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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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Related in: MedlinePlus

Temporal and spatial distribution of pHluorin fluorescent signals within S. mutans biofilms.(A) Dynamic analysis of surface-expressed pHluorin fluorescence signal changes in biofilm after addition of 2% sucrose under phosphate buffered condition (pH 7.5) (upper panel) and unbuffered condition (lower panel), respectively. (B) pHluorin signals after addition of 2% sucrose under buffered or unbuffered conditions is quantified. The proportion was calculated as the amount of pHluorin signal at each time point vs. the initial signal intensity (0 min). The plots show the average of three duplicate tests. The corresponding changes in medium pH are also included.
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pone-0057182-g003: Temporal and spatial distribution of pHluorin fluorescent signals within S. mutans biofilms.(A) Dynamic analysis of surface-expressed pHluorin fluorescence signal changes in biofilm after addition of 2% sucrose under phosphate buffered condition (pH 7.5) (upper panel) and unbuffered condition (lower panel), respectively. (B) pHluorin signals after addition of 2% sucrose under buffered or unbuffered conditions is quantified. The proportion was calculated as the amount of pHluorin signal at each time point vs. the initial signal intensity (0 min). The plots show the average of three duplicate tests. The corresponding changes in medium pH are also included.

Mentions: The dynamic changes of pHluorin-SpaP fusion protein fluorescence intensity within S. mutans biofilms were monitored after addition of 2% sucrose under phosphate buffered (pH 7.5) and unbuffered condition (Fig. 3). Results showed that, under buffered condition, the medium pH remained stable at 7.5 and the biofilms displayed bright fluorescence throughout the 60 min incubation period even in the presence of 2% sucrose. In unbuffered medium in contrast, the addition of sucrose induced a pH drop from 7.5 to below 6.0 within 60 min accompanied by a striking decrease in the fluorescent signals within S. mutans biofilms. Interestingly, after 30 min of incubation, the pHluorin fluorescence intensity decreased by nearly 50%, although the medium pH only dropped to about 7.0. According to the change in fluorescent intensity observed in response to pH reduction in Figure 2A, the microenvironment pH of microcolonies decreases to almost pH 5.5. This suggests that the pH in the microenvironment decreases prior to the pH drop in the medium following sucrose fermentation.


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)

Temporal and spatial distribution of pHluorin fluorescent signals within S. mutans biofilms.(A) Dynamic analysis of surface-expressed pHluorin fluorescence signal changes in biofilm after addition of 2% sucrose under phosphate buffered condition (pH 7.5) (upper panel) and unbuffered condition (lower panel), respectively. (B) pHluorin signals after addition of 2% sucrose under buffered or unbuffered conditions is quantified. The proportion was calculated as the amount of pHluorin signal at each time point vs. the initial signal intensity (0 min). The plots show the average of three duplicate tests. The corresponding changes in medium pH are also included.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057182-g003: Temporal and spatial distribution of pHluorin fluorescent signals within S. mutans biofilms.(A) Dynamic analysis of surface-expressed pHluorin fluorescence signal changes in biofilm after addition of 2% sucrose under phosphate buffered condition (pH 7.5) (upper panel) and unbuffered condition (lower panel), respectively. (B) pHluorin signals after addition of 2% sucrose under buffered or unbuffered conditions is quantified. The proportion was calculated as the amount of pHluorin signal at each time point vs. the initial signal intensity (0 min). The plots show the average of three duplicate tests. The corresponding changes in medium pH are also included.
Mentions: The dynamic changes of pHluorin-SpaP fusion protein fluorescence intensity within S. mutans biofilms were monitored after addition of 2% sucrose under phosphate buffered (pH 7.5) and unbuffered condition (Fig. 3). Results showed that, under buffered condition, the medium pH remained stable at 7.5 and the biofilms displayed bright fluorescence throughout the 60 min incubation period even in the presence of 2% sucrose. In unbuffered medium in contrast, the addition of sucrose induced a pH drop from 7.5 to below 6.0 within 60 min accompanied by a striking decrease in the fluorescent signals within S. mutans biofilms. Interestingly, after 30 min of incubation, the pHluorin fluorescence intensity decreased by nearly 50%, although the medium pH only dropped to about 7.0. According to the change in fluorescent intensity observed in response to pH reduction in Figure 2A, the microenvironment pH of microcolonies decreases to almost pH 5.5. This suggests that the pH in the microenvironment decreases prior to the pH drop in the medium following sucrose fermentation.

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