Lactobacillus Salivarius Extracellular Proteins Indirectly Promote The Adhesion Mechanism Of Staphylococcus Aureus And The Expression Of Effector Protein

At present,food-borne pathogens are still a major factor in harming public health,and have attracted widespread attention from the community.Staphylococcus aureus is one of the most common food-borne pathogens.Adhesion is the first step in the colonization and infection of pathogenic bacteria in the host and is considered to be one of the important threats.It is generally believed that probiotics can inhibit the adhesion of pathogenic bacteria and reduce their accumulation in the body through steric hindrance.However,studies had shown that some Lactobacilli can promote the adhesion of pathogenic bacteria.Some people thought that the probiotics adhered to the intestine on the one hand and the probiotics adhered to the pathogens on the other hand,but the specific mechanism is still unclear.Therefore,this study used pre-screened probiotic Lactobacillus salivarius and Staphylococcus aureus as research objects,trying to study whether probiotics promote adhesion of Staphylococcus aureus through extramembrane protein from the perspective of extramembrane protein.First,we studied the direct adhesion of Lactobacillus salivarius to Staphylococcus aureus.The strains were separately centrifuged and diluted,and Lactobacillus salivarius and Staphylococcus aureus were combined for 4 hours.Microscopic observation showed whether Lactobacillus salivarius and Staphylococcus aureus directly adhered to each other,and further extracted extracellular proteins of Lactobacillus saliva,through electron microscopy and protein coating to study the effects of adhering to Staphylococcus aureus.Caco-2 cells were cultured,and it was confirmed by crystal violet staining and plate counting whether the extracellular proteins indirectly promoted the adhesion of Staphylococcus aureus to cells.In order to analyze the molecular mechanism of Lactobacillus licheniformis extracellular protein promoting S.aureus adhesion,the highest scoring bacterial substrate binding protein ABC transporter substrate-binding protein was selected by pre-mass spectrometry to clone and express the gene.Purification of the target protein by Ni column method.The adhesion of the purified protein to S.aureus was verified by the coating method.Caco-2 cells were cultured again to verify whether the purified protein indirectly promoted the adhesion of S.aureus to cells by plate counting.Finally,the temperature and pH were controlled to study the factors affecting the adhesion of purified protein to Staphylococcus aureus.The results showed that there was co-aggregation of Lactobacillus salivarius and Staphylococcus aureus which was confirmed by microscopic observation.The number of Lactobacillus salivarius adhering to the extracellular protein was decreased and the amount of S.aureus was increased after re-adding extramembrane protein which were observed by electron microscope.It was proved that the main role of adhesion was extramembranous protein.S.aureus was inoculated into 96-well plates coated with extra-membrane proteins,and the amount of S.aureus adhesion was increased.Cellular assays showed that the extracellular protein of Lactobacillus saliva first added to Caco-2 cells,and the adhesion of S.aureus was increased by 20%.The ABC transporter substrate-binding protein was further expressed and purified,and the purified protein was 30 kDa.In the protein coating and cell assays,purified proteins increased S.aureus adhesion values by 40% and 26.7%,respectively.When the temperature was 37 °C,the purified protein promoted the strongest adhesion of S.aureus.