Host-derived pentapeptide affecting adhesion, proliferation, and local pH in biofilm communities composed of Streptococcus and Actinomyces species

Abstract

Salivary proline-rich proteins (PRPs) attach commensal Actinomyces and Streptococcus species to teeth. Here, gel filtration, mass spectrometry and Edman degradation were applied to show the release of a pentapeptide, RGRPQ, from PRP-1 upon proteolysis by Streptococcus gordonii. Moreover, synthetic RGRPQ and derivatives were used to investigate associated innate properties and responsible motifs. The RGRPQ peptide increased 2.5-fold the growth rate of S. gordonii via a Q-dependent sequence motif and selectively stimulated oral colonization of this organism in a rat model in vivo. In contrast, the growth of Streptococcus mutans, implicated in caries, was not affected. While the entire RGRPQ sequence was required to block sucrose-induced pH-decrease by S. gordonii and S. mutans, the N-terminal Arg residue mediated the pH increase (i.e., ammonia production) by S. gordonii alone (which exhibits Arg catabolism to ammonia). Strains of commensal viridans streptococci exhibited PRP degradation and Arg catabolism, whereas cariogenic species did not. The RGRPQ peptide mediated via a differential Q-dependent sequence motif, adhesion inhibition, and desorption of PRP-1-binding strains of A. naeslundii genospecies 2 (5 of 10 strains) but not of S. gordonii (n=5). The inhibitable A. naeslundii strains alone displayed the same binding profile as S. gordonii to hybrid peptides terminating in RGRPQ or GQSPQ, derived from the middle or C-terminal segments of PRP-1. The present findings indicate the presence of a host-bacterium interaction in which a host peptide released by bacterial proteolysis affects key properties in biofilm formation.

FIG. 1.

Identification of a pentapeptide, RGRPQ, derived from proteolysis of PRP-1 by S. gordonii SK12. (A) Isolation of the RGRPQ peptide (marked) by gel filtration of the PRP-1 proteolysis mixture. Synthetic RGRPQ was used as a reference to localize the RGRPQ peptide. (B) Nano-electrospray mass spectrometry of the RGRPQ peptide recovered from gel filtration reveals the expected monoisotopic mass 613.46 Da. (C) Edman degradation of the fraction from gel filtration reveals the sequence RGRPQ.

FIG. 2.

Illustration of RGRPQ innate properties and tentative motifs responsible for each property. (A) Effect of RGRPQ derivatives on the growth of S. gordonii SK12 when cultured in a minimum growth medium in the presence or absence (control) of peptide. The bacterial cell numbers were estimated from measuring the A₅₅₀ of the cell culture. The mean absorbance values and the SEM from one experimental run with triplicate determinations are shown. (B) Effect of RGRPQ derivatives on the adhesion (percent binding bacteria out of total added) of A. naeslundii T14V to PRP-1-coated hydroxyapatite beads. (C) RGRPQ innate properties and tentative RGRPQ motifs for each property. Lines indicate each tentative motif with suggested key determinants marked by a broadened boldface line. The proliferation XXXQ motif is marked by a dotted line to emphasize its unique nature compared to the XXXQ adhesion motif. The potential AT hook marks the GRP motif present in some transcriptional factors.

FIG. 3.

(A and B) Promotion of colonization of S. gordonii SK12 (A), but not of S. mutans NG8 (B), by oral administration of RGRPQ peptide (○) compared to control (sterile water ▪) in a rat model. Rats (n = 14 peptide group, n = 14 control group) were coinfected with S. gordonii SK12 and S. mutans NG8 at the start of the study (day 0). Bacterial numbers (in CFU) are presented as means of the 14 animals in each group, and bars show the SEM. Asterisks indicate statistically significant differences at given time points (***, P < 0.001) based on comparisons between pairs of groups at each time point using the Wilcoxon rank-sum test. Also shown in panel A are the median and the individual S. gordonii CFU values for the RGRPQ-treated rats at the 4- and 11-day time points.

FIG. 4.

(A) Selective inhibition of the adhesion of certain A. naeslundii genospecies 2 strains by the RGRPQ peptide. Adhesion was determined by measuring the aggregation of the bacteria with PRP-1-coated latex beads in the presence (+RGRPQ) or absence (−RGRPQ) of peptide. Score 5 indicates clumping of aggregates in clear solution, and score 0 indicates no aggregates (see Materials and Methods). (B and C) Adhesion of A. naeslundii and S. gordonii strains to hybrid peptides with a RGRPQ or GQSPQ terminus. Adhesion (percent binding bacteria of total added bacteria) of ³⁵S-labeled A. naeslundii strains T14V and LY7 and S. gordonii Blackburn to a hybrid/RGRPQ peptide (B) or a hybrid/GQSPQ peptide (C) coated onto hydroxyapatite beads. The hybrid peptides are designed with the calcium binding 15-amino-acid N-terminal segment of statherin and a proline residue followed by RGRPQ or GQSPQ, derived from the middle or C-terminal segments of PRP-1, respectively.