Screening Of Antibacterial Substances Targeting The Outer Membrane Proteins BamA/BamD Of Gram-negative Bacteri

[Research Purpose and Significance]Antibiotic resistance has become a significant global health threat due to the increasingly serious issue of bacterial resistance.Gram-negative bacteria pose a greater threat than gram-positive bacteria due to the unique membrane structure of their inner and outer membranes.This makes their drug resistance more serious than that of Gram-positive bacteria.Antibiotic drug development has nearly stopped in recent years,and it is a big concern that no new mechanistic antibiotics targeting Gram-negative bacteria have been approved in the last 50 years.This particularly creates a significant challenge in addressing drug resistance in Gram-negative bacteria.The outer membrane of Gram-negative bacteria contains many important proteins that are crucial for bacterial survival and host infection.One such example is the β-barrel assembly machinery(BAM),which plays a critical role in both the folding and membrane insertion of outer membrane proteins.The proper function of this complex is essential for the biogenesis of the outer membrane of Gram-negative bacteria.Intimin is an important virulence factor of pathogenic Escherichia coli that plays a crucial role in its pathogenic process.The special location and critical function of these outer membrane proteins make them highly desirable to serve as targets for the development of new antibiotics.Directly targeting crucial outer membrane proteins,such as the β-barrel assembly machinery,to compromise the inherent obstacle of Gram-negative bacteria is not only highly valuable for the identification of innovative antibiotics with novel mechanisms that are not prone to clinical resistance,but also for the reuse of current antibiotics.[Methods](1)The β-barrel assembly machinery,as well as its related subunit proteins,were purified respectively.Using a combination of affinity ultrafiltration and high-performance liquid chromatography/mass spectrometry,natural products that can interact with this complex were screened.(2)the anti-bacterial activity of the compounds was assessed through drug sensitivity and cell membrane destruction tests.(3)To investigate whether the β-barrel assembly machinery is required for the transport of Intimin,theversatile in vitro membrane protein investigation system was used.Additionally,this system was adaptedto test whether natural products can affect the folding and integration of outer membrane proteins by the β-barrel assembly machinery.[Results](1)Screening with affinity ultrafiltration/high-performance liquid chromatography/mass spectrometry idendified that licochalcone A has a good affinity toward BamA/BamD and inhibit the integration of outer membrane proteins by theβ-barrel assembly machinery at a concentration of 8 μg/mL.The inhibition rate increased as the concentration of licochalcone A increased.At a concentration of 64 μg/mL,the inhibition rate was 50%,and at a concentration of 128 μg/mL,the inhibition rate was 80%.(2)Licochalcone Alacks an obvious direct antibacterial effect in the drug sensitivity test.However,it was observed to cause some levels of damage to the outer membrane.(3)The β-barrel assembly machinery is required for the transport of intimin across the outer membrane.[Conclusion]In vitro analysis demonstrates that licochalcone A,examined by use of affinity ultrafiltration/high performance liquid chromatography/mass spectrometry,effectively inhibits the assembly function of the β-barrel assembly machinery.Subsequent cell membrane destruction experiments revealed that licochalcone A is able to cause a certain level of damage to the cell membrane.However,no significant bacteriostatic or bactericidal effects were observed in these experiments.Moreover,the versatile in vitro membrane protein investigation system demonstrated that the β-barrel assembly machinery is required for the transport of virulence protein Intimin.