| Antimicrobial resistance(AMR)is a current global public health issue that affects the health of humans,animals,and environmental hygiene.The emergence and spread of AMR have challenged the effectiveness of antimicrobial agents in the treatment of infections and the prevention of surgical site infections,exacerbating the difficulty and cost of disease management.Methicillin-resistant Staphylococcus aureus(MRSA)is a severe drug-resistant organism that causes serious infections ranging from mild skin and soft tissue infections to more serious diseases such as endocarditis,sepsis,pneumonia,and osteomyelitis,among others.Although the incidence of MRSA has decreased in many countries in recent years,the high mortality rate associated with its complications remains a significant threat in clinical practice,posing a public health problem worldwide.As the scarcity of new antimicrobial agents becomes increasingly prominent,urgent action is needed to find new strategies to combat the growing and evolving threat of multidrug resistant pathogens.Fortunately,effective antimicrobial techniques are being developed with antivirulence targeting becoming more prevalent.Virulence factors play a crucial role in bacterial adhesion,colonization,invasion,dissemination,and immune evasion.Compared to traditional antibiotics that directly kill or limit bacterial growth,antivirulence therapy aims to disarm the pathogen and reduce bacterial pathogenicity.Additionally,antivirulence therapy avoids imposing survival pressure on bacteria,which can partly prevent the emergence of antibiotic resistance.Therefore,antivirulence therapy has become a new strategy against multidrug-resistant pathogens.Alpha-hemolysin(Hla),also known as alpha-toxin(AT),is a pore-forming toxin produced by almost all virulent bacterial strains.Hla requires self-assembly into a heptamer on the surface of susceptible cells,leading to leakage of cellular contents and ultimately cell death.It is associated with various S.aureus diseases,including skin and soft tissue infections and pneumonia.Due to its critical role in the pathogenesis of S.aureus infection,Hla has emerged as a major candidate target for combating MRSA and is currently the preferred target for developing S.aureus vaccines.Natural products and their derivatives account for a significant proportion of drug development and clinical medicine.According to statistics,as of 2021,a considerable number of molecules(over 60%)in FDA-approved drugs are derived from or directly sourced from natural products.In this study,we screened for compounds with hemolysis-inhibiting properties from a natural small molecule library and further investigated their mechanism of action.We evaluated their therapeutic effects on a mouse model of MRSA-infected pneumonia in vivo,providing new candidates for combating MRSA.The main findings are as follows:(1)Hemolysis experiments demonstrated that schisandrone at a concentration of 32μg/m L effectively inhibited hemolytic activity of MRSA,methicillin-sensitive Staphylococcus aureus(MSSA),and clinical isolates,with hemolysis inhibition rates of 83.06±0.19%,68.27±0.68%,and 92.17±0.27%,respectively.(2)The formation of heptameric pore complexes is closely related to Hla-mediated cell invasion.Oligomerization assays confirmed that schisandrone does not affect the Hla oligomerization induced by deoxycholic acid.Neutralization assays and cellular thermal shift assays(CETSA)indicated that schisandrone does not directly interact with Hla,suggesting that it is not a direct inhibitor of Hla and does not affect heptamer synthesis.(3)The expression of Hla is controlled by a regulatory network composed of multiple regulatory elements,among which the Auxiliary gene regulation(Agr)system plays a central role.Results from RT-qPCR and western blot experiments indicate that schisandrone,in a dose-dependent manner,inhibits the transcription and protein levels of Hla.When the concentration of schisandrone is 32μg/m L,the m RNA and protein levels of Hla decrease by approximately 10-fold and 4-fold,respectively.Additionally,schisandrone inhibits the transcriptional levels of RNAIII and agrA,genes associated with the Agr system,with m RNA levels decreasing by 5-fold and 6-fold at 32μg/m L.These results suggest that schisandrone may regulate the expression of Hla by interfering with the Agr quorum-sensing system,thereby affecting its hemolytic activity.However,it cannot be ruled out that other regulatory elements may also be affected by these compounds.(4)By constructing the pET28a-agrA prokaryotic expression vector and inducing expression with IPTG,agrA protein was obtained.Subsequently,various experiments such as thermal stability assay(TSA),fluorescence quenching,electrophoretic mobility shift assay(EMSA),molecular docking,and kinetic simulations confirmed the direct interaction between schisandrone and agrA.The TSA experiment results showed that the binding of schisandrone to agrA resulted in aΔTm value of 3℃,indicating a direct interaction between the two.Fluorescence quenching revealed a binding constant(K_A)of 8.2×10~4 L/mol between schisandrone and agrA,indicating a strong interaction between the two.In addition,molecular simulation results showed a total binding free energy of-42.588 k J/mol,where ASP157,LYS167,ASP176,and ASN177 were identified as key amino acid sites for the binding of schisandrone to agrA.(5)Ensuring the safety of a drug is the foundation for its further development.Cell toxicity experiments showed that at a concentration of 64μg/m L,schisandrone did not exhibit significant toxicity to A549,HEK293T,and HepG2 cells derived from human lung,liver,and kidney,respectively,which was higher than the concentration that inhibited hemolysis.In addition,further acute toxicity studies in mice showed that a single intraperitoneal injection of200 mg/kg of schisandrone did not affect the survival of mice.Long-term toxicity experiments have also confirmed the safety of schisandrone.(6)Staphylococcus aureus can cause pneumonia infection,and it first needs to break through the barrier of lung epithelial cells.When schisandrone was added to a co-culture system of A549 lung epithelial cells and Staphylococcus aureus,it significantly reduced A549 cell damage and the release of LDH,showing a protective effect on the lung epithelial cells.This suggests that schisandrone may have a protective effect against infections caused by Staphylococcus aureus.(7)In a mouse model of MRSA-induced pneumonia infection,the group treated with schisandrone showed a significant improvement in survival rate of mice infected with a lethal dose of MRSA.The number of colonizing bacteria and the degree of lung damage in the lung tissue were also significantly reduced.The levels of inflammatory cytokines TNF-α,IL-1β,and IL-6 also showed significant changes,which may be achieved by regulating the quorum sensing system to weaken the virulence of MRSA.(8)One current strategy for combating multi-drug resistant pathogens is to use combination therapy,which involves the use of both traditional antimicrobial agents and anti-virulence drugs.This approach has shown significant advantages,as it reduces the amount of each drug used and therefore decreases the risk of pathogen resistance due to antibiotic use.Additionally,the use of anti-virulence drugs can weaken bacterial virulence,making it easier for drugs to clear the infection.Our research utilized a checkerboard assay and time-kill curve analysis to demonstrate and confirm the synergistic effect of schisandrone and cefotiam against MRSA(with a fractional inhibitory concentration index of 0.125).This result was further validated in a mouse model of schisandrone-induced pneumonia.Overall,schisandrone,as an S.aureus virulence inhibitor,significantly reduces MRSA pathogenicity both in vitro and in vivo.It can also be used as an adjuvant with ceftiour to restore its sensitivity to MRSA.This study provides a solid foundation for elucidating the mechanism of schisandrone’s anti-infective effects and innovative anti-infective drug development. |
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