Lactoferrin: A Roadmap to the Borderland between Caries and Periodontal Disease

Abstract

Lactoferrin is one of a number of multifunctional proteins that are present in or on all mucosal surfaces throughout the body. Levels of lactoferrin are consistently elevated in inflammatory diseases such as arthritis, inflammatory bowel diseases, corneal disease, and periodontitis. Single-nucleotide polymorphisms (SNPs) in lactoferrin have been shown to be present in individuals susceptible to Escherichia coli-induced travelers' diarrhea and in tear fluid derived from virally associated corneal disease. Here, we review data showing a lactoferrin SNP in amino acid position 29 in the antimicrobial region of lactoferrin that acts against caries associated bacteria. This SNP was initially discovered in African American subjects with localized aggressive periodontitis (LAP) who had proximal bone loss but minimal proximal caries. Results were confirmed in a genetic association study of children from Brazil with this same SNP who showed a reduced level of caries. In vitro data indicate that lactoferrin from whole saliva derived from subjects with this SNP, recombinant human lactoferrin containing this SNP, or an 11-mer peptide designed for this SNP kills mutans streptococci associated with caries by >1 log. In contrast, the SNP has minimal effect on Gram-negative species associated with periodontitis. Moreover, periodontally healthy subjects homozygous for this lysine (K) SNP have lactoferrin in their saliva that kills mutans streptococci and have reduced proximal decay. The review summarizes data supporting the ecologic plaque hypothesis and suggests that a genetic variant in lactoferrin with K in position 29 when found in saliva and crevice fluid can influence community biofilm composition. We propose that, for caries, this SNP is ethnicity independent and protective by directly killing caries-provoking bacteria (reducing proximal decay). However, the clinical effect of this SNP in LAP is ethnicity dependent, destructive (increases LAP incidence), and complex with mechanisms still to be determined.

Keywords: aggressive periodontitis; dental caries; ecosystem; saliva; single nucleotide polymorphisms; streptococcus mutans.

Figure 1.

Structure of lactoferrin in 3 dimensions. Region in green represents the N-terminus where the antimicrobial activity is seen (residues 1 to 47). Two lobes show the iron-binding region (identified by the purple ball) in pit area. N-terminus is labeled (N-term), and the amino acid of interest is the lysine region (K) in position 29. Single-nucleotide polymorphism described in text occurs as shift from K to R in position 29 (not shown in this illustration). Illustration also depicts arginine (R) in position 31. Amino acids 1 to 32 are presented in letter form to allow for orientation of N-terminus. LF-1 region is seen as amino acids 1 to 11, and the lactoferricin region occupies amino acids 22 to 47; the region of interest extends from 21 to 31.

Figure 2.

Illustration of proposed direct effect of lactoferricin on Gram-negative cell wall. Gram-negative cell wall is composed of 3 units: an outer O-specific polysaccharide that determines antigenic specificity, a core polysaccharide, and a lipid A inner structure (also known as endotoxin). Lactoferrin initially depolarizes the membrane by attacking the region where fatty acid tail joins polar head of lipid A. Lipid bilayer is composed of upper region head and tail and a lower region that consists of a tail-to-head configuration. Head is represented as circle or hexagon; fatty acid/hydrophobic tail is represented as squiggly lines beneath head. Lactoferricin insertion between leaflets of the bilayer creates holes in membrane and increased membrane permeability resulting in disturbance of membrane function. Lactoferricin participates in creating physical holes (red arrows) and induces production of hydrolases that degrade the cell wall. This activity causes destabilization of the membrane and leads to redistribution of membrane lipids between leaflets of the bilayer. Lf, lactoferrin.

Figure 3.

Indirect effect of lactoferrin by means of host modulation. (A) Lactoferrin can interfere with the ability of lipopolysaccharide-binding protein (LBP) found in serum and emanating from the liver to bind to soluble lipopolysaccharide (LPS). Lactoferrin can also interfere with the transfer of LPS to soluble CD14. (B) Lactoferrin can also interfere with LPS transfer from LBP to membrane bound mCD14. CD14 receptors are found on the surface of monocytes, polymorphonuclear neutrophils, dendritic cells. (C) Transfer of LBP bound LPS from CD14 to myeloid differentiating protein 2 (MD-2) occurs. LPS transfer to MD-2 allows for association of LPS with Toll-like 4 receptors for dimerization. (D) This dimerization of Toll-like receptors causes membrane perturbation and cell signaling resulting in cytokine production. Lf, lactoferrin; TLR4, Toll-like receptor 4.

Figure 4.

Isolation of active anti–Streptococcus mutans fraction from whole saliva from a subject with localized aggressive periodontitis. (A) Peak obtained when fraction 19 was eluted from saliva derived from a subject with localized aggressive periodontitis with salivary activity against S. mutans. Whole saliva was applied to S. mutans affinity column, and then fractions were collected after treatment with glycine hydrochloride for elution of fractions bound to S. mutans. (B) Activity of fraction 19 and its ability to prevent the growth of S. mutans by almost 2 logs as compared to phosphate buffered saline (PBS) control. Fraction 23 reduces growth by <1 log, and fraction 26 shows no reduction. (C) Fraction 19 was applied to a filter paper disc, which was then placed over a confluent inoculum of S. mutans grown on a mitis salivarius agar plate. Two days later, a zone of inhibition was seen around the filter disc coated with fraction 19 added to either PBS or normal saliva. Note that “normal” saliva without fraction 19 added has no anti–S. mutans activity. CFU, colony-forming unit; LAP, localized aggressive periodontitis.

Figure 5.

Proposed effect of lactoferrin single-nucleotide polymorphism (SNP) on dental diseases. Left side of panel (A) illustrates proposed direct effect of the lysine variant of lactoferrin on mutans streptococci and other Gram-positive acid-producing bacteria. This SNP also affects Candida species implicated in caries (data not shown). (B) Right side illustrates proposed effect of lysine variant on aggressive periodontitis. Here, both direct antibacterial and host-modulated immunity effects are seen. Lf, lactoferrin.