Identification of the Binding Domain of Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase for Porphyromonas gingivalis Major Fimbriae

Abstract

Porphyromonas gingivalis forms communities with antecedent oral biofilm constituent streptococci. P. gingivalis major fimbriae bind to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) present on the streptococcal surface, and this interaction plays an important role in P. gingivalis colonization. This study identified the binding domain of Streptococcus oralis GAPDH for P. gingivalis fimbriae. S. oralis recombinant GAPDH (rGAPDH) was digested with lysyl endopeptidase. Cleaved fragments of rGAPDH were applied to a reversephase high-pressure liquid chromatograph equipped with a C 18 column. Each peak was collected; the binding activity toward P. gingivalis recombinant fimbrillin (rFimA) was analyzed with a biomolecular interaction analysis system. The fragment displaying the strongest binding activity was further digested with various proteinases, after which the binding activity of each fragment was measured. The amino acid sequence of each fragment was determined by direct sequencing, mass spectrometric analysis, and amino acid analysis. Amino acid residues 166 to 183 of S. oralis GAPDH exhibited the strongest binding activity toward rFimA; confocal laser scanning microscopy revealed that the synthetic peptide corresponding to amino acid residues 166 to 183 of S. oralis GAPDH (pep166-183, DNFGVVEGLMTTIHAYTG) inhibits S. oralis-P. gingivalis biofilm formation in a dose-dependent manner. Moreover, pep166-183 inhibited interbacterial biofilm formation by several oral streptococci and P. gingivalis strains with different types of FimA. These results indicate that the binding domain of S. oralis GAPDH for P. gingivalis fimbriae exists within the region encompassing amino acid residues 166 to 183 of GAPDH and that pep166-183 may be a potent inhibitor of P. gingivalis colonization in the oral cavity.

Bacterial Virulence in the Moonlight: Multitasking Bacterial Moonlighting Proteins Are Virulence Determinants in Infectious Disease

“…The GAPDH of the bacterium responsible for causing pneumonia and meningitis, Streptococcus pneumoniae, also acts as a plasminogen binding protein (24), which is important in the ability of this organism to cross endothelial and epithelial cell barriers (11). In Streptococcus agalactiae, GAPDH is reported to function as a virulence factor with B lymphocyte-modulatory activity (120), and the same enzyme from Streptococcus oralis binds to the major fimbriae of Porphyromonas gingivalis and appears to be important in the colonization of the latter organism (130). In Streptococcus suis serotype 2, the GAPDH enzyme functions as an albumin binding protein (147) and also as an adhesin for cell binding (28).…”

“…For example, the number of 10-kDa (100-amino-acid residue) proteins that can, in theory, be produced is 20 100 or 10 130 . To put 10 130 in perspective, it is estimated that the number of stars in the universe is around 10 24 .…”

Identification and Characterization of Porphyromonas gingivalis Client Proteins That Bind to Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase

“…The plasmid pQE30-Sogap, expressing S. oralis recombinant His-tagged GAPDH (HTrGAPDH), was prepared as described previously (22). A mutation in the P. gingivalis FimA fimbria binding domain of rGAPDH [residues 166 to 183; termed rGAPDH(⌬166-183)] was introduced into pQE30-Sogap using the primers Sogapdh⌬166-183F and Sogapdh⌬166-183R (Table 1).…”

“…However, multiple functions have been reported recently for GAPDH, including roles in membrane fusion, microtubule binding, phosphotransferase activity, nuclear RNA export, DNA replication and repair, apoptosis, and viral pathogenesis (20,21). Our previous study demonstrated that the P. gingivalis FimA fimbria binding domain in S. oralis ATCC 9811 GAPDH was within amino acid residues 166 to 183, and that the peptide corresponding to this domain (DNFGVVEGLMTTIHAYTG) exhibited strong binding activity toward recombinant FimA fimbriae (rFimA) by BIAcore analysis (K A ϭ 4.51 ϫ 10 7 M Ϫ1 ) (22). The synthetic peptide inhibited biofilm formation between various streptococci and P. gingivalis strains with different types of FimA fimbriae in a dose-dependent manner.…”

Role of Mycoplasma pneumoniae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in mediating interactions with the human extracellular matrix

“…Besides their glycolytic activity in the cytosol, these enzymes have been found at the surface of micro-organisms, where they act as binding partners of proteins of the human Abbreviations: ECM, extracellular matrix; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMR, multiple mutation reaction. Microbiology (2011Microbiology ( ), 157, 2328 As a surface-associated protein, GAPDH can interact with not only one or more specified host factors like fibronectin, fibrinogen, albumin, laminin, collagen and plasminogen but also human colonic mucin, human epithelial and endothelial cells or fimbriae of other bacterial species in biofilms (Pancholi & Fischetti, 1992;Gozalbo et al, 1998;Bergmann et al, 2004;Barbosa et al, 2006;Egea et al, 2007;Kinoshita et al, 2008;Nagata et al, 2009;Purves et al, 2010;Tunio et al, 2010a).…”