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, 14 (1), 015001

Origins of Heterogeneity in Streptococcus Mutans Competence: Interpreting an Environment-Sensitive Signaling Pathway

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Origins of Heterogeneity in Streptococcus Mutans Competence: Interpreting an Environment-Sensitive Signaling Pathway

Stephen J Hagen et al. Phys Biol.

Abstract

Bacterial pathogens rely on chemical signaling and environmental cues to regulate disease-causing behavior in complex microenvironments. The human pathogen Streptococcus mutans employs a particularly complex signaling and sensing scheme to regulate genetic competence and other virulence behaviors in the oral biofilms it inhabits. Individual S. mutans cells make the decision to enter the competent state by integrating chemical and physical cues received from their microenvironment along with endogenously produced peptide signals. Studies at the single-cell level, using microfluidics to control the extracellular environment, provide physical insight into how the cells process these inputs to generate complex and often heterogeneous outputs. Fine changes in environmental stimuli can dramatically alter the behavior of the competence circuit. Small shifts in pH can switch the quorum sensing response on or off, while peptide-rich media appear to switch the output from a unimodal to a bimodal behavior. Therefore, depending on environmental cues, the quorum sensing circuitry can either synchronize virulence across the population, or initiate and amplify heterogeneity in that behavior. Much of this complex behavior can be understood within the framework of a quorum sensing system that can operate both as an intercellular signaling mechanism and intracellularly as a noisy bimodal switch.

Figures

FIGURE 1
FIGURE 1
(A) Overview of genetic competence signaling in S. mutans (Smith; Spatafora, 2012). ComC is synthesized, processed and exported as CSP, an extracellular signal peptide. ComD detects CSP and phosphorylates ComE to ComE-P, which acts as a transcriptional activator for several bacteriocin genes. By a mechanism not yet understood, the bacteriocins stimulate the ComRS system. ComR (with XIP or its precursor ComS) forms a transcriptional activator for synthesis of ComX, the master competence regulator. ComX is an alternative sigma factor that stimulates transcription of the late competence genes such as comY that lead to DNA uptake. ComX also drives positive feedback to the upstream comCDE system, as the comE promoter possesses a ComX binding site (Son et al., 2015a). The circuit also integrates a variety of other environmental and physiological inputs such as environmental pH, carbohydrate catabolism, oxidative stress and other mechanisms (Ahn et al., 2007; Ahn et al., 2014; Kaspar et al., 2015). (B)–(C) Overlaid fluorescence (green) and phase contrast (gray scale) microscopy images of S. mutans responding to exogenous signal peptides (B) CSP (1 μM in complex medium) and (C) XIP (500 nM in defined medium). The green fluorescence from a PcomX-gfp reporter shows that CSP and XIP elicit a bimodal and unimodal response respectively from comX (Son et al., 2012).
FIGURE 2
FIGURE 2
(A)–(B): Model for two modes of ComRS function. (A) In the case of purely extracellular feedback, the cell exports ComS and processes it to XIP which accumulates extracellularly as a quorum sensing signal. XIP enters the cell via the permease Opp and forms (with ComR) an activator complex for comS and comX. Supplying exogenous XIP to a population of S. mutans therefore activates comX in all cells, giving a unimodal distribution of comX activity. (B) In the purely intracellular feedback case the cell does not readily import or export ComS or XIP. However basal levels of endogenously produced ComS can interact with ComR to stimulate further comS activity, forming an intracellular feedback loop. Exogenous XIP then has little effect on comX activity. Stochasticity in basal ComS production leads to heterogeneous (bimodal) comX activity in the population. (C) Microfluidic data show that growth medium controls signal and response dynamics of the competence circuit. In a medium containing assorted small peptides (complex medium), exogenous CSP (1 μM, blue) induces a bimodal distribution of comX activity (left) in a microfluidic flow chamber. Medium lacking such small peptides (defined medium) quenches comX response to CSP (right). However exogenous XIP (500 nM, red) produces a unimodal response from comX in defined medium (right) and no response in complex medium (left). A hybrid behavior appears in an admixture of 2–5% complex/95–98% defined medium (center), with a weakly bimodal response to either CSP or XIP. These data suggest that ComRS can operate with either extracellular (A) or intracellular (B) feedback, depending on the presence of assorted small peptides in the growth environment. (D) Results of stochastic simulation of PcomX activity in the bimodal model. Model parameters and equations are given in the Supplemental Information. Histograms show activity of PcomX in an ensemble of cells as calculated in COPASI assuming stimulation with exogenous CSP (300 nM, red) or XIP (1 μM, blue). As described in the Supplemental Information, the change from complex (left) to defined medium (right) is modeled by faster importation of exogenous XIP, but a shorter lifetime for intracellular XIP (or ComS), in the defined medium.
FIGURE 3
FIGURE 3
(A) Experimental data, adapted from (Mashburn-Warren et al., 2010), show that exogenous XIP (in defined medium) stimulates more transformation in the wild-type S. mutans than in a ΔcomS mutant. Studies by Mashburn-Warren et al. were performed in triplicate, leading to uncertainties (standard deviation) similar to the size of the plotting symbols. (B) Simulation of a hybrid of the two feedback models in FIGURE 2, where extracellular XIP can cross the cell membrane but ComS cannot. Environmental XIP enters the cell and stimulates the intracellular ComS feedback loop, leading to greater activation of comS and comX in the wildtype (blue curve) than in ΔcomS (red curve). (C) Diagram of hybrid model. Model details and parameters are given in Supplemental information.
FIGURE 4
FIGURE 4
(A) Response of comX to CSP (blue) and XIP (red) is acutely sensitive to local extracellular pH. In microfluidic studies the median comX activity in a population of S. mutans is maximal near pH 7 and declines sharply away from pH neutral conditions. Dashed lines show spline fit. (B)–(C) Mechanisms downstream of comX introduce additional heterogeneity. When exogenous XIP is supplied to a dual reporter strain carrying PcomX-gfp and PcomY-rfp, all cells activate comX (as indicated by the green reporter fluorescence) (B), but only a subset also activates comY (indicated by the red fluorescence) (C) and other late competence genes. Thus an additional noisy switch lies between comX and comY.

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