| Staphylococcus aureus is a gram-positive opportunistic pathogen responsible for skin and soft tissue infection,mastitis,pneumonia and sepsis in livestock,posing a serious threat to animal husbandry and human health.At present,drug-resistant S.aureus,especially methicillin-resistant S.aureus(MRSA),has become the main drugresistant pathogen that causes animal infections and spreads through the food chain under the pressure of antibiotic.On the other hand,the livestock breeding industry in our country has entered the era of "restricted use of antibiotics",which makes it more difficult to curb S.aureus infection.Despite some antibiotic alternative strategies such as antibodies,phage lysins,vaccine and antimicrobial peptides have made positive progress in recent years,no new drugs appear to have been developed in clinical practice.Thus,new strategies and drugs are urgently in need to combat S.aureus infection.S.aureus exploits various virulence factors to cause host damage and mediate escape immune,and one of the most well-characterized exotoxins is ?-toxin,a typical ?-barrel pore-forming cytotoxin expressed by almost all clinical isolates.Upon attaching cell membrane,?-toxin binds to it's specific cellular receptor ADAM10(disintegrin and metalloproteinase 10)and form perforating channels that causing cell lysis and other cell events.?-toxin plays critical roles in S.aureus pneumonia,skin infection,sepsis and other diseases.In the early stage of infection,the recognition of S.aureus by TLR2 results in the recruitment of the cytoplasmic adaptor My D88 to TLR2 receptor domains,initiating downstream signaling events that induce hyperinflammatory response through the nuclear translocation of nuclear factor ?B(NF-?B)and activation of MAPK.Inflammatory response is an important defense mechanism,which plays a key role in the regulation of the host immune system.However,when inflammation is out of control,host cells and tissues might be damaged in direct or indirect ways,which seriously affects the recovery of tissue homeostasis,or even aggravates infection.Therefore,bacterial-targeted ?-toxin and host-targeted TLR2 are important potential targets against S.aureus infection.Traditional Chinese herbs have been widely used for prevention and treatment of infectious diseases in livestock industry,which represents the best choice after "restricted antibiotics" ban on feed additives.The active ingredients of traditional herbs are also potent source of lead compounds in the research and development of new drugs for antibiotic alternatives.In the present study,we first performed dual-target inhibitor screening from a natural compound library to obtain potential dual-action drug candidates that simultaneously targets S.aureus pathogenicity via inhibiting the critical virulence Hla and the accompanying inflammatory response by blocking TLR2 signaling;then,the mechanism action of the potential candidates was verified both in vitro tissue cell infection model and in vivo animal infection model;finally,the mechanisms of the candidate targeting ?-toxin and TLR2 were further revealed by molecular biology experiments and TLR2 knockout mice.From a chemical library consisting of more than 500 natural compounds extracted from traditional Chinese medicine,we successfully obtained a variety of active inhibitors.In the initial screening,myricetin,asiatic acid and cyanidin were identified as effective ?-toxin inhibitors,while isolinderalactone,kaempferol and cinnamaldehyde exhibited potential inhibitory effect on TLR2 signaling.Amentoflavone and licorice chalcone could inhibit both the two targets,and amentoflavone was the more effective one.The hemolytic activity of ?-toxin was completely inhibited by amentoflavone at the concentration of 4 ?g/m L,which had a IC50 value of 2.326 ?g/m L.Further study demonstrated that the growth of S.aureus and the expression of ?-toxin were not affected by amentoflavone in the tested range,suggesting that amentoflavone had weak growth pressure and gene selectivity.In vitro analysis of cell infection showed that amentoflavone effectively inhibited ?-toxinmediated netic cell death and phagocytic escape in macrophages during S.aureus infection.In addition,amentoflavone also reduced the biofilm formation of S.aureus through targeting ?-toxin.Simultaneously,amentoflavone suppressed TLR2 signaling by interfering with the interaction between TLR2 and its adapter myeloid differentiation primary response gene 88(My D88),thereby blocking the activation of downstream NF-?B and MAPK signaling pathways.Importantly,amentoflavone showed a dosedependent inhibitory effect on the inflammatory response stimulated by various TLR2 ligands(Pam3CSK4,Pam2CSK4 and S.aureus teichoic acid(LTA))and several bacterial TLR2 agonist(heat-killed S.pneumoniae(HKSP)and heat-killed L.monocytogenes(HKLM)),rather than the heat-inactivated E.coli,suggesting that amentoflavone had therapeutic potential in other gram-positive bacterial infections or some inflammatory diseases associated with TLR2 dysfunction.Next,we further evaluated the in vivo effect of amentoflavone on S.aureus pneumonia both on wild-type mice and tlr2-deficient mice.The results demonstrated that amentoflavone could significantly improve the survival rate of wild-type mice by alleviating pathological of lung tissue and the level of inflammatory cytokines.In contrast,amentoflavone had no significant therapeutic effect in tlr2-deficient mouse model.Although amentoflavone failed to reduced the mortality and inflammation level in the lung tissues of tlr2-deficient mouse model,amentoflavone significantly reduced the hemorrhagic injury,which was consistent with the results of in vitro experiments.This alleviation might be attributed to the inhibition of ?-toxin activity by amentoflavone.Collectively,these results further confirmed that amentoflavone conferred protection against S.aureus infection through a dual mechanism in vivo.In conclusion,the present study showed that the natural flavonoid,amentoflavone,was an effective dual inhibitor of ?-toxin and TLR2 signaling.Without growth pressure on S.aureus,amentoflavone specifically prevented the pore formation of ?-toxin and thus effectively inhibited its damage to cells or tissues.At the same time,amentoflavone significantly suppressed acute excessive inflammation that might be detrimental to the host,by suppressing TLR2 signaling through interfering with the interaction between TLR2 and My D88.Our study suggests that simultaneous inhibition of key bacterial virulence factors and important inflammatory pathways represents a potential antibacterial treatment strategy.Moreover,our research provids a lead compound for the development of new drugs based on anti-virulence and anti-inflammatory dual-target. |
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