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. 2012 Jul;85(2):361-77.
doi: 10.1111/j.1365-2958.2012.08116.x. Epub 2012 Jun 15.

Role of DNA base excision repair in the mutability and virulence of Streptococcus mutans

Kaisha Gonzalez  1 , Roberta C Faustoferri, Robert G Quivey Jr
Affiliations

Role of DNA base excision repair in the mutability and virulence of Streptococcus mutans

Kaisha Gonzalez et al. Mol Microbiol. 2012 Jul.

Abstract

The oral pathogen, Streptococcus mutans, possesses inducible DNA repair defences for protection against pH fluctuations and production of reactive oxygen metabolites such as hydrogen peroxide (H(2) O(2) ), which are present in the oral cavity. DNA base excision repair (BER) has a critical role in genome maintenance by preventing the accumulation of mutations associated with environmental factors and normal products of cellular metabolism. In this study, we examined the consequences of compromising the DNA glycosylases (Fpg and MutY) and endonucleases (Smx and Smn) of the BER pathway and their relative role in adaptation and virulence. Enzymatic characterization of the BER system showed that it protects the organism against the effects of the highly mutagenic lesion, 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-dG). S. mutans strains lacking a functional Fpg, MutY or Smn showed elevated spontaneous mutation frequencies; and, these mutator phenotypes correlated with the ability of the strains to survive killing by acid and oxidative agents. In addition, in the Galleria mellonella virulence model, strains of S. mutans deficient in Fpg, MutY and Smn showed increased virulence as compared with the parent strain. Our results suggest that, for S. mutans, mutator phenotypes, due to loss of BER enzymes, may confer an advantage to virulence of the organism.

