WHY TEETH?

The deterioration of oral health in the elderly is accompanied by an increased prevalence of caries and periodontal disease which are risk factors for some systemic diseases and nutrition problems (1), whereas the status of dental hygiene has been recognized as an important determinant of psychological well-being and its aggravation has been associated with an elevated incidence of depressive symptoms (2). In our project, we designed a biosynthetic device to serve as an alternative in preventative dental care for the elderly.

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 Streptococcus mutans (3). 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 designed 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 (4). 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 (5).
The biosynthetic locus was designed by our team in a form allowing for high-yield and fine-tuned expression of mutacin III (Fig. 1). 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.

The information on the design of the mutacin III biosynthetic device can be found here

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 (12). To reflect this, our device could be used to transform E. coli cells and employed as an anti-cariogenic strategy in replacement therapy (Fig. 3). 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 (13). Moreover, the fact that a closely related lantibiotic nisin has been shown to exhibit low in vivo toxicity levels (14) and has been widely used as food preservative from as early as mid 1940s (15) further encourages the prospect of considering the employment of mutacin III as an anti-cariogenic agent.

LanA

In addition to the proposed device, we created a separate Registry entry for lanA, a structural mutacin 1140 propeptide gene which could be used as the first step towards the assembly of an alternative bacteriocin-producing device.

References

1. Gil-Montoya JA, de Mello ALF, Barrios R, Gonzalez-Moles MA, Bravo M. Oral health in the elderly patient and its impact on general well-being: a nonsystematic review. Clin Interv Aging. 2015;10:461–7.
2. Rouxel P, Tsakos G, Chandola T, Watt RG. Oral Health-A Neglected Aspect of Subjective Well-Being in Later Life. J Gerontol B Psychol Sci Soc Sci. 2016 Mar 12.
3. Anderson MH. Changing paradigms in caries management. Curr Opin Dent. 1992 Mar;2:157–62.
4. Abee T. Pore-forming bacteriocins of Gram-positive bacteria and self-protection mechanisms of producer organisms. FEMS Microbiol Lett. 1995 Jun 1;129(1):1–9.
5. 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.
6. Draper LA, Cotter PD, Hill C, Ross RP. Lantibiotic Resistance. Microbiol Mol Biol Rev. 2015 Jun;79(2):171–91.
7. 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.
8. Delves-Broughton J. Nisin and its application as a food preservative. Int J Dairy Technol. 1990 Aug;43(3):73–6.