Abstract
Streptococcus mutans is an important pathogen in the human oral biofilm. It expresses virulent behaviors that are linked to its genetic competence regulon, which is controlled by comX. Expression of comX is modulated by two diffusible signaling peptides, denoted CSP and XIP, and by other environmental cues such as pH and oxidative stress. The sensitivity of S. mutans competence to environmental inputs that may vary on microscopic length scales raises the question of whether the biofilm environment creates microniches where competence and related phenotypes are concentrated, leading to spatial clustering of S. mutans virulence behaviors. We have used two-photon microscopy to characterize the spatial distribution of comX expression among individual S. mutans cells in biofilms. By analyzing correlations in comX activity, we test for spatial clustering that may suggest localized competence microenvironments. Our data indicate that both competence-signaling peptides diffuse efficiently through the biofilm. XIP elicits a population-wide response. CSP triggers a Poisson-like, spatially random comX response from a subpopulation of cells that is homogeneously dispersed. Our data indicate that competence microenvironments if they exist are small enough that the phenotypes of individual cells are not clustered or correlated to any greater extent than occurs in planktonic cultures.
Highlights
Oral biofilms are complex microbial communities that may be inhabited by pathogenic as well as commensal species
competence stimulating peptide (CSP) elicits a bimodal response from S. mutans comX in complex growth media such as Brain Heart Infusion (BHI), whether cells are grown under uniform, microfluidic flow conditions (Son et al, 2012) or in a biofilm (Aspiras et al, 2004)
As described in section “Materials and Methods,” biofilms were grown for 5 h in BHI and incubated for 2 h in fresh medium that contained 1 μM synthetic CSP (Figures 1A,B), or no added CSP (Figures 1C,D), prior to a buffer wash and imaging
Summary
Oral biofilms are complex microbial communities that may be inhabited by pathogenic as well as commensal species. In S. mutans, the activation of comX is modulated by inputs such as pH, oxidative stress, and the availability of carbohydrate and peptide nutrients It is controlled by two diffusible signaling peptides, denoted sigX inducing peptide (XIP) and competence stimulating peptide (CSP). If activation of comX requires exchange of XIP or other signals that diffuse poorly through the matrix or are degraded in transit, cells activating pathogenic behaviors linked to comX could exhibit some tendency to collocate or cluster (Parsek and Greenberg, 2005; Decho et al, 2010) We test this hypothesis using twophoton confocal microscopy to probe at single-cell resolution the expression of competence genes throughout the depth of S. mutans biofilms. Our data allow us to evaluate the diffusibility of the signaling peptides XIP and CSP and characterize the length scale of any microniches of competence activity
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