Oxidative Stressors Modify the Response of Streptococcus mutans to Its Competence Signal Peptides

Abstract

The dental caries pathogen is continually exposed to several types of stress in the oral biofilm environment. Oxidative stress generated by reactive oxygen species has a major impact on the establishment, persistence, and virulence of Here, we combined fluorescent reporter-promoter fusions with single-cell imaging to study the effects of reactive oxygen species on activation of genetic competence in Exposure to paraquat, which generates superoxide anion, produced a qualitatively different effect on activation of expression of the gene for the master competence regulator, ComX, than did treatment with hydrogen peroxide (HO), which can yield hydroxyl radical. Paraquat suppressed peptide-mediated induction of in a progressive and cumulative fashion, whereas the response to HO displayed a strong threshold behavior. Low concentrations of HO had little effect on induction of or the bacteriocin gene, but expression of these genes declined sharply if extracellular HO exceeded a threshold concentration. These effects were not due to decreased reporter gene fluorescence. Two different threshold concentrations were observed in the response to HO, depending on the gene promoter that was analyzed and the pathway by which the competence regulon was stimulated. The results show that paraquat and HO affect the competence signaling pathway differently, and that some portions of the competence signaling pathway are more sensitive to oxidative stress than others. inhabits the oral biofilm, where it plays an important role in the development of dental caries. Environmental stresses such as oxidative stress influence the growth of and its important virulence-associated behaviors, such as genetic competence. competence development is a complex behavior that involves two different signaling peptides and can exhibit cell-to-cell heterogeneity. Although oxidative stress is known to influence competence, it is not understood how oxidative stress interacts with the peptide signaling or affects heterogeneity. In this study, we used fluorescent reporters to probe the effect of reactive oxygen species on competence signaling at the single-cell level. Our data show that different reactive oxygen species have different effects on competence, and that some portions of the signaling pathway are more acutely sensitive to oxidative stress than others.

Environmental Triggers oflrgAExpression inStreptococcus mutans

“…To test our P lrgA-gfp fluorescent reporter strain and characterize lrgA expression in static cultures, we monitored the optical density and fluorescence of the reporter strain growing in well plates containing defined medium (FMC (30, 31)) that was prepared with different initial concentrations of glucose. Figures 1A and 1B show growth curves for the UA159 background and the P lrgA - gfp reporting strain respectively, growing anaerobically under a layer of mineral oil.…”

“…This enzymatic system rapidly consumes O2 from the medium by breaking down glucose to yield gluconic acid and H2O as products. Although the glucose oxidase generates H2O2 as an intermediate product (which is then broken down by the catalase), S. mutans is tolerant of low to moderate concentrations of H2O2 far higher than would be present during this reaction (31).…”

Competence inhibition by the XrpA peptide encoded within the comX gene of Streptococcus mutans

“…Nevertheless, if we accept the premise that xrpA translation is not as efficient as that of comX when the comX promoter is activated, then it can be envisioned that the negative feedback loop created by XrpA acting on ComR modulates transcriptional initiation at the comX promoter; with the feedback loop contributing to fine tuning ComX levels in response to cellular physiology and environment and/or serving as a primary pathway to turn off the competence circuit. The former would be consistent with the observation that signal perception, induction of comX and progression to the competent state are all exquisitely sensitive to key environmental inputs that include pH (Guo et al ., ; Son et al ., ), oxidative stressors (De Furio et al ., ) and carbohydrate source and availability (Moye et al ., ). The latter model would be consistent with the fact that, while the MecA‐Clp pathway can serve as a mechanism to shut off competence through degradation of ComX, the cells also need a way to shut down the ComRS circuit so as not to produce nascent ComX during competence while committing to turning off the competence regulon.…”

Competence Inhibition by the XrpA Peptide Encoded Within thecomXGene ofStreptococcus mutans

“…Nevertheless, if we accept the premise that xrpA translation is not as efficient as that of comX when the comX promoter is activated, then it can be envisioned that the negative feedback loop created by XrpA acting on ComR modulates transcriptional initiation at the comX promoter; with the feedback loop contributing to fine tuning ComX levels in response to cellular physiology and environment and/or serving as a primary pathway to turn off the competence circuit. The former would be consistent with the observation that signal perception, induction of comX and progression to the competent state are all exquisitely sensitive to key environmental inputs that include pH (Guo et al ., 2014; Son et al ., 2015), oxidative stressors (De Furio et al ., 2017) and carbohydrate source and availability (Moye et al ., 2016). The latter model would be consistent with the fact that, while the MecA-Clp pathway can serve as a mechanism to shut off competence through degradation of ComX, the cells also need a way to shut down the ComRS circuit so as not to produce nascent ComX during competence while committing to turning off the competence regulon.…”