Global Transcriptional Regulator Mga-like Regulating The Biosynthesis Of Capsular Polysaccharide And Teichoic Acids In Streptococcus Pneumoniae

Objective:Global transcriptional regulators are prevalent in Gram-positive pathogens.The Mga-like protein encoded by spd＿1587 is a member of the Mga/AtxA global transcriptional regulator family.Members of the Mga/AtxA transcriptional regulatory family bind directly or indirectly to the regulatory regions of target genes to regulate the expression of target genes and are sensitive to changes in peripheral environmental signals.Both capsular polysaccharide and teichoic acids are important virulence factors located on the surface of S.pneumoniae.The capsule is involved in the optophagocytosis of S.pneumoniae,assisting the bacteria to escape clearance by the host immune system.Teichoic acids plays an important role in the adhesion of S.pneumoniae to the host upper respiratory tract and internalization into non-immune cells.In previous studies,we screened out Mga-like as a candidate transcriptional regulator of CPS,and found that it also specifically binds to the teichoic acid synthesis-related gene lic1.Therefore,this study aims to clarify the regulatory mechanism of Mga-like on the capsular and teichoic acid of Streptococcus pneumoniae,and to provide a new idea for understanding the pathogenesis of streptococcus pneumoniae from colonization to invasive pathogenicity.Methods:Firstly,we used erythromycin to replace mga-like gene to construct mga-like defective strain(JH1101).Ppepz,an heterotopic plasmid integration,was used to construct Mga-like complement strain(JH1104).The growth of WT D39(JH1900),JH1101 and JH1104 within 12 hours was observed and the growth curve was drawn.After that,the morphology and size of bacterial colonies were observed on the blood plate.The surface capsule thickness was observed by FITC-dextran exclusion test and transmission electron microscopy,and the band pattern of the capsule was analyzed by Western blot.The contents of teichoic acids and phosphocholine were determined by flow cytometry and Western blot.EMSA and DNase I footprints were used to explore the binding characteristics of Mga-like with cps and lic1 promoters.The downstream genes transcription of cps and lic1 promoter was analyzed by RT-PCR.The changes of anti-phagocytosis and adhesion ability of JH1101 were detected by in vitro toxicity test,and the colonization of JH1101 in nasopharynx and systemic infection caused by JH1101 were detected by in vivo toxicity test.Results: We successfully constructed JH1101 and JH1104 strains.We found that mga-like did not affect the growth of S.pneumoniae through the growth curve.JH1900,JH1101 and JH1104 had no significant differences in colony morphology,cell size,fission and autolysis time.Transmission electron microscopy and FITC-dextran exclusion experiments showed that the surface capsule thickness of JH1101 was not significantly increased compared with that of JH1900.However,Western blot analysis showed that the band pattern of JH1101 was changed,and the small molecule capsule of JH1101 was more than that of JH1900.Flow cytometry and Western blotting showed that increased concentrations of teichoic acid and phosphorylcholine modified on teichoic acid of JH1101 were observed.EMSA and DNase I footprints showed that Mga-like specifically binds to cps and lic1 promoter.RT-PCR results showed that the expression of downstream genes of cps and lic1 promoter was increased after mga-like deficiency.In the process of resisting macrophage phagocytosis,JH1101 does not show obvious advantages,and JH1900,JH1101 and JH1104 have the same ability to resist macrophage phagocytosis.In the adhesion and invasion experiment,mga-like defective strain showed stronger invasion ability.In the mouse model of pneumonia,JH1101 has the strongest nasal colonization ability,and can enter the spleen through blood invasion and cause systemic infection.Conclusion:The global transcription regulator Mga-like inhibits the synthesis of capsule and teichoic acids by binding to cps and lic1 promoters.Knockout of mga-like resulted in the increase of low-molecular-size capsule and total teichoic acids content in the lysate of whole bacteria.The ability of mga-like defective strain to cause systemic infection is increased.