Analysis of a Functionally Important Sequence in the Glucosyltransferases of Streptococcus mutans

• Main Authors: Chia Jean-San, 賈景山
• Other Authors: Chen Jen-Yang
• Format: Others
• Language: zh-TW
• Published: 1993
• Online Access: http://ndltd.ncl.edu.tw/handle/7sfr6h

博士 === 國立臺灣大學 === 微生物學系 === 81 === Streptococcus mutans has been identified as the principal pathog- en of the human dental caries。One of its virulence traits is the synthesis of glucan polymers which mediate the attachment of bac- teria to smooth surfaces and formation of dental plaque。Glucosy- ltransferases(GTFs) are enzymes responsible for the synthesis of water-soluble and -insoluble glucan。GtfB(162kDa) :GtfC(149kDa) synthesis primarily insoluble glucan in a primer-independent ma- nner，while GtfD(155kDa) synthesis water-soluble glucan exclusi- vely in a peimer-dependent manner。Using restriction fragment le- ngth polymorphisms(RFLP) analysis，we have initially identified genetic variations existed in the gtfB and gtfC genes。DNA sequ- ence analysis revealed that while several nucleotide changes were identified, accounting for the ploymorphisms，the amino acid that they code for remain unchanged。A synthetic peptide of 19 amino acids from this region (435-45)3 of GtfC was able to reverse the inhibitory effect of a polyclonal antibody which neutralized the GTFs'' activities。The 19-amino- acid peptide was conjugated to bovine serum albumin(BSA) and immunized BALB/c mice。Six IgG se- creting hybridoma clones specifically reacted with peptide，GtfC and GtfD but not BSA。 Aall six mAbs inhibited GtfC''s enzymatic activity，but failed to inhibit the GtfD，although GtfD contains the same peptide sequence。Affinity column purified CJSml8-S3 specifically inhibited GtfC''s enzymatic activity for insoluble glucan synthesis in a dose dependent manner. Furthermore，CJSml8- S3 also inhibited in vitro sucrose-dependent adherence of the Streptococcus mutans in a dose dependent manner。 These results indicated that the peptide sequence within a N-terminal conserved is important for the enzymatic activities and the bacteria。The 19 amino-acid peptide may have value for inclusion in a synthetic dental caries vaccine。