Limits...
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.

Show MeSH

Related in: MedlinePlus

pH-responsivity of surface-expressed pHluorin in S. mutans.(A) Fluorescence signal intensity of the pHluorin-SpaP fusion protein in S. mutans strain O87 at different pH values. Planktonic cells of strain O87 were adjusted to the indicated pH values. The ratio was calculated as the amount of pHluorin signal at each pH value vs. the initial signal at pH 7.5. The plots show the average of triplicate samples, and the error bars correspond to the standard deviations. (B) Time-course of surface-expressed pHluorin fluorescence signal in S. mutans biofilms at different pH values. CLSM analysis of surface-expressed pHluorin signals in S. mutans O87 biofilms is shown in (a). The fluorescence of biofilms was monitored at pH 7.5 (left), shifted to pH 5.5 for 10 min (middle) followed by a shift back to pH 7.5 for 10 min (right). Quantification of pHluorin signals in biofilms at different pH is shown in (b). The ratio was calculated as the amount of pHluorin signal at each time point vs. initial amount (0 min). The plots represent the average of three duplicate tests.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585301&req=5

pone-0057182-g002: pH-responsivity of surface-expressed pHluorin in S. mutans.(A) Fluorescence signal intensity of the pHluorin-SpaP fusion protein in S. mutans strain O87 at different pH values. Planktonic cells of strain O87 were adjusted to the indicated pH values. The ratio was calculated as the amount of pHluorin signal at each pH value vs. the initial signal at pH 7.5. The plots show the average of triplicate samples, and the error bars correspond to the standard deviations. (B) Time-course of surface-expressed pHluorin fluorescence signal in S. mutans biofilms at different pH values. CLSM analysis of surface-expressed pHluorin signals in S. mutans O87 biofilms is shown in (a). The fluorescence of biofilms was monitored at pH 7.5 (left), shifted to pH 5.5 for 10 min (middle) followed by a shift back to pH 7.5 for 10 min (right). Quantification of pHluorin signals in biofilms at different pH is shown in (b). The ratio was calculated as the amount of pHluorin signal at each time point vs. initial amount (0 min). The plots represent the average of three duplicate tests.

Mentions: To examine the in vivo pH-responsivity of the ecliptic pHluorin when expressed on the cell surface of S. mutans as a pHluorin-SpaP fusion protein, planktonic cells of strain O87 were suspended in buffer with different pH values and their fluorescence signals were monitored. A reduction in fluorescence signal intensity corresponding to the decrease in pH was observed (Fig. 2A), confirming the pH-responsivity of the surface displayed ecliptic pHlourin in S. mutans strain O87 and its potential for detecting pH changes in real time.


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)

pH-responsivity of surface-expressed pHluorin in S. mutans.(A) Fluorescence signal intensity of the pHluorin-SpaP fusion protein in S. mutans strain O87 at different pH values. Planktonic cells of strain O87 were adjusted to the indicated pH values. The ratio was calculated as the amount of pHluorin signal at each pH value vs. the initial signal at pH 7.5. The plots show the average of triplicate samples, and the error bars correspond to the standard deviations. (B) Time-course of surface-expressed pHluorin fluorescence signal in S. mutans biofilms at different pH values. CLSM analysis of surface-expressed pHluorin signals in S. mutans O87 biofilms is shown in (a). The fluorescence of biofilms was monitored at pH 7.5 (left), shifted to pH 5.5 for 10 min (middle) followed by a shift back to pH 7.5 for 10 min (right). Quantification of pHluorin signals in biofilms at different pH is shown in (b). The ratio was calculated as the amount of pHluorin signal at each time point vs. initial amount (0 min). The plots represent the average of three duplicate tests.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057182-g002: pH-responsivity of surface-expressed pHluorin in S. mutans.(A) Fluorescence signal intensity of the pHluorin-SpaP fusion protein in S. mutans strain O87 at different pH values. Planktonic cells of strain O87 were adjusted to the indicated pH values. The ratio was calculated as the amount of pHluorin signal at each pH value vs. the initial signal at pH 7.5. The plots show the average of triplicate samples, and the error bars correspond to the standard deviations. (B) Time-course of surface-expressed pHluorin fluorescence signal in S. mutans biofilms at different pH values. CLSM analysis of surface-expressed pHluorin signals in S. mutans O87 biofilms is shown in (a). The fluorescence of biofilms was monitored at pH 7.5 (left), shifted to pH 5.5 for 10 min (middle) followed by a shift back to pH 7.5 for 10 min (right). Quantification of pHluorin signals in biofilms at different pH is shown in (b). The ratio was calculated as the amount of pHluorin signal at each time point vs. initial amount (0 min). The plots represent the average of three duplicate tests.
Mentions: To examine the in vivo pH-responsivity of the ecliptic pHluorin when expressed on the cell surface of S. mutans as a pHluorin-SpaP fusion protein, planktonic cells of strain O87 were suspended in buffer with different pH values and their fluorescence signals were monitored. A reduction in fluorescence signal intensity corresponding to the decrease in pH was observed (Fig. 2A), confirming the pH-responsivity of the surface displayed ecliptic pHlourin in S. mutans strain O87 and its potential for detecting pH changes in real time.

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