A lactotransferrin single nucleotide polymorphism demonstrates biological activity that can reduce susceptibility to caries

Abstract

Streptococcus mutans is prominently linked to dental caries. Saliva's influence on caries is incompletely understood. Our goal was to identify a salivary protein with anti-S. mutans activity, characterize its genotype, and determine genotypic variants associated with S. mutans activity and reduced caries. An S. mutans affinity column was used to isolate active moieties from saliva obtained from a subject with minimal caries. The bound and eluted protein was identified as lactotransferrin (LTF) by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis and confirmed by Western blotting with LTF antibody. A single nucleotide polymorphism (SNP) that produced a shift from arginine (R) to lysine (K) at amino acid position 47 in the LTF antimicrobial region (rs: 1126478) killed S. mutans in vitro. Saliva from a subject with moderate caries and with the LTF "wild-type" R form at position 47 had no such activity. A pilot genetic study (n = 30) showed that KK subjects were more likely to have anti-S. mutans activity than RR subjects (P = 0.001; relative risk = 3.6; 95% confidence interval [95% CI] = 1.5 to 11.13). Pretreatment of KK saliva with antibody to LTF reduced S. mutans killing in a dose-dependent manner (P = 0.02). KK subjects were less likely to have caries (P = 0.02). A synthetic 11-mer LTF/K peptide killed S. mutans and other caries-related bacteria, while the LTF/R peptide had no effect (P = 0.01). Our results provide functional evidence that the LTF/K variant results in both anti-S. mutans activity and reduced decay. We suggest that the LTF/K variant can influence oral microbial ecology in general and caries-provoking microbes specifically.

Fig 1

Isolation of anti-S. mutans activity from saliva from an LAP subject and its associated activity. (A) Elution profile of fractions collected from the S. mutans affinity column, with the protein peak at fraction 19. (B) Activity against S. mutans derived from fraction 19. (C) Disk diffusion assay. Anti-S. mutans activity is shown as a zone of inhibition at the edge of the filter disk. The zone is seen in the middle panel (fraction 19 added to PBS) and the right panel (addition of fraction 19 to normal, nonactive saliva).

Fig 2

Identification of LTF as a salivary “factor” with anti-S. mutans activity. (A) SDS-PAGE gel (lanes 1 and 2) and Western blot (lanes 3 and 4). Lane 1 contains the LTF standard, while lane 2 contains fraction 19. Lanes 3 and 4 show that the 78-kDa bands transferred from lanes 1 and 2 reacted with antibody to LTF. (B) Graph showing the 2-log reduction in S. mutans growth with salivary fraction 19 and the increase in S. mutans growth when this fraction was pretreated with a commercially available antibody to LTF (compare the two middle bars). Pretreatment of fraction 19 with BSA had no effect.

Fig 3

Antibacterial activity of saliva from subjects with LTF genotypes. (A) The leftmost bar shows the anti-S. mutans activity of saliva from subjects homozygous for lysine (KK). The next bar shows results for heterozygous (RK) subjects, the following bar shows anti-S. mutans activity of saliva from subjects homozygous for arginine (RR), and the last bar shows the PBS control. Those homozygous for lysine (n = 8) showed an approximately 1.3-log reduction (−1.3 ± 0.10) in survival of S. mutans. Those with a heterozygous genotype had about a 1.1-log reduction, while those homozygous for arginine (n = 10) had no effect (P < 0.01; ANOVA). (B) Dose-dependent reduction observed by diluting saliva from KK subjects. S, undiluted saliva; S1, 1/10 dilution of saliva; S4, 1/2 dilution of saliva. Saliva for dilution testing was derived from 5 KK subjects. Assays were performed in triplicate.

Fig 4

Anti-S. mutans activity in saliva derived from subjects with KK, RK, and RR genotypes, and effect of pretreatment of this saliva with antibodies to LTF. The figure shows the anti-S. mutans activity of 10 RR, 12 RK, and 8 KK individuals and the effect of pretreatment of saliva with antibody to LTF. The KK bars show results for 8 individuals: the left bar shows growth of S. mutans after treatment with antibody, while the second bar shows a significant reduction in S. mutans growth in untreated saliva with active LTF. While 8 of 10 subjects in the RR group had no anti-S. mutans activity, 2 RR subjects had some activity. It is particularly informative that the 2 individuals with activity (RR-X) were not affected by pretreatment of saliva with LTF antibody, suggesting that the anti-S. mutans activity seen was not related to lactotransferrin. The RK bars show a significant reduction in anti-S. mutans activity when saliva was pretreated with antibody to LTF (ANOVA; P ≤ 0.01).