (MD) simulations were employed. The initial structure of Hla was obtained from thehomology modeling. A standard docking procedure for Hla and baicalin wasperformed with AutoDock4.0, while the Lamarckian genetic algorithm (LGA) wasapplied in the docking calculations to obtain optimal conformation. MD simulationswere performed with the Gromacs4.5.3software package to analyze the moleculardocking data. The binding free energies were calculated using MM-GBSA approachsupplied by Amber10package. The results showed that baicalin could bind to the"triangle region" of the Hla monomer, while Tyr148, Pro151and Phe153were themajor binding sites. The hydrophobic interaction was the principal binding mode; thebinding of bacicalin restrained the conformational change of "triangle region",thereby inhibiting the self-assembly of the heptameric transmembrane pore.Site-directed mutagenesis and fluorescence quenching assays were applied toverify the binding sites of baicalin on Hla. The Tyr148, Pro151and Phe153in Hlawere mutated to Alanine (Ala). We constructed plasmids encoding wild-type(WT)-Hla, Y148A-Hla, P151A-Hla and F153A-Hla. Further, WT-Hla, Y148A-Hla,P151A-Hla and F153A-Hla were expressed and purified. The purified proteins wereused for determination of fluorescence quenching constants. The results showed thatmutations of Hla led to reduction of baicalin binding. The binding constants of theinteraction between baicalin and Hla decrease in the following order: WT-Hla>F153A-Hla> P151A-Hla> Y148A-Hla. Moreover, we used thedeoxycholate-induced oligomerization assay and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) to assess the influence ofbaicalin on the formation of Hla heptamer. Apparently, the addition of baicalinresulted in concentration-dependent reduction of Hla heptamer formation.Alveolar epithelial cells (A549) were co-cultured with S. aurues. Followingtreatment with baicalin, cell viability was measured either using the confocal laserscanning microscope or by measuring LDH. The data indicated that baicalin couldprotect Hla mediated injury of A549cells, suggesting the potential therapeutic effectof baicalin in staphylococcal pneumonia.The pharmacokinetic characteristics of baicalin in mice were determined by highperformance liquid chromatography (HPLC). The maximum concentrations of baicalin in plasma (Cmax) were4.5,14.81and24.74μg/ml for single subcutaneousdoses of25,50and100mg/kg, respectively. The half-time (t1/2) is about1.29h. Thepharmacokinetics of baicalin conformed to one-compartment open model with firstorder absorption.The mouse model of S. aurues pneumonia was established to assess the in vivoperformance of baicalin, and the dosage regimen was designed according to thepharmacokinetic characteristics of baicalin in mice. The results showed that treatedmice with baicalin significantly decreased the mortality and the colony forming units(CFUs) in the lung tissues. The lung wet-dry ratio was also reduced, indicating thatthe pulmonary edema was alleviated. Histopathologic analysis revealed extenuatedhyperemia, decreased inflammatory cell infiltration and normal alveolar architectureof lung tissue following treatment with baicalin. Analysis of bronchoalveolar lavage(BAL) fluid revealed that treated mice with baicalin led to significantly lessneutrophil influx into the airway, a marked reduction in IL-1β, TNF-α and IL-6concentrations in BAL fluids.In summary, baicalin inhibits the cell lytic activity of Hla via preventing theself-assembling of Hla heptameric pore; baicalin prevents Hla mediated A549cellinjury; baicalin protects mice from S. aurues pneumonia. Our study will lay thefoundation for clarifying the anti-infective mechanism of baicalin and developmentof innovative anti-infective drugs.