Streptococcus Mutans Adhesin Biotypes that Match and Predict Individual Caries Development

Abstract

Dental caries, which affects billions of people, is a chronic infectious disease that involves Streptococcus mutans, which is nevertheless a poor predictor of individual caries development. We therefore investigated if adhesin types of S.mutans with sucrose-independent adhesion to host DMBT1 (i.e. SpaP A, B or C) and collagen (i.e. Cnm, Cbm) match and predict individual differences in caries development. The adhesin types were measured in whole saliva by qPCR in 452 12-year-old Swedish children and related to caries at baseline and prospectively at a 5-year follow-up. Strains isolated from the children were explored for genetic and phenotypic properties. The presence of SpaP B and Cnm subtypes coincided with increased 5-year caries increment, and their binding to DMBT1 and saliva correlated with individual caries scores. The SpaP B subtypes are enriched in amino acid substitutions that coincided with caries and binding and specify biotypes of S. mutans with increased acid tolerance. The findings reveal adhesin subtypes of S. mutans that match and predict individual differences in caries development and provide a rationale for individualized oral care.

Keywords: Adhesion; Chronic infections; Dental caries; SpaP; Streptococcus mutans; Virulence.

Fig. 1

Determination of S. mutans adhesin types in whole saliva. Children infected (+) or not infected (−) with A) a dominant or mixed SpaP A, B or C adhesin types and B) a Cnm or Cbm adhesin type of S. mutans by quantitative PCR of whole salivas (pg DNA responses, 95% CI). C) Total colony-forming units, CFU, of S. mutans (ms counts) in whole saliva of infected children as shown by box plots (*p = 0.003, Mann-Whitney U test).

Fig. 2

S. mutans biotypes A, B and C with corresponding SpaP adhesin subtypes of high (B-1) and low (A-1) cariogenicity. Clustering of S. mutans isolates from 35 caries (+) or 35 caries-free (−) extremes of infected children with caries status (+, − or DeFS at 12 years of age) based on neighbor joining and clonal complex analyses of spaP and housekeeping gene sequences. The isolates grouped both into biotypes A, B, and C with distinct SpaP A, B, and C adhesin types and clonal complexes C1–C9, and into genetically related subtypes that coincided with high caries (i.e. B-1, B-2 and C6, C7) or low caries (i.e. A-1 and C1) cases. The high- and low-cariogenic types also differed in acid tolerance and adhesion to DMBT1 and saliva. The clonal complexes share at least six identical alleles with at least one member in the group, all reflecting housekeeping alleles except for C8 which also shared the same spaP B allele. A total of 55 sequence types (ST) occurred among the 70 children. Numbers in parentheses mark STs shared in 15 children or identical STs in 25 children from which 74 additional strains were analyzed (mean 3, range 2 to 8 isolates/child).

Fig. 3

SpaP B- and Cnm-mediated binding to DMBT1 correlates with individual caries scores. A) Correlation of binding of SpaP B to DMBT1 with individual numbers of caries DeFS lesions (binding to saliva generated virtually identical results, data not shown). B) Binding of Cnm-positive and Cnm-negative isolates to collagen, DMBT1, and saliva. Each isolate is marked by a dot and mean adhesion by a black line (Mann-Whitney U test). C) Correlation of binding of Cnm isolates (no. 1–12) to DMBT1 and collagen (r = 0.62, p = 0.031, respectively; data not shown). D) Deduced Cnm protein sequences of isolates no. 1–12. Substitutions are indicated with (|), translational stop codons with (*), and numbers of β-repeats with (o). LP = leading peptide. CWA = cell-wall anchoring motif.

Fig. 4

Adhesins SpaP A, B and C of different cariogenicity. A) Domain (A, V, P and C) structure and sequence identity of SpaP A, B and C and orthologs in various streptococci (reference SpaP A). The SpaP A, B and C adhesins differ by clustered substitutions in the V-domain and by single substitutions in the A-, P- and C- domains. LP = leading peptide, CWA = cell wall anchoring region. B) The clustered amino acid substitutions specific to SpaP A, B, and C localize on opposite sides of a pocket in the V-domain; A as reference, B and C share 29 substitutions (side chains in red), C holds 32 unique substitutions (side chains in yellow). C) SpaP structure with a tip-localized V-domain, intertwined P and A domains followed by C- and N-terminal complexes. Marked are all substitutions associated positively with caries and adhesion (red) or with caries alone (blue) in SpaP B isolates. D) Amino acid substitutions in SpaP B isolates (marked by a row) that coincide positively with caries and adhesion (to DMBT1 and saliva) or with caries alone upon PLS analyses. The substitutions are enriched in B-1 and B-2 isolates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)