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Hyperosmotic response of streptococcus mutans: from microscopic physiology to transcriptomic profile.

Liu C, Niu Y, Zhou X, Zhang K, Cheng L, Li M, Li Y, Wang R, Yang Y, Xu X - BMC Microbiol. (2013)

Bottom Line: We found that the hyperosmotic condition was able to initiate S. mutans biofilm dispersal by reducing both microbial content and extracellular polysaccharides matrix.Further analysis of microarray data showed significant up-regulation of genes and pathways involved in carbohydrate metabolism.Hyperosmotic condition induces significant stress response on S. mutans at both phenotypic and transcriptomic levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, PR China. nixux1982@hotmail.com.

ABSTRACT

Background: Oral streptococci metabolize carbohydrate to produce organic acids, which not only decrease the environmental pH, but also increase osmolality of dental plaque fluid due to tooth demineralization and consequent calcium and phosphate accumulation. Despite these unfavorable environmental changes, the bacteria continue to thrive. The aim of this study was to obtain a global view on strategies taken by Streptococcus mutans to deal with physiologically relevant elevated osmolality, and perseveres within a cariogenic dental plaque.

Results: We investigated phenotypic change of S. mutans biofilm upon hyperosmotic challenge. We found that the hyperosmotic condition was able to initiate S. mutans biofilm dispersal by reducing both microbial content and extracellular polysaccharides matrix. We then used whole-genome microarray with quantitative RT-PCR validation to systemically investigate the underlying molecular machineries of this bacterium in response to the hyperosmotic stimuli. Among those identified 40 deferentially regulated genes, down-regulation of gtfB and comC were believed to be responsible for the observed biofilm dispersal. Further analysis of microarray data showed significant up-regulation of genes and pathways involved in carbohydrate metabolism. Specific genes involved in heat shock response and acid tolerance were also upregulated, indicating potential cross-talk between hyperosmotic and other environmental stress.

Conclusions: Hyperosmotic condition induces significant stress response on S. mutans at both phenotypic and transcriptomic levels. In the meantime, it may take full advantage of these environmental stimuli to better fit the fluctuating environments within oral cavity, and thus emerges as numeric-predominant bacterium under cariogenic conditions.

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Phenotypic characteristics of S. mutans after short-term and long-term hyperosmotic stimuli. (A) Representative Scanning Electronic Microscopy images of S. mutans biofilm on glass surfaces. Images shown were taken at 1000 ×, 5000 × and 10000 × magnification. (B) Representative 3D rendering images of S. mutans biofilms without NaCl for 24 h (upper left), versus with 0.4 M NaCl for either 15 min (upper right) or 24 h (lower left). Bacterial cells and EPS are in situ labelled. Green, the bacteria (SYTO 9); red, the EPS (Alexa Fluor 647). At the right of each panel, the two channels are displayed separately, while the merged image is displayed at the left. Lateral (side) views of each biofilm are displayed at the bottom. Quantitative determination of S. mutans biofilms (lower right) confocal image stacks analyzed by the image-processing software COMSTAT. Results were averaged from 3 independent experiments and are presented as mean ± standard deviation. *, P ≤ 0.05.
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Figure 2: Phenotypic characteristics of S. mutans after short-term and long-term hyperosmotic stimuli. (A) Representative Scanning Electronic Microscopy images of S. mutans biofilm on glass surfaces. Images shown were taken at 1000 ×, 5000 × and 10000 × magnification. (B) Representative 3D rendering images of S. mutans biofilms without NaCl for 24 h (upper left), versus with 0.4 M NaCl for either 15 min (upper right) or 24 h (lower left). Bacterial cells and EPS are in situ labelled. Green, the bacteria (SYTO 9); red, the EPS (Alexa Fluor 647). At the right of each panel, the two channels are displayed separately, while the merged image is displayed at the left. Lateral (side) views of each biofilm are displayed at the bottom. Quantitative determination of S. mutans biofilms (lower right) confocal image stacks analyzed by the image-processing software COMSTAT. Results were averaged from 3 independent experiments and are presented as mean ± standard deviation. *, P ≤ 0.05.

Mentions: By constructing the growth curve of S. mutans under increasing concentrations of NaCl, we found that 0.4 M of NaCl provided the sub-inhibitory level of osmolality that slightly retarded the growth rate of S. mutans (Figure 1A). We thus chose this concentration of NaCl for the rest of study. We investigated the short-term and long-term effects of 0.4 M of NaCl on the biofilm configuration of S. mutans. Hyperosmotic conditions significantly inhibited the biomass of S. mutans biofilm, and this inhibitory effect was time and concentration-dependent (Figure 1B and C). In addition, we performed live/dead fluorescence stain of biofilm and enumerated the biofilm colony forming unit (CFU), and we found that either the percentage or absolute number of viable cells after exposure to 0.4 M NaCl was comparable to that of non-treated control (Figure 1D and E). These data indicate that the observed biomass reduction after hyperosmotic exposure was less likely caused by growth inhibition, but more likely attributed to the dispersal of biofilm under adversary conditions. The osmolality-provoked biofilm dispersal was further confirmed with fluorescence double-labeling and scanning electronic microscopy (Figure 2). Exposure to sub-inhibitory level of hyperosmotic stimuli not only inhibited cellular components within the biofilm, but also reduced the extracellular polysaccharides (EPS) matrix synthesized.


