Study Of Interaction Between GtfA And GtfB And Its Regulation Of Glycosylation Of SraP In Staphylococcus Aureus And PsrP In Streptococcus Pneumoniae

Objective SraP（Serine-rich adhesin for platelets,SraP）is a glycoprotein in the cell wall of staphylococcus aureus,which mediates the binding of staphylococcus aureus to platelets and respiratory epithelial cells.PsrP（pneumococcal serine-rich repeat protein,PsrP）is a macromolecular glycoprotein in the cell wall of streptococcus pneumoniae,which mediates the combination of streptococcus pneumoniae and respiratory epithelial cells.Glycosylation is the premise and foundation of the adhesion function of SraP and PsrP.Both operons of SraP-SecY2A2 and PsrP-SecY2A2 contain GtfA（Glycosyltransferase A）and GtfB（Glycosyltransferase B）.In vitro experiments with staphylococcus aureus and streptococcus pneumoniae confirmed that the coexistence of GtfA and GtfB can glycosylate SraP and PsrP.This study intends to investigate whether GtfA interact with GtfB to form enzyme complex GtfAB to regulate SraP and PsrP glycosylation.Methods The glycosylation models of SraP and PsrP in Escherichia coli were constructed respectively.The effects of GtfA and GtfB on the expression and the glycosylation of SraP_91-5131-513 and PsrP_1-374-374 were detected by western blot assay and sWGA（succinylwheat germ lectin）blot.The interaction between GtfA and GtfB in staphylococcus aureus was detected by GST pull-down and protein affinity co-purification assay,while the interaction between GtfA and GtfB in streptococcus pneumoniae was detected by GST pull-down and yeast two-hybrid assay.Results Western blot and lectin imprinting experiments showed that:（1）when the SraP_91-513,GtfA and GtfB of staphylococcus aureus coexist,the relative molecular weight of SraP_91-5131-513 is significantly higher than when it coexists with GtfA or GtfB,and SraP_91-5131-513 can specifically bind to sWGA only when co-expressed with GtfA and GtfB;（2）when PsrP_1-374,GtfA and GtfB of streptococcus pneumoniae coexisted,the relative molecular weight of PsrP_1-374-374 was significantly higher than that when it coexisted with GtfA or GtfB,and PsrP_1-374-374 could specifically bind to sWGA only when co-expressed with GtfA and GtfB.GST Pull-down experiments showed that GtfA of staphylococcus aureus and streptococcus pneumoniae could directly bind to their corresponding GtfB.Two yeast hybridization experiments showed that AH109/（pGADT7-GtfA+pGBKT7-GtfB）and AH109/（pGADT7-GtfB+pGBKT7-GtfA）could grow on SD-LTHA plate.Protein affinity co-purification experiments showed interactions between GtfA and GtfB of staphylococcus aureus.Conclusions Both GtfA and GtfB are required for the glycosylation of SraP in staphylococcus aureus and PsrP in streptococcus pneumoniae.GtfA interact with GtfB to form enzyme complex GtfAB to initiate and regulate glycosylation of SraP and PsrP.GtfAB can transfer GlcNAc to the peptide chains of SraP and PsrP.