| Objectives:The biofilm is mainly responsible for Staphyloccocus aureus drug-resistance and immune evasion,which cause persistent infection and bring the big challenge for the clinical treatment.The garlic extract proved to be effective on the inhibition S aureus biolfilm formation,but the reason remains unclearied.This paper intends to use network pharmacology,molecular docking and experimental verification methods to explore the anti-biofilm formation mechanism of main antibacterial components in garlic,and lay a theoretical foundation for further clinical treatment.Methods:1.Based on extensive reading of literature,databases search and preliminary work of our research team,the active ingredients of garlic were selected.2.Target library of garlic active ingredients was constructed by retrieving Stitch,Pharm Mapper,TCMSP and other databases.3.The antibacterial target library was constructed by mapping Genecards,Drugbank,and TTD other databases combined with literature retrieval.Venny2.1.0 was used to obtain the intersection of garlic active ingredient target and antibacterial and anti-S.aureus target.4.The intersection target was imported into Cytoscape3.7.1 software to construct the active component-target gene network.The key targets were determined.5.Upload key targets to String platform to build protein interaction network（PPI）and analyze key core targets related to biofilm formation.6.KEGG metabolic pathway and GO biological process analysis were performed on key targets.7.Docking allicin with key core targets by using Auto Dock1.5.6 software,analyzing the binding energy,stability and potential binding sites.8.Finally,the effects of allicin on selected core targets were experimentally verified.Results:1.398 effect targets for the three active components of garlic,and 1924 antimicrobial targets were found by retrieving databases and related literature.The intersection of active component target gene and antibacterial target gene was 33.（See Appendix B for targets）2.The compound-target network showed that allicin may be the main antibacterial and anti-biofilm component in garlic sulfur-containing compounds.3.Protein interaction network（PPI）showed that allicin interacts with ica gene family and ribosome large subunit,causing anti-biofilm and antibacterial effects respectively.In contrast,other sulfur-containing compounds with the disulfide or trisulfide functional groups interact with ribosome large subunit but ica operons.The result implies the structure of sulfonic ester in allicin may be a essential functional group against SA biofilm formation.4.Key targets were analyzed by KEGG metabolic pathway and GO biological process,and 11 KEGG targets and 4 GO targets were obtained.According to the protein database notes,allicin may cause cell death by interfering with basic processes such as DNA replication,protein translation,and the integrity of cell walls and membranes.5.Molecular docking showed that allicin has a good affinity with Ica A,Ica B and Ica C proteins.Its pharmacophore of sulfonic ester may form hydrogen bond with Arg-52 site of Ica B,by which deactivate Ica B from catalytic synthesis functional protein and blocking SA biofilm formation.Allicin can also complex with Ica A and Ica C,disturbing their auxiliary catalytic function,which also contribute to anti-biofilm effect.6.Some experiments were performed to verify allicin’s inhibition on SA biofim formation: the MIC and MBC were 13.9 μg/m L and 445.3 μg/m L,respectively;the silver staining test produced the result of confirmation allicin’s anti-SA biofilm effect,with a positive correlation between concentration and effect.Crystal violet staining was used to determine the growth curve of SA biofilm,and it was found that allicin inhibited the biofilm formation at 1-4 h,namely,the adhesion stage of SA biofilm formation.Congo red plate method was used to determine the inhibitory effect of allicin on the synthesis of PIA,and the results showed a positive correlation between concentration and effect.Gene level of ica family was also measured by RT-q PCR: compared with the blank group,gene expression of ica A,ica B,ica C and ica D were down-regulated by 64.0%,58.6%,56.7% and 67.2%,while ica R was up-regulated by 27.6%.Conclusions:Allicin can inhibit the formation of S.aureus biofilm in the adhesion stage.The possible mechanism is that allicin binds to Ica B and other proteins through hydrogen bonds through sulfonic acid ester functional groups,resulting in deactivaion Ica B function as the enzyme of synthesis PIA.It may also complex Ica A and Ica C proteins and disrupt with their auxiliary catalytic function to block biofilm synthesis. |
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