Study On The Antibacterial Effect Of Isoliquiritigenin On Staphylococcus Xylosus With IGPD As Target

Cow mastitis is recognized as the most common disease that affects the health of cow in the world.Among them,cow mastitis caused by coagulase-negative staphylococcus infection is increasing year by year.As an important clinical isolate of coagulase negative staphylococcus infection,Staphylococcus xylosus（S.xylosus）shows a variety of drug-resistant phenotypes,which makes the treatment of this kind of mastitis very difficult.In 2017-2018,the Ministry of agriculture and rural areas of China successively issued the national action plan to curb the drug resistance of bacteria from animal sources（2017-2020）and the pilot work plan for the use of Antibacterials for animal use.The notice specified explicitly stipulated the withdrawal of all drug feed additives except traditional Chinese medicine.Therefore,based on the epidemic trend of mastitis in dairy cows and the policy of our country,it is of great significance to develop new antibacterial drugs with traditional Chinese medicine as the main body in the treatment of mastitis in dairy cows.Several studies have shown that Imidazole glycerophosphate dehydratase（IGPD）is an essential enzyme in plants and microorganisms,and thus it is an attractive target for the development of herbicides and antibacterial agents,for which there are presently a limited number of biological targets.Isoliquiritigenin is a kind of flavonoids,which has good antibacterial activity and medicinal properties.Although isoliquiritigenin can inhibit the growth of S.aureus and its biofilm formation,it has not been seen about studies on the effects of S.xylosus growth and biofilms.Therefore,this study explored the regulation of IGPD expression by isoliquiritigenin to reveal the antibacterial mechanism of isoliquiritigenin to S.xylosus,and to further evaluate the therapeutic effect of isoliquiritigenin on S.xylosus infection on animal mastitis model.The specific results are as follows:（1）The minimal inhibitory concentration（MIC）of isoliquiritigenin to S.xylosus was measured by microdilution method,and the time sterilization curve was established to evaluate the effect of isoliquiritigenin on the growth of S.xylosus.The results showed that the MIC was 80μg·m L^-1,and the growth of S.xylosus was completely inhibited when isoliquiritigenin was more than 2 MIC.（2）The effects of isoliquiritigenin on the biofilm formation of S.xylosus were evaluated by crystal violet staining and scanning electron microscopy.The results showed that 1/2 MIC(40μg·m L^-1),1/4 MIC(20μg·m L^-1)and 1/8 MIC(10μg·m L^-1)isoliquiritigenin could significantly interfere with the biofilm formation of S.xylosus and destroy the integrity of the biofilm.But isoliquiritigenin could not interfere with the formation of biofilm in S.xylosus.（3）Real-time q PCR,Westren blot,and determination of L-histidine were used to reveal the effect of isoliquiritigenin on IGPD.It was found that isoliquiritigenin can significantly affect the his B transcription level and IGPD expression by significantly reducing L-histidine synthesis.（4）The direct binding between IGPD and isoliquiritigenin was confirmed by the Bio-layer interferometry technology,enzyme activity analysis and molecular docking technology.The results showed that isoliquiritigenin can directly bind to IGPD and inhibit IGPD activity.（5）The therapeutic effect of isoliquiritigenin against S.xylosus virulence was further determined in an animal mastitis model.In vivo studies confirmed that isoliquiritigenin effectively attenuated the damage in the mammary gland tissues of the infected mice and prevented the occurrence of inflammatory symptoms in the mammary gland tissues.In summary,based on the target IGPD,this study revealed the effects of isoliquiritigenin on the growth and biofilm formation of S.xylosus from two aspects including regulation and direct binding.At the same time,a mouse mastitis model was constructed to further evaluate the therapeutic effect of isoliquiritigenin on the infection of S.xylosus.This study will provide a scientific basis for the future research and development of targeted IGPD drugs and the treatment of cow mastitis caused by S.xylosus.