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. 2008 Jul;76(7):3273-80.
doi: 10.1128/IAI.00366-08. Epub 2008 May 12.

Interaction of Porphyromonas gingivalis with oral streptococci requires a motif that resembles the eukaryotic nuclear receptor box protein-protein interaction domain

Carlo Amorin Daep  1 , Richard J Lamont, Donald R Demuth
Affiliations

Interaction of Porphyromonas gingivalis with oral streptococci requires a motif that resembles the eukaryotic nuclear receptor box protein-protein interaction domain (VSports)

VSports手机版 - Carlo Amorin Daep et al. Infect Immun. 2008 Jul.

Abstract

Porphyromonas gingivalis initially colonizes the oral cavity by interacting with organisms in supragingival plaque, such as the oralis group of oral streptococci. This interaction involves the association of the streptococcal antigen I/II with the minor fimbrial antigen (Mfa1) of P. gingivalis. Our previous studies showed that a peptide (BAR) derived from antigen I/II inhibits P. gingivalis adherence and subsequent biofilm formation on streptococcal substrates. In addition, screening a combinatorial peptide library identified select amino acid substitutions in the NITVK active region of BAR that increased the adherence of P. gingivalis to streptococci. Here we report that incorporating these residues in a synthetic peptide results in more-potent inhibition of P. gingivalis adherence and biofilm formation (I(50) [50% inhibition] at 0 VSports手机版. 52 microM versus I(50) at 1. 25 microM for BAR). In addition, a second structural motif in BAR, comprised of the amino acids KKVQDLLKK, was shown to contribute to P. gingivalis adherence to streptococci. Consistent with this, the KKVQDLLKK and NITVK motifs are conserved only in antigen I/II proteins expressed by the oralis group of streptococci, which interact with P. gingivalis. Interestingly, the primary and secondary structures and the functional characteristics of the amphipathic VQDLL core alpha-helix resemble the consensus nuclear receptor (NR) box protein-protein interacting domain sequence (LXXLL) of eukaryotes. BAR peptides containing amino acid substitutions with the potential to disrupt the secondary structure of VQDLL were less-effective inhibitors of P. gingivalis adherence and biofilm formation, suggesting that the alpha-helical character of VQDLL is important. Furthermore, replacing the lysines that flank VQDLL with acidic amino acids also reduced inhibitory activity, suggesting that the association of VQDLL with Mfa1 may be stabilized by a charge clamp. These results indicate that the Mfa1-interacting interface of streptococcal antigen I/II encompasses both the KKVQDLLKK and NITVK motif and suggest that the adherence of P. gingivalis to streptococci is driven by a protein-protein interaction domain that resembles the eukaryotic NR box. Thus, both motifs must be taken into account in designing potential peptidomimetics that target P. gingivalis adherence and biofilm formation. .

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Figures

FIG. 1.
FIG. 1.
Inhibition of P. gingivalis-streptococcus biofilm formation by BAR peptide analogs. (A) Peptides of 20 amino acids that contain the VQDLL and NITVK motifs of BAR (BAR-VII) or the corresponding regions from the antigen I/II protein of S. mutans (BAR-VIII) were incubated with P. gingivalis cells and inoculated onto adherent streptococci. The resulting biofilms were visualized by confocal laser scanning microscopy and analyzed as described in Materials and Methods. A statistically significant difference (*) in microcolony numbers (P < 0.001) was observed when a pairwise comparison of specific inhibitory activity was made for BAR-VII, BAR-VIII, and untreated cells. Light-gray and dark-gray bars represent 0.845 μM and 3.38 μM peptide, respectively. (B) Decapeptides containing either the VQDLL or NITVK motifs of BAR (BAR-IV and BAR-V, respectively) or the control peptide comprising the region corresponding to NITVK in the antigen I/II protein of S. mutans (BAR-VI) did not inhibit the formation of P. gingivalis biofilms, even when P. gingivalis cells were treated with the peptides at a concentration of 16.9 μM. Light-gray and dark-gray bars represent 3.38 μM and 16.9 μM peptide, respectively. (C) Amino acid substitutions in the VXXLL motif influence biofilm inhibitory activity of the BAR peptide. Analogs of the BAR peptide that were altered at the variable positions in the core VXXLL sequence (BAR-X), the lysine residues flanking VXXLL (BAR-XI), or at the hydrophobic Val and Leu residues of VXXLL (BAR-IX) were synthesized and analyzed for biofilm inhibition as described in Materials and Methods. A functional VXXLL motif requires the maintenance of α-helicity and positive charge through the region. A single asterisk designates a significant difference where the P value is <0.001; a double asterisk indicates a P value of <0.01. For all experiments, percent inhibition was calculated as follows: average number of microcolonies per frame (control peptide) − average number of microcolonies per frame (experimental peptide)/average number of microcolonies per frame (control peptide). At least 30 independent frames from three separate biofilm cultures were analyzed for each peptide sample. Error bars show standard errors of the means.
FIG. 2.
FIG. 2.
Comparison of the BAR region sequences in antigen I/II-related polypeptides of streptococci and lactococci. The VXXLL and NITVK motifs are conserved only in S. gordonii, S. oralis, and S. sanguinis (blue and yellow boxes, respectively). Sequences corresponding to NITVK are also present in the antigen I/II of the other organisms but contain amino acid substitutions that are incompatible with P. gingivalis adherence (see text). NR box-like motifs (underlined) similar to VXXLL and containing hydrophobic residues at the +1, +4, and +5 positions occur in most of the other antigen I/II sequences. A VXXML motif is conserved in antigen I/II of S. downei, S. sobrinus, and S. criceti (red box), and VXXVL is present in S. intermedius and L. lactis (green boxes). Furthermore, these NR box-like motifs are flanked by charged amino acid residues. In all antigen I/II sequences, with the exception of that of Streptococcus pyogenes, proline and alanine residues occupy positions −3 and +8, respectively, where +1 represents the first hydrophobic residue in the NR box-like motif. A consensus sequence of the BAR region of antigen I/II is shown on the last line. The consensus residues shown were present in at least 8 of the 14 antigen I/II sequences analyzed. Residues that were not conserved at this level are indicated by dashes.
FIG. 3.
FIG. 3.
Schematic representation of the interaction of SspB and Mfa1. The adherence of P. gingivalis to streptococci requires the VXXLL and NITVK motifs of SspB. The initial interaction between SspB (bottom) and Mfa1 (top) may occur via the amphipathic, α-helical VXXLL motif. This interaction may be stabilized by a charge clamp that involves hydrogen bonding or electrostatic interactions between the lysine residues that flank VXXLL and adjacent amino acids in Mfa1. Our current and previous results suggest that the specificity of the Mfa interaction with antigen I/II proteins from oralis streptococci is dictated by the downstream NITVK motif and that the alteration of Asn1182 and Val1185 can either facilitate or inhibit the association of SspB and Mfa1 (20).
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References (VSports app下载)

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