This revised competing renewal application proposes studies that will further develop replacement therapy as an approach for the prevention of dental caries in humans. The first of three proposed projects details methods for the construction of an effector strain of Streptococcus mutans that combines the properties of low virulence and strong colonization potential. The former property is fulfilled by lactate dehydrogenase deficiency which renders the strain less acidogenic. The latter property depends on the production of elevated levels of a small bacteriocin-like molecule that is inhibitory to the growth of other Strep. mutans strains. Construction of the effector strain will use recombinant DNA methodology to yield a genetically well-defined effector strain that is completely stable to reversion. The second project describes methods to analyze the recombinant effector strain with regard to its ability to serve in the replacement therapy of dental caries. The spectrum and levels of its fermentation end-products will be determined, as will the specificity and potency of its bacteriocin- like activity. The cariogenic potential of the effector strain will be analyzed in three different rat models. Its effects on the ecology of plaque and on the general health of the animals will be studied. A rat model will also be used to test the ability of the recombinant effector strain to preemptively colonize the Strep. mutans ecological niche on teeth, and to aggressively displace indigenous, disease-causing strains of this microorganism. In the third project, the chemical composition of the purified bacteriocin- like activity will be determined. The purified activity will be compared to the activity produced in culture with regard to its spectrum of activity. The molecular mechanism of the bacteriocin-like activity will be investigated by determining its site of action in target microorganisms. A rat model will be used to compare the purified activity to sodium fluoride as a topical anti-caries agent. The effects of the bacteriocin-like activity on the microbial composition of plaque and general health of the animals will be analyzed as measures of its safety.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
2R01DE004529-13A2
Application #
3219094
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Project Start
1976-06-01
Project End
1991-12-31
Budget Start
1991-05-01
Budget End
1991-12-31
Support Year
13
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Forsyth Institute
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02142
Hillman, J D; McDonell, E; Cramm, T et al. (2009) A spontaneous lactate dehydrogenase deficient mutant of Streptococcus rattus for use as a probiotic in the prevention of dental caries. J Appl Microbiol 107:1551-8
Smith, Leif; Zachariah, Cherian; Thirumoorthy, Ramanan et al. (2003) Structure and dynamics of the lantibiotic mutacin 1140. Biochemistry 42:10372-84
Wu, Yi; Lee, Seok-Woo; Hillman, Jeffrey D et al. (2002) Identification and testing of Porphyromonas gingivalis virulence genes with a pPGIVET system. Infect Immun 70:928-37
Hillman, J D; Brooks, T A; Michalek, S M et al. (2000) Construction and characterization of an effector strain of Streptococcus mutans for replacement therapy of dental caries. Infect Immun 68:543-9
Smith, L; Novak, J; Rocca, J et al. (2000) Covalent structure of mutacin 1140 and a novel method for the rapid identification of lantibiotics. Eur J Biochem 267:6810-6
Cvitkovitch, D G; Gutierrez, J A; Behari, J et al. (2000) Tn917-lac mutagenesis of Streptococcus mutans to identify environmentally regulated genes. FEMS Microbiol Lett 182:149-54
Gutierrez, J A; Crowley, P J; Cvitkovitch, D G et al. (1999) Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress. Microbiology 145 ( Pt 2):357-66
Hillman, J D; Novak, J; Sagura, E et al. (1998) Genetic and biochemical analysis of mutacin 1140, a lantibiotic from Streptococcus mutans. Infect Immun 66:2743-9
Crowley, P J; Gutierrez, J A; Hillman, J D et al. (1997) Genetic and physiologic analysis of a formyl-tetrahydrofolate synthetase mutant of Streptococcus mutans. J Bacteriol 179:1563-72
Hillman, J D (1996) Principles of microbial ecology and their application to xerostomia-associated opportunistic infections of the oral cavity. Adv Dent Res 10:66-8

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