title: A 60-kilodalton immunodominant glycoprotein is essential for cell wall integrity and the maintenance of cell shape in Streptococcus mutans.
abstract: We have demonstrated previously by Western blotting that in naturally sensitized humans, the serum or salivary antibody response to Streptococcus mutans was directed predominantly to a protein antigen with a size of approximately 60-kDa. To identify this immunodominant antigen, specific serum antibodies were eluted from immunoblots and five positive clones with inserts ranging in length from 3 to 8 kb from identical chromosomal loci were obtained by screening a genomic expression library of Streptococcus mutans GS-5. Amino acid sequencing established the identity of this immunodominant antigen, a 60-kDa immunodominant glycoprotein (IDG-60), to be a cell wall-associated general stress protein GSP-781, which was originally predicted to have a molecular mass of approximately 45 kDa based on the derived nucleotide sequence. Discrepancy in the molecular mass was also observed in recombinant his-tagged IDG-60 (rIDG-60) expressed from Escherichia coli. Glycosylation, consisting of sialic acid, mannose galactose, and N-acetylgalactosamine, was 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 wall, and pleiomorphic cell shape with multifold ingrowth of cell wall. In addition, 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 from plasmid pDL277 complemented the growth defect and restored normal cell shape. 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.
authors.affiliation: Graduate Institute of Microbiology, College of Medicine National Taiwan University, National Taiwan University Hospital, Taipei, Taiwan, Republic of China. chiajs@ha.mc.ntu.edu.tw
authors.forename: J S
authors.initials: JS
authors.lastname: Chia
authors.forename: L Y
authors.initials: LY
authors.lastname: Chang
authors.forename: C T
authors.initials: CT
authors.lastname: Shun
authors.forename: Y Y
authors.initials: YY
authors.lastname: Chang
authors.forename: Y G
authors.initials: YG
authors.lastname: Tsay
authors.forename: J Y
authors.initials: JY
authors.lastname: Chen
mesh_terms: D000328:Adult; D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000302:isolation & purification; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D001483:Base Sequence; D002473:Cell Wall / Q000502:physiology; D004269:DNA, Bacterial; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D016056:Immunodominant Epitopes / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D017422:Sequence Analysis, DNA / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods; D013290:Streptococcal Infections / Q000097:blood / Q000276:immunology / Q000382:microbiology; D013295:Streptococcus mutans / Q000276:immunology / Q000502:physiology*
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006023:Glycoproteins; C439649:IDG-60 protein, Streptococcus mutans; D016056:Immunodominant Epitopes; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase