Identification of functional domains of the minor fimbrial antigen involved in the interaction of Porphyromonas gingivalis with oral streptococci

Abstract

Porphyromonas gingivalis is associated with chronic periodontitis and may initially colonize the oral cavity by adhering to streptococci. Adhesion to streptococci is driven by interaction of the minor fimbrial antigen (Mfa1) with streptococcal antigen I/II. We identified the region of antigen I/II required for this interaction and developed small molecule mimetics that inhibited P. gingivalis adherence. However, the functional motifs of Mfa1 involved in the interaction with antigen I/II remain uncharacterized. A series of N- and C-terminal peptide fragments of Mfa1 were expressed and tested for inhibition of P. gingivalis adherence to S. gordonii. This approach identified residues 225-400 of Mfa1 as essential for P. gingivalis adherence. Using the three-dimensional structure of Mfa1, a putative binding cleft was identified using SiteMap and five small molecule mimetics could dock in this site. Site-specific mutation of residues in the predicted cleft, including R240A, W275A, D321A and A357P inhibited the interaction of Mfa1 with streptococci, whereas mutation of residues not in the predicted cleft (V238A, I252F and ΔK253) had no effect. Complementation of an Mfa1-deficient P. gingivalis strain with wild-type mfa1 restored adherence to streptococci, whereas complementation with full-length mfa1 containing the R240A or A357P mutations did not restore adherence. The mutations did not affect polymerization of Mfa1, suggesting that the complemented strains produced intact minor fimbriae. These results identified specific residues and structural motifs required for the Mfa1-antigen I/II interaction and will facilitate the design of small molecule therapeutics to prevent P. gingivalis colonization of the oral cavity.

Keywords: P. gingivalis; Mfa1; adherence; biofilm; streptococci.

Figure 1

Schematic representation of the series of Mfa1 peptide fragments. The full‐length Mfa1 lacking the signal peptide (21–563aa residues), and N‐terminal peptide fragments N194, N225, N279 and N400 encoding residues 21–194, 21–225, 21–279 and 21–400, respectively, are shown. The C‐terminal peptide fragment, C280, is comprised of Mfa1 residues 280–563

Figure 2

(A) Peptide mediated inhibition of P. gingivalis adherence in a dual species biofilm model comprising S. gordonii (red) and P. gingivalis (green). Panels (a) PBS treated, (b), (c), (d), (e), (f) and (g) were treated with peptides N194, N225, N279, N400, full‐length Mfa1 and C280, respectively. (B) Quantification of relative adherence of P. gingivalis and S. gordonii was determined by VOLOCITY software. Comparisons of biofilms formed in PBS (control) with peptide‐treated biofilms were carried out using an unpaired T test. *p < .05

Figure 3

(a) Three‐dimensional structure of the Mfa1 with a composite of five peptidomimetic adherence inhibitory compounds docked in a putative binding cleft. The residues that comprise the predicted binding cleft shown in “a” are shown in red underlined text in the Mfa1 sequence (b) or highlighted in red in the Mfa1 structure (c). The positions of residues R240 and W275 (see text) are shown in green and cyan, respectively

Figure 4

Inhibition of P. gingivalis adherence to S. gordonii by mutated Mfa1 peptides. Biofilms treated with parent and mutated peptides were compared and analyzed using an unpaired T test. ***p < .05, ns, not statistically significant

Figure 5

Complementation of P. gingivalis SMF1 with wild‐type and site‐specific mutants of Mfa1. (a) Cell surface expression of Mfa1 was determined by ELISA using polyclonal anti‐Mfa1 antibodies. Cell surface expression was normalized to the level of Mfa1 expression in wild‐type P. gingivalis 33277. (b) Adherence of P. gingivalis to streptococci was determined using a two species biofilm model as described in Materials and Methods. Adherence data were normalized to the level of adherence of the wild‐type P. gingivalis 33277 and data were analyzed using an unpaired T test. ***p < .001, **p < .05, ns, not statistically significant

Figure 6

Denaturation of P. gingivalis minor fimbriae. P. gingivalis cells were suspended in 1× LDS buffer and incubated either at (a) 100°C or (b) 60°C for 10 min. Extracts were electrophoresed in a 12% Bis‐Tris gel and after transfer, Mfa1 was visualized using polyclonal anti‐Mfa1 antibodies. Lanes 1, P. gingivalis ATCC 33277; 2, P. gingivalis SMF1; 3, P. gingivalis cSMF1; 4, P. gingivalis cMF1‐R240A; and 5, P. gingivalis cSMF1‐A357P; M, size markers