GlmS and NagB regulate amino sugar metabolism in opposing directions and affect streptococcus mutans virulence

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Abstract

Streptococcus mutans is a cariogenic pathogen that produces an extracellular polysaccharide (glucan) from dietary sugars, which allows it to establish a reproductive niche and secrete acids that degrade tooth enamel. While two enzymes (GlmS and NagB) are known to be key factors affecting the entrance of amino sugars into glycolysis and cell wall synthesis in several other bacteria, their roles in S. mutans remain unclear. Therefore, we investigated the roles of GlmS and NagB in S. mutans sugar metabolism and determined whether they have an effect on virulence. NagB expression increased in the presence of GlcNAc while GlmS expression decreased, suggesting that the regulation of these enzymes, which functionally oppose one another, is dependent on the concentration of environmental GlcNAc. A glmS-inactivated mutant could not grow in the absence of GlcNAc, while nagB-inactivated mutant growth was decreased in the presence of GlcNAc. Also, nagB inactivation was found to decrease the expression of virulence factors, including cell-surface protein antigen and glucosyltransferase, and to decrease biofilm formation and saliva-induced S. mutans aggregation, while glmS inactivation had the opposite effects on virulence factor expression and bacterial aggregation. Our results suggest that GlmS and NagB function in sugar metabolism in opposing directions, increasing and decreasing S. mutans virulence, respectively. © 2012 Kawada-Matsuo et al.

Figures

  • Table 1. Strains and plasmids used in this study.
  • Figure 1. Streptococcus mutans growth curves in various bacterial media. A small aliquot of an overnight culture of WT (circle), glmS mutant (square), nagB mutant (triangle), glmS complement strain (square, dashed line) or nagB complement strain (triangle, dashed line) cells was inoculated
  • Table 2. Doubling times of UA159, glmS or nagB deletion mutant and its complement strains grown in TSB or CDM-G50.
  • Figure 2. Effect of GlcNAc on GlmS and NagB expression. After washing a WT UA159 overnight culture, a small aliquot was inoculated into CDM-G50 containing various concentrations of GlcNAc and then incubated at 37uC with 5% CO2. When the sample reached an OD660 of 0.5, the cells were collected. Next, the samples were prepared for immunoblotting (A), quantitative PCR (B), and Northern blotting (C) as described in the Materials and Methods. Panel B: The diamonds and squares represent glmS and nagB expression, respectively.
  • Figure 3. Time course of GlmS and NagB expression after the addition of GlcNAc. Streptococcus mutans UA159 was grown in CDM-G50 at 37uC with 5% CO2. When the cells reached an OD660 of 0.5, GlcNAc was added at 2.5 or 25 mM. Cells were collected 10, 30, and 60 min after the addition of GlcNAc. Next, the cells were prepared for immunoblotting (A) and quantitative PCR (B) as described in the Materials and Methods. Panel B: The symbols represent samples without GlcNAc (diamonds), with 3 mM GlcNAc (squares), and with 25 mM GlcNAc (triangles). Normal and dashed lines represent glmS and nagB expression, respectively. doi:10.1371/journal.pone.0033382.g003
  • Figure 4. GlmS, NagB, and virulence factor expression in WT UA159 cells, its deletion mutants, and complementation strains. After washing overnight cultures of WT and mutant cells, a small aliquot of each was inoculated into CDM-G50 containing various concentrations of GlcNAc and incubated at 37uC with 5% CO2. At an OD660 of 0.5, the cells were collected and prepared for immunoblotting (A) and quantitative PCR (B) as described in the Materials and Methods. *p , 0.05, as determined by Tukey’s HSD; **p , 0.005, as determined by Tukey’s HSD. doi:10.1371/journal.pone.0033382.g004
  • Figure 5. Association of VicRK with virulence factor expression in the glmS and nagB mutants. After washing overnight cultures of WT and mutant cells, a small aliquot of each was inoculated into TSB containing 50 mM GlcNAc and incubated at 37uC with 5% CO2. When the samples reached an OD660 of 0.5, cells were collected and prepared for quantitative PCR as described in the Materials and Methods. (A) vicR expression in WT UA159, vicK mutant, glmS deletion mutant, nagB deletion mutant, glmS+vicK double mutant, nagB+vicK double mutant, glmS complement strain, and nagB complement strain cells. *p , 0.05, as determined by Tukey’s HSD; **p , 0.005, as determined by Tukey’s HSD. (B) gtfB, gtfC, and spaP expression in UA159, vicK mutant, glmS+vicK double mutant, and nagB+vicK double mutant strains. *p , 0.05, as determined by Tukey’s HSD; **p , 0.005, as determined by Tukey’s HSD. doi:10.1371/journal.pone.0033382.g005
  • Figure 6. glmS and nagB expression in ccpA mutant cells. After washing overnight cultures of WT and ccpA mutant cells, a small aliquot of each

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Kawada-Matsuo, M., Mazda, Y., Oogai, Y., Kajiya, M., Kawai, T., Yamada, S., … Komatsuzawa, H. (2012). GlmS and NagB regulate amino sugar metabolism in opposing directions and affect streptococcus mutans virulence. PLoS ONE, 7(3). https://doi.org/10.1371/journal.pone.0033382

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