Study On Two-component Systems YycF/YycG Regulating The Biosynthesis Of Capsular Polysaccharide And Teichoic Acids In Streptococcus Pneumoniae

Objective:Bacterial two-component system?TCS?is a signal transduction system which can sense and transmit signals to maintain their survival in complex environment.YycF/YycG two-component system found in all low G+C gram-positive bacteria,such as Staphylococcus aureus and Streptococcus pneumonia,plays a central role in cell wall metabolism,biofilm formation,cell division and other physiological regulation.In Streptococcus pneumoniae?S.pn?,YycF/YycG is participated in cell wall metabolism,fatty acid metabolism and virulence regulation.The gene encoding response regulator YycF protein is essential for bacterial growth.Capsular polysaccharide?CPS?is an important virulence factor of Streptococcus pneumoniae.Griffith,a British bacteriologist,discovered that the capsular polysaccharide of S.pn is regulated by environmental signals at the beginning of the last century,but the specific regulatory mechanism is still unclear.Teichoic acid?TAs?is the main component of cell wall of S.pn,which participates in many biological activities such as bacterial division,adhesion and invasion.It is also an important virulence factor of S.pn.In the research of Bacillus subtilis and Staphylococcus aureus,YycF/YycG two-component system plays an important role in cell wall metabolism,polysaccharide synthesis,bacterial division and so on.This study was aimed to explore whether YycF/YycG two-component system is involved in the synthesis regulation of CPS and TAs of S.pn,and to confirm its comprehensive influence on the virulence of S.pn.Methods:The pcsB constitutive expression strain JH1002?Pc-PcsB⁺?was constructed by JC?Janus cassette?counter selection,the yycF gene in JH1002 was replaced with an erythromycin resistance gene?erm?by long flanking homology polymerase chain reaction?LFH-PC?.The?yycG mutant strain JH1005 without insertion gene was constructed by JC?Janus cassette?reverse selection,with D39 streptomycin resistant strain?D39rpsl41?as background strain.The growth of the D39rpsl41,JH1002,JH1003,JH1005 strain in C+Y medium was monitored by continuous detection of bacterial absorbance(OD₆₀₀).Morphologies of JH1003,JH1005strains were observed by phase-contrast microscopy.The change of capsular polysaccharide and teichoic acid in mutant strains were detected by Western Blotting and ELISA.The ability of bacteria to resist macrophage phagocytosis and bacterial adhesion invasion was tested in vitro.The pneumonia model in mice was constructed for the detection of pneumococcal colonization and virulence.Results:We successful constructed a yycF-deficient mutant strain based on JH1002?Pc-PcsB⁺?,and a yycG-deficient mutant strain based on D39rpsl41,and found that a slower growth was displayed in JH1003 than that in JH1002.The slower growth of yycG mutant was found than that of D39rpsl41 strains;The abnormal division of JH1003 and JH1005 were discovered by phase-contrast microscopy.The increase of small molecule capsular polysaccharide and teichoic acid in lysate of JH1003 and JH1005were detected by Western Blotting.The results of EMSA showed that there was a specific binding between YycF protein and lic1 promoter.But no specific binding between YycF protein and CPS gene cluster promoter was measured.The results of RT-PCR showed that the transcription level of tarI gene in the downstream of lic1 promoter increased significantly in JH1003.In vitro results showed that the adhesion ability of yycF-deficient strain was significantly declined?P=0.006?.The adhesion ability?P=0.002?and invasion ability?P=0.005?of yycG-deficient strain were weaker than those of D39rpsl41.The virulence test suggested that all mice infected with D39rpsl41 died,while the mortality rates of mice challenged with JH1002,JH1003 were decreased to 91.7%and 75%respectively,though no statistical significance between them was observed?P=0.183?.The mortality of JH1005 infection group was significantly lower than that of wild-type strain infection group?P=0.0005?.The colonization study revealed that the bacterial burden of JH1003 in the lung tissue was significantly lower than that of JH1002?P=0.033?,the bacterial burden of JH1005 in the lung tissue was also significantly lower than that of D39rpsl41?P=0.006?.Conclusion:The YycF in Streptococcus pneumoniae,as a transcription regulator,negatively regulate the precursor synthesis of TAs by binding with the promoter region of TAs synthesis related genes.And negatively regulate the precursor synthesis of CPS.After deficiency of yycF and yycG,the pathogenicity of bacteria decreased...