Investigating Acid Production by <em>Streptococcus mutans</em> with a Surface-Displayed pH-Sensitive Green Fluorescent Protein

<div><p>Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium <i>Streptococcus mutans</i>. 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 <i>S. mutans</i> 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 <i>S. mutans</i> as a fusion protein with SpaP. The resulting strain (O87) was used to monitor temporal and spatial pH changes in the microenvironment of <i>S. mutans</i> cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of <i>S. mutans</i> microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within <i>S. mutans</i> biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the <i>S. mutans</i> acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate <i>in vivo</i> and <i>in situ</i> acid production and distribution by the cariogenic species <i>S. mutans</i>.</p>