短小芽孢杆菌ClpX蛋白对金黄色葡萄球菌的抑菌活性分析OA

[Objective]The purpose of current study was to analyze the antibacterial activity of the ClpX protein of Bacillus pumi-lus against Staphylococcus aureus.[Methods]The culture supernatant of B.pumilus LG-1 was concentrated using the ammo-nium sulfate precipitation method.The physicochemical properties of the antibacterial substances in the supernatant were ana-lyzed via proteinase k digestion.The substances in the concentrated supernatant were identified using high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).The ClpX protein was enriched using immunoprecipitation.The minimum inhibitory concentration(MIC)of the target protein was determined by the microbroth method.The influence of the target protein on the viability of S.aureus was analyzed by the ATP bioluminescence assay.The influence of the target pro-tein on the membrane potential of S.aureus was detected using the membrane potential probe DiBAC4(3).The influence of the target protein on the morphology of S.aureus was analyzed using field emission scanning electron microscopy(FE-SEM).[Results]Experiments proved that the antibacterial substance in the supernatant of LG-1 was a protein.Mass spectrometry analysis identified a total of 13 proteins with a matching peptide count≥2.Among these,three proteins were members of the Clp protease complex,including the regulatory subunit ClpX protein.Microbroth method results showed that the minimum in-hibitory concentration of ClpX protein against S.aureus was 0.75 mg/mL;ATP bioluminescence detection results showed that the inhibition rate of bacterial viability reached 87.42%,indicating an significant inhibitory effect.The DiBAC4(3)probe detection results indicated that S.aureus treated with 2 MIC of ClpX protein exhibited hyperpolarization,suggesting cell mem-brane damage.FE-SEM results showed that after treatment with 2 MIC of ClpX protein,the cell walls of some S.aureus were damaged or missing,suggesting bacterial cell wall damage.[Conclusion]The ClpX protein of B.pumilus LG-1 significantly inhibited the proliferation and viability of S.aureus and damage the membrane and cell wall.This study was the first to report the antibacterial effect of ClpX again S.aureus,providing a theoretical basis for further research and application of ClpX in con-trolling S.aureus infection and drug resistance.