WHY TEETH?

Add something about the link between teeth and healthy ageing.

The problem: Biofilm formation and cariogenesis

The oral cavity is inhabited by a wide range of interacting communities of metabolically and structurally organized microorganisms which synthesize an extracellular polysaccharide matrix (EPS) enabling them to adhere to the surface of the teeth and assemble in matrix-embedded biofilms. Progressing biofilm accumulation puts the bacteria under increasing metabolic stress which leads to localized metabolite and acid accumulation and a shift in the dynamic homeostasis towards acid-tolerating species such as Gram-positive (1) A resultant decrease in pH causes tooth demineralization and constitutes a mechanism of dental caries.

Our approach: a bacteriocin producing device

A decrease in biofilm formation caused by interference with the viability of certain bacterial species presents an approach towards limiting cariogenesis. Our team engineered a locus capable of producing and exporting a mature form of an antimicrobial peptide known as mutacin III, first identified in Streptococcus mutans UA787 isolated from a caries-active white female patient in the late 1980s (2). Mutacin III is effective against a wide range of Gram positive bacteria implicated in dental caries, e.g. other strains of Streptococcus mutans and Actinomyces naeslundii, while Gram-negative bacteria are resistant to inhibition (3). The biosynthetic locus was engineered by our team in a form allowing for high-yield and fine-tuned expression of mutacin III. We placed a strong T7 promoter upstream of mutA to obtain high levels of the propeptide, a repressible pTet promoter upstream of the mutBCDP co-transcription unit and an inducible araBAD promoter for the mutT gene, coding for the ATP-binding-cassette-like transporter of mutacin III. All illegal restriction sites were removed from the endogenous sequences by silent mutagenesis.

Mutacin III is a ribosomally synthesized 22 amino acid screw-shaped lanthionine-containing peptide. The biosynthesis of mutacin III involves the expression of the structural gene mutA to make a prepropeptide, comprising a C-terminal propeptide and an N-terminal leader peptide from which the former undergoes processing and the latter is cleaved off before export into the medium (2). The specific post-translational modifications make mutacin III distinct from other bacteriocins and are introduced by enzymes coded for by other genes in the locus (mutBCDP). Basing on sequence homologies of genes in the locus with those of other lantibiotic biosynthetic loci it can be inferred that these enzymes catalyze the dehydration (mutB) and cyclization (mutC) of the propeptide serine and threonine residues which can condense with a neighboring cysteine residue (4) leading to the formation of lanthionine or methyllanthionine (thioether) bridges, respectively. The enzyme coded by mutD catalyzes the oxidative decarboxylation of the C-terminal cysteine residue (5) while the product of mutP is a serine protease which cleaves the leader peptide and is likely the last step in the biosynthesis (6). The mature mutacin III is composed of rings connected by flexible linkers (Fig. 1) which may be important in the mechanism of bacteriocidal activity (7). Following export, the peptide is believed to form transmembrane pores as monomer aggregates leading to membrane disruption and efflux of cellular components (8). The content of anionic phospholipids in the membrane has been suggested to be an important factor influencing initial binding – mutacin III has a net positive charge whereas Gram-positive bacteria have a high relative amount of anionic lipids (9).

In one investigation of the activity of mutacin-related lantibiotic gallidermin it became clear that lantibiotics are more effective in preventing biofilm formation rather than in exterminating microorganisms already embedded in biofilms (10). To reflect this, our device could be used to transform E. coli cells and employed as an anti-cariogenic strategy in replacement therapy. Such a novel bacterial strain would demonstrate features of a successful effector strain as it would not cause disease by itself and because it could displace the host pathogenic bacteria. Importantly, there are very few existing examples of lantibiotic resistance compared with antibiotics and only one mechanism of resistance to mutacin III, known as CprRK in Clostridium difficile, has been established (11). Moreover, the fact that a closely related lantibiotic nisin has been shown to exhibit low in vivo toxicity levels (12) and has been widely used as food preservative from as early as mid 1940s (13) further encourages the prospect of considering the employment of mutacin III as an anti-cariogenic agent.

LanA TBC

References

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10. Saising J, Dube L, Ziebandt A-K, Voravuthikunchai SP, Nega M, Gotz F. Activity of Gallidermin on Staphylococcus aureus and Staphylococcus epidermidis Biofilms. Antimicrob Agents Chemother. 2012 Nov 1;56(11):5804–10.
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12. Hagiwara A, Imai N, Nakashima H, Toda Y, Kawabe M, Furukawa F, et al. A 90-day oral toxicity study of nisin A, an anti-microbial peptide derived from Lactococcus lactis subsp. lactis, in F344 rats. Food Chem Toxicol. 2010 Aug;48(8-9):2421–8.
13. Delves-Broughton J. Nisin and its application as a food preservative. Int J Dairy Technol. 1990 Aug;43(3):73–6.