Antibacterial Activities And Mechanism Of N-glycome Of Human Milk Glycoprotein Against Staphylococcus Aureus

Staphylococcus aureus is an opportunistic human pathogen.As multiple antibioticresistant Staphylococcus aureus have been emerging and spreading,it is considered to be one of the most serious human pathogens.The previous work of our group demonstrated that the human milk N-glycome exerted an excellent efficacy in inhibiting the growth of pathogenic bacteria,especially for Staphylococcus aureus,and its antibacterial effect can even be comparable to the corresponding antibiotics.Based on these results,we speculate that the human milk N-glycome has a good potential for research and development and is a novel candidate to replace or reduce antibiotic use for the treatment of Staphylococcus aureus.Therefore,exploring the multifaceted antibacterial functions of human milk Nglycome and further clarifying its mechanism of antibacterial action has great significance to explore its use as an antibacterial substance for the treatment of Staphylococcus aureus infections.In this study,human milk N-glycome was extracted from human milk and was consequently purified.The antibacterial effect of human milk N-glycome was analyzed with Staphylococcus aureus as the target strain.The antibacterial mechanism of human milk N-glycome was preliminary elucidated at the cellular level,molecular level and transcriptional level,respectively,in order to provide new ideas and options for the development of antibiotic alternatives or synergistic substances.The main results of the study are as follows:1.Purification and antibacterial activity of human milk N-glycomeThe N-glycosidase-treated human milk glycoprotein was purified by combined ethanol precipitation,activated carbon solid-phase extraction and gel column chromatography,which can effectively remove impurities and obtain human milk N-glycome retaining intact structure.The minimum inhibitory concentrations of human milk N-glycome against Staphylococcus aureus was 256 μM,and the minimum bactericidal concentrations was 512μM.1024 μM of human milk N-glycome could reach the maximum bactericidal amount within 5 min.The human milk N-glycome could inhibit the adhesion of Staphylococcus aureus to Caco-2 cells,and also inhibit the formation of biofilm by Staphylococcus aureus,and kill the live bacteria in the mature biofilm.The human milk N-glycome is a potential antibacterial potentiator with synergistic antibacterial effects with ribosome-targeted antibiotics and additive or indifferent effects with cell wall-targeted antibiotics.2.Effect of human milk N-glycome on the morphology and cell surface structure of Staphylococcus aureusScanning electron microscopy observed that human milk N-glycome led to irreversible damage such as morphological damage and cell surface wrinkling of Staphylococcus aureus.The treatment with human milk N-glycome resulted in the leakage of intracellular ions,DNA and proteins,depolarization of transmembrane potential and enhancement of PI uptake in Staphylococcus aureus,indicating that human milk N-glycome led to the increase of cell membrane permeability and disruption of cell membrane integrity in Staphylococcus aureus.It was preliminarily speculated that human milk N-glycome exerted antibacterial effects by disrupting the cell membrane structure.The binding of human milk N-glycome decreased the cell surface zeta potential and hydrophobicity,and changed the physical and chemical properties of the cell surface.It was found that the binding ability of Staphylococcus aureus to human milk glycoproteins was significantly higher than that of human milk deglycosylated protein by fluorescence labeling technique,and the results of competitive binding inhibition assay showed that the binding ability of bacteria pre-incubated with human milk N-glycome to human milk glycoproteins were significantly decreased.It was demonstrated at the cellular level that the human milk Nglycome could bind to the surface of Staphylococcus aureus.Native-PAGE analysis showed that the human milk N-glycome lagged the bands of bacterial surface proteins,and endogenous fluorescence spectroscopy of bacterial surface proteins analysis showed that the human milk N-glycome caused fluorescence reduction and red-shift effect.LC-MS/MS identified that the major surface protein bound to the human milk N-glycome was TRAP.It was demonstrated at the molecular level that human milk Nglycome can bind to cell surface proteins and alter the tertiary structure of the proteins.3.Effect of human milk N-glycome on intracellular biomacromolecules of Staphylococcus aureusSDS-PAGE showed that human milk N-glycome caused disturbance of protein synthesis and metabolism in Staphylococcus aureus,and the content of protein and ATP was significantly reduced,indicating that human milk N-glycome affected the normal intracellular physiological metabolic processes.Five glycosidases were not found in the CAZy database,and although galactosidase activity was detected inside and outside Staphylococcus aureus cells,the addition of human milk N-glycome inhibited the galactosidase activity.While,the UPLC peak spectrum showed that the enzyme solution inside and outside of Staphylococcus aureus cells could not degrade human milk N-glycome,indicating that Staphylococcus aureus could not utilize human milk N-glycome,which acted on Staphylococcus aureus in its intact structural form.A database search revealed that multiple sugar transport systems exist on the cell membrane of Staphylococcus aureus,and fluorescently labeled human milk N-glycome was observed inside the bacteria and overlapped with the position of DNA,suggesting that human milk N-glycome may be transported to the interior of the bacteria by the transporter on the cell membrane.Agarose gel retardation and UV spectroscopy results indicated that human milk N-glycome can bind to DNA in vitro,and molecular docking analysis indicated that human milk N-glycome were bonded to the minor grooves of DNA by hydrogen bonds.The gel retardation results of in vivo binding indicated that human milk N-glycome did not cause intracellular DNA band retardation due to the small amount of human milk N-glycome entering the cell to bind with intracellular DNA,indicating that it may not be the main mode of antibacterial effect of human milk N-glycome.4.Transcriptomics-based study of the antibacterial mechanism of human milk Nglycome against Staphylococcus aureusA total of 956 differentially expressed genes,464 significantly up-regulated genes and 488 significantly down-regulated genes were screened by RNA-Seq after 2.5 h treatment with 256 μM human milk N-glycome.The differentially expressed genes were significantly enriched to 31 GO terms,involving biological processes mainly including cytolysis,interspecies interactions between organisms and pathogenesis,etc.The cellular component was the extracellular region,and the molecular function was mainly transporter activity.Differentially expressed genes were significantly enriched to 14 KEGG pathways,mainly including glycolysis/gluconeogenesis,microbial metabolism in diverse environments and quorum sensing.Human milk N-glycome inhibited the expression of peptidoglycan precursor synthesis gene glmS and cell wall degradation enzyme-related genes(atl,isaA,slel and sdpC),which affected normal bacterial growth and division.The expression of Opu transporter genes was up-regulated,resulting in increased cell membrane permeability.Human milk N-glycome inhibited the expression of genes related to purine and pyrimidine metabolism,interfered with the synthesis of genetic material,and destabilized the bacterial DNA replication and transcription process.The expression of DNA repair genes(NTH,uvrC and mutS2)were upregulated,indicating that human milk N-glycome caused DNA damage.Transcriptomic data also revealed a mechanism by which human milk N-glycome inhibited biofilm formation by downregulating the expression of genes related to cell surface adhesion matrix(ClfA,ClfB,FnbA,SdrC,SdrD,SdrE and Sbi),thereby suppressing the initial adhesion phase of biofilm formation.The expression of the Agr system and secreted virulence factor-related genes were down-regulated,suggesting that human milk N-glycome may control the expression of virulence factors by inhibiting the Agr system.Human milk N-glycome disrupted carbon and amino acid metabolism in Staphylococcus aureus to adapt to the unfavorable environment brought by human milk Nglycome.