Cariogenicity of Streptococcus mutans Strains with Defects in Fructan Metabolism Assessed in a Program-fed Specific-pathogen-free Rat Model

Abstract

To define the role of dental plaque fructans and the enzymes involved in their metabolism in the initiation and progression of dental caries, we constructed otherwiseisogenic mutants of Streptococcus mutans defective in the ability to synthesize fructans, to degrade fructans, or to do both. The cariogenic potential of these organisms was evaluated in a specific-pathogen-free rat model in which the feeding patterns of the animals were controlled by means of a Konig-Hofer programed feeder. Specifically, rats were infected with wild-type S. mutans UA159 or derivatives of this strain which contained an insertionally-inactivated fructanase (fruA) gene, fructosyltransferase (ftf) gene, or which had both genes inactivated. The animals were fed 17 meals per day of Diet 2000 containing 56% sucrose at 70-minute intervals for five weeks, and caries experience was evaluated. Animals infected with S. mutans with a mutated fruA gene only had statistically significant decreases in sulcal caries severity. Such a decrease was not observed in previous studies with ad libitum-fed animals (Wexler et al., 1992). The manifestation of diminished virulence in the programed feeding model, but not in ad libitum-fed animals, supports the concept that the primary contribution of FruA to virulence is through the utilization of fructans storage polysaccharides. Animals infected with strains carrying the ftf mutation or simultaneous mutations in ftf and fruA did not display decreased virulence, perhaps indicating that sucrose utilization pathways may compete for substrate in vivo, or that accumulation of fructans may affect the ecology or the physicochemical characteristics of dental plaque in such a way as to reduce its cariogenic potential. The results of this study also emphasize that the contribution of a particular virulence determinant to the caries process may be highly dependent on the experimental design, feeding regimen and diet, and the presence or absence of other enzymatic activities.

Keywords

genes (bacterial)polysaccharides dental caries sucrose Streptococcus mutans.

Get full access to this article

View all access options for this article.

References

Ausubel FM , Brent R. , Kingston RE , Moore DD , Seidman JG , Smith JA , et al. (1989). Current protocols in molecular biology . New York, NY: J. Wiley and Sons .

Birkhed D. , Rosell KG , Granath K. (1979). Structure of extracellular water-soluble polysaccharides synthesized from sucrose by oral strains of Streptococcus mutans, Streptococcus salivarius, Streptococcus sanguis, and Actinomyces viscosus. Arch Oral Biol 24:53-61.

Bowen WH , Amsbaugh SM , Monell-Torrens S. , Brunelle J. (1983). Effects of varying intervals between meals on dental caries in rats. Caries Res 17:466-471.

Burne RA (1991). Oral ecological disasters: the role of short-term extracellular storage polysaccharides. In: Cariology for the nineties. Bowen WH , Tabak LA , editors. Rochester, NY: University of Rochester Press, pp. 351-364.

Burne RA , Penders JE (1992). Characterization of the Streptococcus mutans GS-5 fruA gene encoding exo-beta-D-fructosidase. Infect Immun 60:4621-4632.

Burne RA , Rubinfeld B. , Bowen WH , Yasbin RE (1986). Cloning and expression of a Streptococcus mutans glucosyltransferase gene in Bacillus subtilis. Gene 47:201-209.

DaCosta T. , Gibbons RJ (1968). Hydrolysis of levan by human plaque streptococci . Arch Oral Biol 13:609-617.

Ebisu S. , Kato K. , Kotani S. , Misaki A. (1975). Structural differences in the fructans elaborated by Streptococcus mutans and Strep. salivarius. J Biochem 78:879-887.

Freedman ML , Tanzer JM (1982). Use of mutants to study the glucan-associated pathophysiology of Streptococcus mutans. In: Microbiology-1982 . Schlessinger D , editor. Washington, DC: American Society for Microbiology, pp. 186-190.

10.

Harris GS , Michalek SM , Curtiss R Iii (1992). Cloning of a locus involved in Streptococcus mutans intracellular polysaccharide accumulation and virulence testing of an intracellular polysaccharide-deficient mutant. Infect Immun 60:3175-3185.

11.

Keyes PH (1958). Dental caries in the molar teeth of rats. I. Distribution of lesions induced by high carbohydrate, low-fat diets. J Dent Res 37:1077-1087.

12.

Konig KG , Schmid P. , Schmid R. (1968). An apparatus for frequency controlled feeding of small rodents and its use in dental caries experiments. Arch Oral Biol 13:13-26.

13.

Kuramitsu HK (1987). Utilization of a mini mu transposon to construct defined mutants in Streptococcus mutans. Molec Microbiol 1:229-232.

14.

Madison KM , Bowen WH , Pearson SK , Falany JL (1991). Enhancing the virulence of Streptococcus sobrinus in rats. J Dent Res 70:38-43.

15.

Maniatis T. , Fritsch EF , Sambrook J. (1982). Molecular cloning: A laboratory manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory .

16.

Munro C. , Michalek SM , Macrina FL (1991). Cariogenicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by allelic exchange. Infect Immun 59:2316-2323.

17.

Murchison HH , Barrett JF , Cardineau GA , Curtiss R Iii (1986). Transformation of Streptococcus mutans with chromosomal and shuttle plasmid DNAs. Infect Immun 54:273-282.

18.

Schroeder VA , Michalek SM , Macrina FL (1989). Biochemical characterization and evaluation of virulence of a fructosyltransferase-deficient mutant of Streptococcus mutans V403. Infect Immun 57:3560-3569.

19.

Senghas E. , Jones JM , Yamamoto M. , Gawron-Burke C. , Clewell DB (1988). Genetic organization of the bacterial conjugative transposon Tn916. J Bacteriol 170:245-249.

20.

Shern RJ , Monell-Torrens E. , Couet KM , Kingman A. , Bowen WH (1981). Post-mortem evaluation of dental plaque in rats . In: Animal models in cariology. Tanzer JM , editor. Spec suppl, Microbiol Abstr. Washington, DC: IRL Press, pp. 215-221.

21.

Shimamura A. , Tsuboi K. , Nagase T. , Ito M. , Tsumori H. , Mukasa H. (1987). Structural determination of D-fructans from Streptococcus mutans, serotype b, c, e, and f strains, by 13C-n.m.r. spectroscopy. Carbohydr Res 165:150-154.

22.

Shiroza T. , Kuramitsu HK (1988). Sequence analysis of the Streptococcus mutans fructosyltransferase gene and flanking regions. J Bacteriol 170:810-816.

23.

Tanzer JM , Freedman ML , Woodiel FN , Eifert RL , Rinehimer LA , editors ( 1976). Association of Streptococcus mutans virulence with synthesis of intracellular polysaccharide. In: Proceedings, Microbial aspects of dental caries. Spec suppl, Microbiol Abstr. Stiles HM , Loesche WL , O'Brien TL , editors. London, England: Information Retrieval Inc., pp. 597-616.

24.

van Houte J. , Jansen HM (1968). Levan degradation by streptococci isolated from human dental plaque. Arch Oral Biol 13:827-830.

25.

Wexler DL , Penders JE , Bowen WH , Burne RA (1992). Characteristics and cariogenicity of a fructanase-defective Streptococcus mutans strain. Infect Immun 60:3673-3681.

26.

Yamashita Y. , Bowen WH , Burne RA , Kuramitsu HK (1993). Role of the Streptococcus mutans gtf genes in carious induction in the specific-pathogen-free rat model. Infect Immun 61:3811-3817.