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investigating acid production by Streptococcus mutans with a surface-displayed pH-sensitive green fluorescent protein.

Guo L, Hu W, He X, Lux R, McLean J, Shi W - PLoS ONE (2013)

Bottom Line: Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP.Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity.Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, University of California Los Angeles, Los Angeles, California, USA.

ABSTRACT
Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins) to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87) was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.

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Signals of the pHluorin-SpaP fusion protein and metabolic activities of S. mutans in biofilms after addition of sucrose.(A) CLSM image showing pH heterogeneity of different microcolonies (indicated by arrows) within S. mutans biofilm in the presence of sucrose. Biofilms were revealed with CellTracker Orange. CellTracker Orange (blue), GFP (green) and the corresponding overly images are shown. (B) CLSM images showing differences in metabolic activities of microcolonies within S. mutans biofilm after addition of sucrose. (a) CLSM analysis of pHluorin signals of S. mutans strain O87 biofilm after addition of 2% sucrose for 60 min. Bars represent 50 µm. (b) 3D surface plot of fluorescence intensities of pHluorin (green) and the fluorescent metabolic indicator dye 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC - red) in strain O87 biofilm after addition of 2% sucrose for 60 min. (c) and (d) The representative CLSM images of microcolonies within the same biofilm. Bars represent 20 µm.
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pone-0057182-g004: Signals of the pHluorin-SpaP fusion protein and metabolic activities of S. mutans in biofilms after addition of sucrose.(A) CLSM image showing pH heterogeneity of different microcolonies (indicated by arrows) within S. mutans biofilm in the presence of sucrose. Biofilms were revealed with CellTracker Orange. CellTracker Orange (blue), GFP (green) and the corresponding overly images are shown. (B) CLSM images showing differences in metabolic activities of microcolonies within S. mutans biofilm after addition of sucrose. (a) CLSM analysis of pHluorin signals of S. mutans strain O87 biofilm after addition of 2% sucrose for 60 min. Bars represent 50 µm. (b) 3D surface plot of fluorescence intensities of pHluorin (green) and the fluorescent metabolic indicator dye 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC - red) in strain O87 biofilm after addition of 2% sucrose for 60 min. (c) and (d) The representative CLSM images of microcolonies within the same biofilm. Bars represent 20 µm.

Mentions: One intriguing discovery in this study is the pH heterogeneity of different microcolonies within the same S. mutans biofilm in the presence of sucrose (Figure 4A). Within biofilms of S. mutans O87 some microcolonies displayed a much more intense green fluorescence signal than others (indicated by arrows) after addition of sucrose, suggesting the pH in the microenvironment of microcolonies is not homogeneous throughout S. mutans biofilms following sucrose fermentation. To investigate the possible correlation between microenvironment pH heterogeneity and the metabolic status of microcolonies, CTC (5-cyano-2,3-ditolyl tetrazolium chloride) was added to the experimental setup for evaluating the respiratory activity of microcolonies within S. mutans biofilm in the presence of sucrose and a correlation between metabolic status and pH was observed (Fig. 4Ba and 4Bb). Microcolonies with less CTC signal typically exhibited brighter pHluorin fluorescence, while strong CTC signals were often associated with reduced pHluorin fluorescence (Fig. 4Bc and 4Bd).


investigating acid production by Streptococcus mutans with a surface-displayed pH-sensitive green fluorescent protein.

Guo L, Hu W, He X, Lux R, McLean J, Shi W - PLoS ONE (2013)

Signals of the pHluorin-SpaP fusion protein and metabolic activities of S. mutans in biofilms after addition of sucrose.(A) CLSM image showing pH heterogeneity of different microcolonies (indicated by arrows) within S. mutans biofilm in the presence of sucrose. Biofilms were revealed with CellTracker Orange. CellTracker Orange (blue), GFP (green) and the corresponding overly images are shown. (B) CLSM images showing differences in metabolic activities of microcolonies within S. mutans biofilm after addition of sucrose. (a) CLSM analysis of pHluorin signals of S. mutans strain O87 biofilm after addition of 2% sucrose for 60 min. Bars represent 50 µm. (b) 3D surface plot of fluorescence intensities of pHluorin (green) and the fluorescent metabolic indicator dye 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC - red) in strain O87 biofilm after addition of 2% sucrose for 60 min. (c) and (d) The representative CLSM images of microcolonies within the same biofilm. Bars represent 20 µm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585301&req=5

pone-0057182-g004: Signals of the pHluorin-SpaP fusion protein and metabolic activities of S. mutans in biofilms after addition of sucrose.(A) CLSM image showing pH heterogeneity of different microcolonies (indicated by arrows) within S. mutans biofilm in the presence of sucrose. Biofilms were revealed with CellTracker Orange. CellTracker Orange (blue), GFP (green) and the corresponding overly images are shown. (B) CLSM images showing differences in metabolic activities of microcolonies within S. mutans biofilm after addition of sucrose. (a) CLSM analysis of pHluorin signals of S. mutans strain O87 biofilm after addition of 2% sucrose for 60 min. Bars represent 50 µm. (b) 3D surface plot of fluorescence intensities of pHluorin (green) and the fluorescent metabolic indicator dye 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC - red) in strain O87 biofilm after addition of 2% sucrose for 60 min. (c) and (d) The representative CLSM images of microcolonies within the same biofilm. Bars represent 20 µm.
Mentions: One intriguing discovery in this study is the pH heterogeneity of different microcolonies within the same S. mutans biofilm in the presence of sucrose (Figure 4A). Within biofilms of S. mutans O87 some microcolonies displayed a much more intense green fluorescence signal than others (indicated by arrows) after addition of sucrose, suggesting the pH in the microenvironment of microcolonies is not homogeneous throughout S. mutans biofilms following sucrose fermentation. To investigate the possible correlation between microenvironment pH heterogeneity and the metabolic status of microcolonies, CTC (5-cyano-2,3-ditolyl tetrazolium chloride) was added to the experimental setup for evaluating the respiratory activity of microcolonies within S. mutans biofilm in the presence of sucrose and a correlation between metabolic status and pH was observed (Fig. 4Ba and 4Bb). Microcolonies with less CTC signal typically exhibited brighter pHluorin fluorescence, while strong CTC signals were often associated with reduced pHluorin fluorescence (Fig. 4Bc and 4Bd).

Bottom Line: Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP.Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity.Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, University of California Los Angeles, Los Angeles, California, USA.

ABSTRACT
Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins) to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87) was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.

Show MeSH
Related in: MedlinePlus