Abstract
Dental caries is the most prevalent infectious disease of humans and can ultimately result in destruction of affected teeth. Recent studies indicate that there is a resurgence of dental caries occurrence in both developing and developed countries. Current methods of treatment are expensive, and a cost-effective preventative would be of great value. The mutans streptococci (MS) levels present in the human oral cavity provide one measure of the current dental caries activity in the host. Important virulence factors of the MS include their ability to utilise dietary sucrose resulting in: (1) formation of extracellular polysaccharides (EPS) that facilitate their attachment and accumulation within dental plaque; (2) storage of intracellular polysaccharides (IPS) as a latent source of energy and; (3) fermentation with formation of high concentrations of lactic acid. The combination of these activities results in the creation and maintenance of a low plaque pH and subsequent demineralisation of the enamel with associated formation of carious lesions.
Current prevention regimens have not adequately limited the development of dental caries. Prophylactic dosing with classical antibiotics has been investigated for the prevention of dental caries, and while this approach can achieve some reduction in both MS levels and caries development it can also seriously damage the protective normal microflora, enhancing the likelihood that opportunistic infections or resistance to medically-important antibiotics may occur. A more focused alternative to the use of classical chemotherapeutic antibiotics may be the deployment of bacteriocins (proteinaceous anti-bacterial agents) which have much narrower inhibition spectra. A potential impediment however to the use of bacteriocins is their relatively large size (most are >2000 Da) which may limit their penetration of the extracellular polysaccharide (EPS) present in the extracellular matrix of dental plaque.
A primary objective of this study was to use plaque biofilm models to document the extent to which bacterially-produced EPS interferes with the activity of bacteriocins against the target cells of MS. Several in vitro models were developed and applied to assess the effect EPS has on the inhibitory activity of bacteriocins of different sizes. The enzyme dextranase was then tested for its ability to enhance the penetration of anti-MS bacteriocins within the biofilm environment.
Initially, a selection of known bacteriocin-producing bacteria were tested for both their dextranase and anti-MS inhibitory activities. This screen identified Streptococcus salivarius strains JH and M18 as producers of both anti-MS and dextranase activities, with strain JH having particularly strong dextranase activity. Growth of these strains in an in vitro saliva model system enhanced the dextranase activity of both strains when compared to the levels produced in traditional growth media.
Further research was undertaken to identify the cause of the enhanced dextranase activity of strain JH. Analysis of the dextranase sequence in strain JH identified a five amino acid insert not present in the dextranase sequence of the relatively weak dextranase producer S. salivarius JIM8777. Three dimensional protein modeling revealed that this insert resulted in a repositioning of the C-terminal end of the conserved region of the strain JH dextranase, moving it closer to the dextran-binding domain. It is proposed that this conformational change is at least in part responsible for the increase in the dextranase activity of strain JH.
Two novel EPS models were developed to further test the inhibitory effect of EPS on bacteriocin efficacy and the possibility of using dextranase to aid bacteriocin penetration of the EPS. The first, an agar overlay model, enabled several MS bacteria to be tested simultaneously. With use of this model a range of effects specific to the bacteriocin producer strain were demonstrated, ranging from complete loss of inhibitory activity in the presence of EPS to no apparent change in activity. The 27800 Da bacteriocin zoocin A had greatly-reduced activity against MS contained within an EPS matrix. By comparison the 2270 Da mutacin B-Ny266 retained anti-MS activity irrespective of the presence of EPS.
The second model used glass fibre filters to provide a three dimensional matrix for the deposition of EPS. Pretreatment of this EPS-containing matrix with dextranase prior to zoocin A titration enhanced target cell killing, presumably due to the hydrolysis of EPS facilitating penetration of the zoocin A through the matrix.
Another aspect of the study involved attempting to develop a natural derivative of strain JH that would retain the existing dextranase and bacteriocin activities of strain JH but would now be supplemented with the anti-MS activity of strain M18. The objective was to create a candidate probiotic strain comprising both broad-spectrum anti-MS BLIS and strong dextranase activities. Although the attempts to create the proposed strain JH variant were not successful, a potentially similar outcome could be achieved in practice by combined administration of probiotic strains JH and M18.
In summary, this study has shown that bacterial EPS can potentially reduce the inhibitory activity of anti-MS bacteriocins. This reduction appears to specifically relate to larger bacteriocins. Concomitant application of dextranase to hydrolyse dextran (a major glucan present in EPS) could potentially increase the killing efficacy of anti-MS bacteriocins within the plaque ecosystem. A possible probiotic approach to increasing the permeability of plaque was identified through the use of S. salivarius strain JH, which was found to produce higher levels of dextranase activity than any other strain tested. Analysis of the underlying cause of strain JH’s apparently increased dextranase activity identified that an insert in the conserved region of the dextranase repositions this region closer to the dextran binding site, possibly improving the dextran binding efficacy of the enzyme and leading to increased bioactivity.
Therefore, the results of this study support the hypothesis that in situ biosynthesis of dextranase may be a beneficial option to synergistically enhance the killing efficacy of relatively-large anti-MS bacteriocins. Strain JH is recommended as a particularly good candidate for the probiotic control of dental caries, especially if administered in combination with additional strongly anti-MS strains such as M18.