Hyperosmotic response of streptococcus mutans: from microscopic physiology to transcriptomic profile.

Liu C, Niu Y, Zhou X, Zhang K, Cheng L, Li M, Li Y, Wang R, Yang Y, Xu X - BMC Microbiol. (2013)

Phenotypic characteristics of S. mutans after short-term and long-term hyperosmotic stimuli. (A) Representative Scanning Electronic Microscopy images of S. mutans biofilm on glass surfaces. Images shown were taken at 1000 ×, 5000 × and 10000 × magnification. (B) Representative 3D rendering images of S. mutans biofilms without NaCl for 24 h (upper left), versus with 0.4 M NaCl for either 15 min (upper right) or 24 h (lower left). Bacterial cells and EPS are in situ labelled. Green, the bacteria (SYTO 9); red, the EPS (Alexa Fluor 647). At the right of each panel, the two channels are displayed separately, while the merged image is displayed at the left. Lateral (side) views of each biofilm are displayed at the bottom. Quantitative determination of S. mutans biofilms (lower right) confocal image stacks analyzed by the image-processing software COMSTAT. Results were averaged from 3 independent experiments and are presented as mean ± standard deviation. *, P ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219374&req=5

Figure 2: Phenotypic characteristics of S. mutans after short-term and long-term hyperosmotic stimuli. (A) Representative Scanning Electronic Microscopy images of S. mutans biofilm on glass surfaces. Images shown were taken at 1000 ×, 5000 × and 10000 × magnification. (B) Representative 3D rendering images of S. mutans biofilms without NaCl for 24 h (upper left), versus with 0.4 M NaCl for either 15 min (upper right) or 24 h (lower left). Bacterial cells and EPS are in situ labelled. Green, the bacteria (SYTO 9); red, the EPS (Alexa Fluor 647). At the right of each panel, the two channels are displayed separately, while the merged image is displayed at the left. Lateral (side) views of each biofilm are displayed at the bottom. Quantitative determination of S. mutans biofilms (lower right) confocal image stacks analyzed by the image-processing software COMSTAT. Results were averaged from 3 independent experiments and are presented as mean ± standard deviation. *, P ≤ 0.05.
Mentions: By constructing the growth curve of S. mutans under increasing concentrations of NaCl, we found that 0.4 M of NaCl provided the sub-inhibitory level of osmolality that slightly retarded the growth rate of S. mutans (Figure 1A). We thus chose this concentration of NaCl for the rest of study. We investigated the short-term and long-term effects of 0.4 M of NaCl on the biofilm configuration of S. mutans. Hyperosmotic conditions significantly inhibited the biomass of S. mutans biofilm, and this inhibitory effect was time and concentration-dependent (Figure 1B and C). In addition, we performed live/dead fluorescence stain of biofilm and enumerated the biofilm colony forming unit (CFU), and we found that either the percentage or absolute number of viable cells after exposure to 0.4 M NaCl was comparable to that of non-treated control (Figure 1D and E). These data indicate that the observed biomass reduction after hyperosmotic exposure was less likely caused by growth inhibition, but more likely attributed to the dispersal of biofilm under adversary conditions. The osmolality-provoked biofilm dispersal was further confirmed with fluorescence double-labeling and scanning electronic microscopy (Figure 2). Exposure to sub-inhibitory level of hyperosmotic stimuli not only inhibited cellular components within the biofilm, but also reduced the extracellular polysaccharides (EPS) matrix synthesized.

Bottom Line: We found that the hyperosmotic condition was able to initiate S. mutans biofilm dispersal by reducing both microbial content and extracellular polysaccharides matrix.Further analysis of microarray data showed significant up-regulation of genes and pathways involved in carbohydrate metabolism.Hyperosmotic condition induces significant stress response on S. mutans at both phenotypic and transcriptomic levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, PR China. nixux1982@hotmail.com.

ABSTRACT

Background: Oral streptococci metabolize carbohydrate to produce organic acids, which not only decrease the environmental pH, but also increase osmolality of dental plaque fluid due to tooth demineralization and consequent calcium and phosphate accumulation. Despite these unfavorable environmental changes, the bacteria continue to thrive. The aim of this study was to obtain a global view on strategies taken by Streptococcus mutans to deal with physiologically relevant elevated osmolality, and perseveres within a cariogenic dental plaque.

Results: We investigated phenotypic change of S. mutans biofilm upon hyperosmotic challenge. We found that the hyperosmotic condition was able to initiate S. mutans biofilm dispersal by reducing both microbial content and extracellular polysaccharides matrix. We then used whole-genome microarray with quantitative RT-PCR validation to systemically investigate the underlying molecular machineries of this bacterium in response to the hyperosmotic stimuli. Among those identified 40 deferentially regulated genes, down-regulation of gtfB and comC were believed to be responsible for the observed biofilm dispersal. Further analysis of microarray data showed significant up-regulation of genes and pathways involved in carbohydrate metabolism. Specific genes involved in heat shock response and acid tolerance were also upregulated, indicating potential cross-talk between hyperosmotic and other environmental stress.

Conclusions: Hyperosmotic condition induces significant stress response on S. mutans at both phenotypic and transcriptomic levels. In the meantime, it may take full advantage of these environmental stimuli to better fit the fluctuating environments within oral cavity, and thus emerges as numeric-predominant bacterium under cariogenic conditions.

Show MeSH
Related in: MedlinePlus