The Study On LytR Protein Regulates Capsule And Teichoic Acid Anchoring To The Cell Wall In Streptococcus Pneumoniae

ObjectiveCapsule polysaccharide?CPS?plays an important role in pneumococcal virulence,while teichoic acid?TAs?is the characteric component of the cell wall of gram-positive bacteria with function of maintaining morphology,they are both contribute to adherence and invasion of bacteria.But the process of anchoring to the cell wall remains uncleared.The LytR protein belongs to Lyt R-Cps-Psr?LCP?family,which have been shown to be proved with the anchoring of the cell wall-related glycopolymers to peptidoglycan.But it is still not clear whether the LytR protein?SPD₁741?plays the same role in Streptococcus pneumoniae.In this study,we preliminarily explored how the LytR protein works in the process of capsule and teichoic acid anchoring to the cell wall and effects on the phenotype and virulence of cells.The study provides new evidence for the molecular mechanism of the capsule and teichoic acid biosynthesis and benefits finding new target for the treatment of streptococcus pneumoniae infection.MethodsThe different phases expression of LytR protein was detected by Western blot.Fluorescence activated Cell Sorting?FACS?was used to confirm the surface content and location of the LytR protein.The ?lytR mutant was constructed by long flanking homology polymerase chain reaction?LFH-PCR?and the complemented lytR strain was constructed by plasmid pJWV25.The growth curve was plotted by culturing the wild-type bacteria,?lytR mutant,complemented lyt R strain and ?lytR mutant supplemented with recombinant LytR protein in vitro for observing the ability of bacteria reproduction.Western blot,ELISA and Dot blot were used to quantify the amount of bacterial CPS and TAs.The virulence of bacteria was analysed by cell adhesion assay,anti-phagocytosis of macrophages experiment and mouse infection experiment.The transcriptional regulation effect of the lyt A,cps,rfaX operon by LytR were determined by EMSA.ResultsThe LytR protein expression was very high and steady in different phases.The FACS shows the LytR protein which can express at the cell surface.Comparing with wild-type D39,D39?lytR mutant exhibited defection in growth,shortening of the plateau,premature autolysis,incomplete cell wall and reduction of CPS and TAs.The D39 complemented lytR strain and D39?lytR mutant supplemented with recombinant LytR protein were both improved in growth and the CPS along with the cell wall demonsrated no significant difference comparing with the wild type D39.The amounts of the CPS and TAs in the D39? lytR mutant were decreased in cell?p<0.05?but increased in the cultural supernatant?p<0.05?.The adherence ability of R6? lytR mutant was descended comparing with wild-type R6?p<0.05?.And the anti-phagocytosis assay indicates a reduction of adherence of D39? lytR comparing to the wild type?p<0.05?.The mortality of mouse infected the wild-type D39 was 83.3% and the mouse which infected the D39?lytR mutant was 0%,there were obvious differences between them?p<0.05?.The lungs of mouse infected by the wild-type D39 displayed a intensive inflammatory cell infiltration,hyperemia and interstitial hyperplasia.The lungs of the D39? lytR mutant infected mouse showed weaker inflammatory response.The EMSA indicted LytR protein did not bind with the promoter of lytA,cps and rafX.ConclusionThe LytR protein stably expressing in streptococcus pneumoniae.It may be involved in the anchoring of CPS and TAs to the cell wall and affects bacterial morphology,growth,autolysis and virulence in streptococcus pneumoniae.