Casein Phosphopeptide-Amorphous Calcium Phosphate Reduces <i>Streptococcus mutans</i> Biofilm Development on Glass Ionomer Cement and Disrupts Established Biofilms - Fig 3

<p>Representative images of <i>S</i>. <i>mutans</i> biofilm formation on Fuji VII GIC (A) and Fuji VII EP GIC (B) after 16 h growth in a flow cell system.</p

Biometric parameters of 16 h flow cell <i>S</i>. <i>mutans</i> biofilms cultured using Fuji VII or Fuji VII EP as the substratum.

<p>Values are the mean and standard deviation of three biological replicates.</p

<i>Porphyromonas gingivalis</i> Gingipains Display Transpeptidation Activity

<i>Porphyromonas gingivalis</i> is a keystone periodontal pathogen that has been associated with autoimmune disorders. The cell surface proteases Lys-gingipain (Kgp) and Arg-gingipains (RgpA and RgpB) are major virulence factors, and their proteolytic activity is enhanced by small peptides such as glycylglycine (GlyGly). The reaction kinetics suggested that GlyGly may function as an acceptor molecule for gingipain-catalyzed transpeptidation. Purified gingipains and <i>P. gingivalis</i> whole cells were used to digest selected substrates including human hemoglobin in the presence or absence of peptide acceptors. Mass spectrometric analysis of the substrates digested with gingipains in the presence of GlyGly showed that transpeptidation outcompeted hydrolysis, whereas the trypsin-digested controls exhibited predominantly hydrolysis activity. The transpeptidation levels increased with increasing concentration of GlyGly. Purified gingipains and whole cells exhibited extensive transpeptidation activities on human hemoglobin. All hemoglobin cleavage sites were found to be suitable for GlyGly transpeptidation, and this transpeptidation enhanced hemoglobin digestion. The transpeptidation products were often more abundant than the corresponding hydrolysis products. In the absence of GlyGly, hemoglobin peptides produced during digestion were utilized as acceptors leading to the detection of up to 116 different transpeptidation products in a single reaction. <i>P. gingivalis</i> cells were able to digest hemoglobin faster when acceptor peptides derived from human serum albumin were included in the reaction, suggesting that gingipain-catalyzed transpeptidation may be relevant for substrates encountered in vivo. The transpeptidation of host proteins in vivo may potentially lead to the breakdown of immunological tolerance, culminating in autoimmune reactions

<i>Porphyromonas gingivalis</i> Gingipains Display Transpeptidation Activity

<i>Porphyromonas gingivalis</i> is a keystone periodontal pathogen that has been associated with autoimmune disorders. The cell surface proteases Lys-gingipain (Kgp) and Arg-gingipains (RgpA and RgpB) are major virulence factors, and their proteolytic activity is enhanced by small peptides such as glycylglycine (GlyGly). The reaction kinetics suggested that GlyGly may function as an acceptor molecule for gingipain-catalyzed transpeptidation. Purified gingipains and <i>P. gingivalis</i> whole cells were used to digest selected substrates including human hemoglobin in the presence or absence of peptide acceptors. Mass spectrometric analysis of the substrates digested with gingipains in the presence of GlyGly showed that transpeptidation outcompeted hydrolysis, whereas the trypsin-digested controls exhibited predominantly hydrolysis activity. The transpeptidation levels increased with increasing concentration of GlyGly. Purified gingipains and whole cells exhibited extensive transpeptidation activities on human hemoglobin. All hemoglobin cleavage sites were found to be suitable for GlyGly transpeptidation, and this transpeptidation enhanced hemoglobin digestion. The transpeptidation products were often more abundant than the corresponding hydrolysis products. In the absence of GlyGly, hemoglobin peptides produced during digestion were utilized as acceptors leading to the detection of up to 116 different transpeptidation products in a single reaction. <i>P. gingivalis</i> cells were able to digest hemoglobin faster when acceptor peptides derived from human serum albumin were included in the reaction, suggesting that gingipain-catalyzed transpeptidation may be relevant for substrates encountered in vivo. The transpeptidation of host proteins in vivo may potentially lead to the breakdown of immunological tolerance, culminating in autoimmune reactions

Effect of a single CPP-ACP treatment on an established 16 h <i>S</i>. <i>mutans</i> biofilm cultured in a 3-channel flow cell system as determined using COMSTAT analysis of CLSM images.

