Competence-Stimulating-Peptide-Dependent Localized Cell Death and Extracellular DNA Production in Streptococcus mutans Biofilms

Abstract

Extracellular DNA (eDNA) is a biofilm component that contributes to the formation and structural stability of biofilms. Streptococcus mutans, a major cariogenic bacterium, induces eDNA-dependent biofilm formation under specific conditions. Since cell death can result in the release and accumulation of DNA, the dead cells in biofilms are a source of eDNA. However, it remains unknown how eDNA is released from dead cells and is localized within S. mutans biofilms. We focused on cell death induced by the extracellular signaling peptide, competence-stimulating peptide (CSP). We demonstrate that nucleic acid release into the extracellular environment occurs in a subpopulation of dead cells. eDNA production induced by CSP was highly dependent on the lytF gene, which encodes an autolysin. Although lytF expression was induced bimodally by CSP, lytF-expressing cells further divided into surviving cells and eDNA-producing dead cells. Moreover, we found that lytF-expressing cells were abundant near the bottom of the biofilm, even when all cells in the biofilm received the CSP signal. Dead cells and eDNA were also abundantly present near the bottom of the biofilm. The number of lytF-expressing cells in biofilms was significantly higher than that in planktonic cultures, which suggests that adhesion to the substratum surface is important for the induction of lytF expression. The deletion of lytF resulted in reduced adherence to a polystyrene surface. These results suggest that lytF expression and eDNA production induced near the bottom of the biofilm contribute to a firmly attached and structurally stable biofilm. Importance Bacterial communities encased by self-produced extracellular polymeric substances (EPSs), known as biofilms, have a wide influence on human health and environmental problems. The importance of biofilm research has increased as biofilms are the preferred bacterial lifestyle in nature. Furthermore, in recent years, due to the contribution of phenotypic heterogeneity within biofilms to their characteristics, analysis at the single-cell or subpopulation level is required to understand bacterial life strategies. In Streptococcus mutans, a cariogenic bacterium, extracellular DNA (eDNA) contributes to biofilm formation. However, it remains unclear how and where the cells produce eDNA within the biofilm. We focused on LytF, an autolysin that is induced by extracellular peptide signals. We used single-cell level imaging techniques to analyze lytF expression in the biofilm population. Here, we show that S. mutans generates eDNA by inducing lytF expression near the bottom of the biofilm, thereby enhancing biofilm adhesion and structural stability.

The oralome and its dysbiosis: New insights into oral microbiome-host interactions

“…Recently, Nagasawa et al . [105] demonstrated that CSP may be involved into releasing eDNA in S. mutans biofilms, contributing to firmer and more stable S. mutans biofilms. Counterintuitively, S. gordonii mutants, lacking the comC gene, produced more eDNA and more stable biofilms in the presence of C. albicans than the isogenic wild-type counterpart [106] .…”

Effects of lipid emulsions on the formation of Escherichia coli–Candida albicans mixed-species biofilms on PVC

“…This was consistent with previous research 10,27 . Nucleic acids released into the extracellular environment by dead pathogens have been demonstrated to contribute to the formation and structural stability of BFs and provide abundant nutrients for live pathogens to support the sustainable growth of BFs 28 . This result suggested that over time, the number of dead cells at the bottom of the BFs increased, and the structure of the BFs became more stable and robust.…”

Novel Insights into Microbial Behavior Gleaned Using Microfluidics

“…Based on our studies, it appears that eDNA is also an important common factor. We demonstrated that 2D confinement offers the opportunity to test conditions that may otherwise be impossible to recreate in a controlled manner, which may reveal cell affinity for particular environmental geometries (Nagasawa et al, 2020b).…”

“…eDNA is an important component of the EPS matrix in S. mutans biofilms and is necessary for initial cell attachment; however, the mechanisms by which cells release eDNA from within a biofilm are unclear. By culturing S. mutans in our 2D microchambers, we imaged the release of eDNA into the extracellular space, which we were correlated with cell death (Nagasawa et al, 2020b) (Fig. 2D).…”

“…As observed with Leptothrix, microfluidic confinement (Kunoh et al, 2020;Nagasawa et al, 2020b) may facilitate the tracking and analysis of organisms that escape the imaging plane. We described the use of microfluidics to investigate cell polarity and growth in filamentous fungi, which are fundamental processes for all cellular functions (Howard et al, 2011;Zhao et al, 2018).…”