A 60-kilodalton immunodominant glycoprotein is essential for cell wall integrity and the maintenance of cell shape in Streptococcus mutans

We have demonstrated previously by Western blotting that in naturally sensi tized humans, the serum or salivary antibody response to Streptococcus muta ns was directed predominantly to a protein antigen with a size of approxima tely 60-kDa. To identify this immunodominant antigen, specific serum antibo dies were eluted from immunoblots and five positive clones with inserts ran ging in length from 3 to 8 kb from identical chromosomal loci were obtained by screening a genomic expression library of Streptococcus mutans GS-5. Am ino acid sequencing established the identity of this immunodominant antigen , a 60-kDa immunodominant glycoprotein (IDG-60), to be a cell wall-associat ed general stress protein GSP-781, which was originally predicted to have a molecular mass of approximately 45 kDa based on the derived nucleotide seq uence. Discrepancy in the molecular mass was also observed in recombinant h is-tagged IDG-60 (rIDG-60) expressed from Escherichia coli. Glycosylation, consisting of sialic acid, mannose galactose, and N-acetylgalactosamine, wa s detected by lectin binding to IDG-60 in cell wall extracts from S. mutans and rIDG-60 expressed in vivo or translated in vitro. Despite the presence of multiple Asn or Ser or Thr glycosylation sites, IDG-60 was resistant to the effect of N-glycosidase F and multiple O-glycosidase molecules but not to beta -galactosidase. Insertional inactivation of the gene encoding IDG- 60, sagA, resulted in a retarded growth rate, destabilization of the cell w all, and pleiomorphic cell shape with multifold ingrowth of cell wall. In a ddition, distinct from the parental GS-5 strain, the isogenic mutant GS-51 was unable to survive the challenge of low pH and high osmotic pressure or high temperature. Expression of the wild-type gene in trans within GS-51 fr om plasmid pDL277 complemented the growth defect and restored normal cell s hape. These results suggested that IDG-60 is essential for maintaining the integrity of the cell wall and the uniformity of cell shape, both of which are indispensable for bacteria survival under stress conditions.