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Figures

Figure 1
Figure 1. Growth characteristics of BER-defective strains of S. mutans
Growth of the parent strain (□), and fpg (▲), mutY (●), smx (▲), and smn (●) strains was monitored using a Bioscreen C growth reader. Conditions were as follows: (A) unbuffered BHI medium at 37°C; (B) BHI medium titrated to pH 5; (C) BHI medium at 37°C containing 0.50mM hydrogen peroxide. D.T. indicates the doubling time of the strain, in minutes, determined from the exponential growth rate. Error bars represent one standard deviation from the mean. * indicates statistically different values compared to parent strain, using a Student’s t-test, p-value <0.005; n>5.
Figure 2
Figure 2. Analysis of genomic DNA fragmentation in BER-deficient strains
(A) Cultures of parent strain, S. mutans UA159, and BER mutant strains were grown to mid-exponential phase (OD600 ~0.4) in BHI at 37°C in an atmosphere of 5% (vol/vol) CO2/ 95% air, harvested and treated with 5mM H2O2 for 0, 0.5, 3, 6, 12, 24 hours. Genomic DNA was isolated and separated by electrophoresis on a 1.8% agarose gel. An equal amount of samples (2.5 µg chromosomal DNA) was applied in each lane. (B) Bar graphs display quantitative assessment of DNA fragmentation in agarose gels. Expression of band intensities was quantified with the available software NIH Image J (http://rsb.info.nih.gov/ij/) and the fragmented DNA is expressed as a percentage of the total intensities relative to the 0 hr time point on the same gel. The values for BER strains were statistically different (*, p < 0.01**, p < 0.001, ***, p < 0.0001) relative to parent strain based on Student’s t-test. (C) Survival curves of S. mutans parent strain (□), fpg (▲), mutY (●), smx (▲), and smn (●) following exposure to 5mM H2O2. The graph is plotted as (surviving CFU) / (initial CFU) versus time. Values reported are means ± standard deviations from at least three independent cultures.
Figure 3
Figure 3. 8-oxo-dG formation in DNA of BER-deficient strains
The oxidative DNA adduct, 8-oxo-dG, in genomic DNA of parent and BER mutant strains (fpg, mutY, smx, and smn) grown overnight in BHI at 37°C in an atmosphere of 5% (vol/vol) CO2/ 95% air was quantitatively measured by 8-oxo-dG ELISA. The experiments were repeated at least four times. Error bars represent one standard deviation from the mean. The values for parent strain and BER mutant strains were significantly different (***, p < 0.001) based on Student’s t-test.
Figure 4
Figure 4. DNA nicking activities of BER enzymes with mismatched substrates
End-labeled oligonucleotide substrates were mixed with 25 µg cell-free extracts derived from cultures of parent strain (wt), BER mutant strains (fpg, mutY, smx, and smn) or genetically complemented BER strains (fpgc, mutYc, smxc, and smnc) and incubated at 37°C for 30 minutes. Control lane (-) indicates the lack of cell-free extract. The first base indicates the residue in the sense strand oligo radio-labeled at the 5’ end. The second base represents the base-paired residue in the complement strand. The bands corresponding to product (P) and substrate (S) are indicated. The reaction products were separated using 20% denaturing polyacrylamide gels. The experiments were repeated at least three times, and the results are representative of a typical experiment.
Figure 5
Figure 5. Spontaneous mutation frequencies of S. mutans BER-deficient strains
Mutation frequencies of BER mutant strains (A) or genetically complemented BER strains (B) were determined by screening for rifampicin-resistance. The experiments were replicated a minimum of fifteen times with independent cultures. The values indicate the fold-increase in RifR, relative to the parent strain, and the line represents the mean for all replicates. The frequencies were determined by the ratio between the number of resistant colonies and total CFU in the culture. BER mutant strains (fpg, mutY, and smn) were statistically different as compared to parent strain, using Student’s t-test, ***, p < 0.001. (C) Analysis of mutational changes determined by sequencing of the rpoB gene in DNA isolated from S. mutans and BER mutant strains. Mutational changes were classified as follows: transitions (white), transversions (black), insertions (gray), and deletions (hashed). Values represent the means ± standard deviations measured from at least 15 independent cultures; n≥3.
Figure 6
Figure 6. Sensitivity of S. mutans BER-deficient strains to acid and hydrogen peroxide conditions
Survival curve of a long-term acid challenge of BER mutant strains (A) or genetically complemented BER strains (B) determined by measuring growth over 5 days in a medium containing excess glucose. (C) Photographs of bacterial inhibition zones on BHI agar plates grown after exposure to 1M hydrogen peroxide. Arrows indicate resistant colonies. (D) The graph shows the proportion of H2O2 sensitivity in parent strain, compared to the fpg, mutY, smx, and smn strains. The experiments were repeated at least four times. Error bars represent one standard deviation from the mean. Significant differences (**, p < 0.01; ***, p < 0.001) were based on Student’s t-test.
Figure 7
Figure 7. Interspecies competition assay between S. mutans BER-deficient strains, S. gordonii, and S. sanguinis
Cultures of S. sanguinis or S. gordonii were grown in an atmosphere of 5% (v/v) CO2/95% air at 37 °C, spotted on agar media (BHI agar plus 1% sucrose) and incubated for 24 hr. Aliquots of cultures from BER glycosylase-deficient strains (fpg and mutY) (A), or BER endonuclease-deficient strains (smx and smn) (B), were then inoculated next to the pioneer colonizer, and incubated for 24 hours until plates were photographed. (C) BER mutant strains grown in the presence of 8 µg ml−1 catalase; n≥3.
Figure 8
Figure 8. Virulence of BER-deficient strains, as compared to the parent strain, in the Galleria mellonella model
Kaplan-Meier plots of BER mutant strains (A) or genetically complemented BER strains (B) injected at 1 × 107 CFU/larva. Heat-killed S. mutans UA159 (HK) was used as the inoculation control (- - -). The experiments were replicated a minimum of three times with groups of 20 larvae each, and the results are representative of a typical experiment. Significant differences (***, p < 0.001) are based on the two-tailed Log-rank test.
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References

    1. Aertsen A, Michiels CW. Diversify or die: generation of diversity in response to stress. Crit Rev Microbiol. 2005;31:69–78. - PubMed
    1. Ajdic D, McShan WM, McLaughlin RE, Savic G, Chang J, Carson MB, Primeaux C, Tian R, Kenton S, Jia H, Lin S, Qian Y, Li S, Zhu H, Najar F, Lai H, White J, Roe BA, Ferretti JJ. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci U S A. 2002;99:14434–14439. - PMC - PubMed
    1. Aoki H, Shiroza T, Hayakawa M, Sato S, Kuramitsu HK. Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis. Infect Immunity. 1986;53:587–594. - PMC - PubMed
    1. Aperis G, Fuchs BB, Anderson CA, Warner JE, Calderwood SB, Mylonakis E. Galleria mellonella as a model host to study infection by the Francisella tularensis live vaccine strain. Microbes Infect. 2007;9:729–734. - PMC - PubMed
    1. Aubry-Damon H, Galimand M, Gerbaud G, Courvalin P. rpoB mutation conferring rifampin resistance in Streptococcus pyogenes. Antimicrob Agents Chemother. 2002;46:1571–1573. - PMC - PubMed

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