<p>Values are the mean and standard deviation of three biological replicates. The percentage change relative to the control is shown in brackets.</p

<i>Porphyromonas gingivalis</i> Gingipains Display Transpeptidation Activity

<i>Porphyromonas gingivalis</i> is a keystone periodontal pathogen that has been associated with autoimmune disorders. The cell surface proteases Lys-gingipain (Kgp) and Arg-gingipains (RgpA and RgpB) are major virulence factors, and their proteolytic activity is enhanced by small peptides such as glycylglycine (GlyGly). The reaction kinetics suggested that GlyGly may function as an acceptor molecule for gingipain-catalyzed transpeptidation. Purified gingipains and <i>P. gingivalis</i> whole cells were used to digest selected substrates including human hemoglobin in the presence or absence of peptide acceptors. Mass spectrometric analysis of the substrates digested with gingipains in the presence of GlyGly showed that transpeptidation outcompeted hydrolysis, whereas the trypsin-digested controls exhibited predominantly hydrolysis activity. The transpeptidation levels increased with increasing concentration of GlyGly. Purified gingipains and whole cells exhibited extensive transpeptidation activities on human hemoglobin. All hemoglobin cleavage sites were found to be suitable for GlyGly transpeptidation, and this transpeptidation enhanced hemoglobin digestion. The transpeptidation products were often more abundant than the corresponding hydrolysis products. In the absence of GlyGly, hemoglobin peptides produced during digestion were utilized as acceptors leading to the detection of up to 116 different transpeptidation products in a single reaction. <i>P. gingivalis</i> cells were able to digest hemoglobin faster when acceptor peptides derived from human serum albumin were included in the reaction, suggesting that gingipain-catalyzed transpeptidation may be relevant for substrates encountered in vivo. The transpeptidation of host proteins in vivo may potentially lead to the breakdown of immunological tolerance, culminating in autoimmune reactions

Interaction of gingipain catalytic domains with their propeptides.

<p>(<b>A</b>) Model of RgpB highlighting the N-terminus, catalytic Cys and His residues, and residues that differ between strains in red. The residues that form a surface-exposed conserved patch are predicted to interact with the propeptide. (<b>B</b>) Schematic representation of the inhibition of Kgp by its propeptide. Kgp was modelled using Orchestrar from within Sybyl-8.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065447#pone.0065447-Tripos1" target="_blank">[55]</a> and based on the X-ray crystal structure of RgpB 1cvr.pdb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065447#pone.0065447-Eichinger2" target="_blank">[56]</a>. The propeptide is based on the A chain of the X-ray crystal structure of RgpB interacting with its propeptide 4ief.pdb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065447#pone.0065447-deDiego1" target="_blank">[49]</a>.</p

Oligonucleotides used in the course of this study.

a<p>Restriction endonuclease sites are underlined and stop codons in lowercase.</p

Proteolytic activity time course profiles of rKgp and RgpB using chromogenic and fluorescent substrates.

<p>(A) Time course of rKgp measured as change in absorbance (405 nm) without an inhibitor (•) and with 40 mg/L Kgp propeptide (Kgp-PP) with (□) and without the hexahistidine tag (▵) at 1 mM cysteine in the assay with the Lys-specific chromogenic substrate (GPKNA). The final concentration of rKgp per well is 1.16 mg/L. (B) Time course of RgpB using the fluorescent natural substrate DQ-BSA without RgpB-PP (▪), with 10 mg/L RgpB-PP (⋄) and 1 mM TLCK (□).</p

Relative growth inhibition of <i>P. gingivalis</i> in a protein-based minimal medium (MM) by RgB-propeptide (PP) and Kgp-propeptide (PP).

<p>Relative growth inhibition of <i>P. gingivalis</i> in a protein-based minimal medium (MM) by RgB-propeptide (PP) and Kgp-propeptide (PP).</p