The Identification Of Key Functional Domains Of Spd1672and Its Effects On The Synthesis Of Teichoic Acid And Virulence In Streptococcus Pneumoniae

ObjectiveTeichoic acid (teichoic acid, TA) is a polysaccharide polymer thatpresents on the surface of Gram-positive bacteria. It is an important surfaceantigen for Bacteria. spd1672gene was identified as an in-vivo-inducedgene in our previous research. Bioinformatics analysis predicted that thisgene was highly conserved in different Streptococcus pneumoniae havingO-antigen ligase and polymerase domains. Preliminary results showed thatSPD1672exerted a pivotal role in the synthesis of teichoic acid andvirulence in D39(serotype2), it may act as the synthase of teichoic acid,however, the key domains have remained elusive. In this work, the primaryobjectives were to identify the key structural domains in SPD1672, theeffects of SPD1672on the synthesis of teichoic acid, and virulence indifferent serotypes of Streptococcus pneumoniae.Methods The spd1672-deletion mutants of D39（serotype2）, R6(serotype2),203（serotype3）, TIGR4（serotype4）were constructed using LFH-PCRtechnology. The external loop4(EL4) deletion strains and complementedstrains in D39，203，TIGR4were generated by using pEPV3plasmid. Fivemutant strains in external loop4were obtained by PCR. Activity sites inSPD1672were identified by detecting the difference of teichoic acidsub-fractions in wild and deficient bacteria. The effects on the synthesis ofWTA and LTA in different serotypes of Streptococcus pneumoniae werealso analyzed by western blot. The sub-cellular localization of SPD1672was determined with PJWV25and PAE03plasmids which belong tofluorescence reporting system. Cell wall and protoplasm in differentserotypes of Streptococcus pneumoniae were separated; the effect ofspd1672gene on the synthesis of wall teichoic acid (WTA) and lipoteichoicacid (LTA) were elucidated by western blottging. The contribution ofSPD1672in the virulence of different serotypes of S. pn was evaluatedfrom the survival rate and colonization capacity in mice.ResultEL4negative strains were impaired in teichoic acid compared to thatof wild D39according to western blotting analysis, which was quiteconsistent with that of D39△1672strain.These results togetherdemonstrated that EL4is a key loop for SPD1672to function normally.Further enzyme activity analysis showed that there were no differences in the synthesis of teichoic acid between wild type D39and amino acid pointmutants at the sites of266,270,287,306. The mutant at the site of304waswith significantly less teichoic acid than wild type parent. Then, theanti-phosphorylcholine antibody was employed to analyze the synthesis ofphosphocholine. We found that phosphocholine was also decreased asteichoic acid, which further strengthen the involvement of SPD1672insynthesis of teichoic acids. SPD1672appeared to locate in the cellmembrane as revealed by the fluorescence with N-terminal and C-terminalGFP-SPD1672fusion plasmid. Teichoic acid in serotype3and TIGR4(serotype4) was impaired in spd1672deficient strains as compared withwild type strains, suggesting that spd1672gene is conserved in S. pn. Micewhich were intranasally infected with D39△1672，203△1672,TIGR4△1672recovered less bacterial load in blood, nasopharynx, lungand brain than wild type strains. The reduced virulence for spd1672deletion could also be recognized from survival rate after mice challengedwith wild type strains and mutants, where mice infected with mutantssurvived much longer than wild type strains.ConclusionThe present study indicates that SPD1672is located in the bacterialmembrane. The external loop4(EL4) would be a key domain for SPD1672to function normally. The304histidine appear to determine the polymerase activity of SPD1672. spd1672gene appears to involved in the synthesis ofteichoic acid in pneumococcal strains, and importantly contributes topneumococcal colonization and virulence.