Effects Of RmlT Deletion On Biological Characteristics And Membrane Vesicle Protein Composition Of Listeria Monocytogenes

Listeria monocytogenes is a Gram-positive pathogenic bacterium that can cause listeriosis in humans and animals.Listeriosis is a rare but highly fatal disease.Wall teichoic acids（WTAs）,a unique component in the cell wall of Gram-positive bacteria,have a variety of functions,including recognition and adhesion.At the same time,the cell wall is associated with the release of membrane vesicles（MVs）.rml T encodes L-rhamnosyltransferase,a key gene for glycosylation modification of WTAs.In order to explore the effect of glycosylation modification of WTAs on the secretion and function of bacterial MVs,this study was conducted in three parts:（1）construction of rml T deletion mutant strain EGDeΔrml T and rml T complementation strain EGDeΔrml T+p ERL3-rml T,（2）analysis of the effect of rml T deletion on the biological characteristics of L.monocytogenes,and（3）analysis of the effect of rml T deletion on the physical and chemical properties,protein composition,and bacterial binding of MVs.The results of the study are as follows:1.In this study,we successfully constructed rml T deletion mutant strain EGDeΔrml T and complementation strain EGDeΔrml T+p ERL3-rml T using homologous recombination.We performed non-denaturing gel electrophoresis to verify the extraction of mutant strains WTAs.2.We compared the differences in some biological characteristics among the wild-type strain,EGDeΔrml T deletion mutant strain,and EGDeΔrml T+p ERL3-rml T complementation strain.The experimental results are as follows:（1）Bacterial growth:There was no significant difference in the growth of the three strains in BHI medium.（2）Antibiotic sensitivity:Compared with EGDe,EGDeΔrml T showed no significant difference in the minimum inhibitory concentration（MIC）and inhibition zone diameter for cefotaxime sodium,penicillin,gentamicin,and tetracycline（P>0.05）.（3）Bacterial biofilm formation ability:Under the premise of similar growth of the three strains,the amount of biofilm formed by EGDeΔrml T was significantly lower than that of EGDe at 48 h and 72 h（P≤0.001）.At the same time,the biofilm structure formed by EGDeΔrml T was looser than that of EGDe,indicating that the deletion of rml T affected the formation ability of bacterial biofilm.（4）Bacterial virulence:The hemolytic activity of EGDeΔrml T was the lowest among the three strains（P≤0.05）,and the hemolytic activity of EGDe and the complementation strain were similar.Compared with EGDe,the transcription levels of the main virulence genes,including prf A,inl A,inl B,act A,plc B,and hly,were significantly down-regulated in EGDeΔrml T（P≤0.05）,indicating that the deletion of rml T affected the virulence of the bacteria.（5）Transcription levels of major autolysin genes:Compared with EGDe,the transcription levels of three genes,ami,spl,and fla A,were down-regulated in EGDeΔrml T（P≤0.01）,while the transcription level of mur A was significantly up-regulated（P≤0.01）,indicating that the deletion of rml T affected the transcription of bacterial autolysins.3.We analysised the effects of rml T deletion on the physicochemical properties,protein composition,and binding to bacteria of MVs.The experimental results are as follows:（1）Physicochemical properties of MVs:The diameter of EGDeΔrml T-MVs was slightly smaller than that of EGDe-MVs（P≤0.05）.The Zeta potentials of the three strains showed stable single peaks,indicating that the presence of MVs in the three strains was relatively stable.There were no significant morphological differences in the MVs observed under transmission electron microscopy.（2）Protein composition of MVs:A total of 132 different proteins were identified from the MVs of the three strains using LC-MS.Among them,EGDe-MVs contained 33 unique proteins,EGDeΔrml T-MVs contained 11 unique proteins,and EGDeΔrml T+p ERL3-rml T-MVs contained 6 unique proteins.A total of 52 common proteins were identified in the three types of membrane vesicles.Differential expression analysis of the common proteins showed that compared with EGDe-MVs,there were 10 upregulated proteins and 11downregulated proteins in EGDeΔrml T-MVs,while EGDeΔrml T+p ERL3-rml T-MVs showed 5 upregulated proteins and 13 downregulated proteins.Calcium-transporting ATPase was upregulated 45.88-fold in EGDeΔrml T-MVs.Therefore,we compared the growth and intracellular calcium changes of EGDe and EGDeΔrml T under different concentrations of Ca Cl₂,as well as the differences in the transcriptional level of Calcium-transporting ATPase,to preliminarily explore the biological significance of this significantly upregulated protein.（3）Binding of MVs to bacteria:The binding of EGDe-MVs to different species of bacteria showed a certain species specificity.The deletion of rml T significantly increased the binding efficiency of EGDe-MVs to EGDe and Bacillus subtilis（P≤0.001）.To a certain extent,the Derjaguin-Landau-Verwey-Overbeek（DLVO）theory was used to explain the differences in the binding efficiency of MVs to different species of bacteria.In summary,rml T has been shown to affect the formation of the bacterial biofilm,virulence,and expression of autolysins in Listeria monocytogenes.Additionally,rml T plays a crucial role in the size of membrane vesicles（MVs）,the composition of proteins within MVs,and the ability of MVs to bind to bacteria.