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. 2013 Dec 18:2013:460202.
doi: 10.1155/2013/460202. eCollection 2013.

A new gcrR-deficient Streptococcus mutans mutant for replacement therapy of dental caries

Wenting Pan  1 , Tiantian Mao  2 , Qing-an Xu  1 , Jin Shao  1 , Chang Liu  1 , Mingwen Fan  1
Affiliations

A new gcrR-deficient Streptococcus mutans mutant for replacement therapy of dental caries

Wenting Pan et al. ScientificWorldJournal. .

Abstract

Background: gcrR gene acts as a negative regulator related to sucrose-dependent adherence in S. mutans. It is constructive to test the potential capacity of mutans with gcrR gene deficient in bacteria replacement therapy.

Methods: In this study, we constructed the mutant by homologous recombination. The morphological characteristics of biofilms were analyzed by confocal laser scanning microscopy. S. mutans UA159 and the mutant MS-gcrR-def were inoculated, respectively, or together for competitive testing in vitro and in rat model.

Results: Adhesion assay showed that the adhesion ability of the mutant increased relative to the wild type, especially in the early stage. MS-gcrR-def out-competed S. mutans UA159 in vitro biofilm, and correspondingly coinfection displayed significantly fewer caries in vivo. The former possessed both a lower level of acid production and a stronger colonization potential than S. mutans UA159.

Conclusion: These findings demonstrate that MS-gcrR-def appears to be a good candidate for replacement therapy.

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Figures

Figure 1
Figure 1
(a) To confirm that gcrR was deleted, chromosomal DNA was isolated from the selected transformants as well as UA159 progenitor and used as a template for PCR amplification with the P1/P2 and RT-gcrRF/RT-gcrRR primers. The resulting amplicons (the gcrR upstream-kanamycin resistance (KanR) cassette junction and the part of gcrR gene) were analyzed via agarose gel electrophoresis. Nucleotide sequencing of the purified amplicons with the same primer set was conducted to confirm the gcrR knockout and KanR cassette insertion. (b) Lane 1: the resulting amplicons (chromosomal DNA of MS-gcrR-def as template and RT-gcrRF/RT-gcrRR as primers); lane 2: the resulting amplicons (chromosomal DNA of UA159 as template and RT-gcrRF/RT-gcrRR as primers); lane 3: 1 kb marker (Fermentas). (c) Lanes 1, 2, 3, and 4: the resulting amplicons (chromosomal DNA of MS-gcrR-def as template and P1/P2 as primers); Lane 5, 1 kb marker (Fermentas); Lane 6, the resulting amplicons (chromosomal DNA of UA159 as template and P1/P2 as primers).
Figure 2
Figure 2
The adhesion ability of Streptococcus mutans UA159 as well as the MS-gcrR-def was determined based on the absorbance of the cells on the plate. *Significant differences of adhesion between UA159 and MS-gcrR-def (P < 0.05) were found, while there was no variance in cell growth.
Figure 3
Figure 3
(a) IOD (integrated optical density) of the green and red fluorescence from biofilm cells. (b) Biofilm thickness. The Z section was used to record the biofilm thickness. Vertical lines were chosen randomly to analyze each image. The red and green fluorescence intensities of the dead cells and living cells, respectively, were also quantified using Image Pro-plus. *Significant difference was found between green fluorescence of UA159 and MS-gcrR-def (P < 0.05).
Figure 4
Figure 4
For in vitro acid production, the initial pH of the culture was measured at 7.3 by using a pH meter. Each sample was repeated three times. *Significant differences were found between UA159 and MS-gcrR-def (P < 0.05).
Figure 5
Figure 5
The coculture bacteria were harvested and used to extract RNA; expression of biofilm-associated genes was examined by quantifying the gcrR gene using real-time RT-PCR. *Significant difference observed from comparison of the two groups at every testing time (P < 0.05).
Figure 6
Figure 6
The enamel and dentinal caries scores of rats infected with UA159, MS-gcrR-def, and the mixture of both. The caries levels were scored by Keyes' method. The results are expressed as mean ± S.D. values. Symbols for statistical significance: *significantly different from the group UA159 infection alone (P < 0.05).
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