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The VicRK System of Streptococcus mutans Responds to Oxidative Stress
1 Department of Cariology Endodontology and Pedodontology, Academic Centre for Dentistry Amsterdam (ACTA), Louwesweg 1, 1066 EA Amsterdam, The Netherlands; and Correspondence: * corresponding author, d.deng{at}acta.nl
In Streptococcus mutans, virulence and cariogenicity may be modulated via the two-component regulatory system VicRK. Environmental signals, sensed by VicK, inducing this modulation are still unclear, however, and were investigated in the present study. We found that VicRK displays homology with protein-domains that, in other bacteria, are involved in redox-sensing. After constructing a VicRK-promoter GFP-reporter strain, we showed increased fluorescence intensity under oxidative stress. Potential interference of alternative signals and experimental conditions on GFP expression was excluded by the use of negative and positive control strains. Finally, we constructed a clean vicK knockout mutant, which proved to be more sensitive to H2O2 than the wild-type. In conclusion, this study showed that the VicRK system responds to and protects against oxidative stress. As a result, a link between oxidative/redox stress and the cariogenic nature of S. mutans can be hypothesized.
Key Words: two-component system • oxidative stress • Streptococcus mutans • signaling • green fluorescence protein
In caries, Streptococcus mutans has been implicated as the primary etiological agent, due to its sucrose-dependent biofilm formation, high aciduricity, and potent acidogenesis (Loesche, 1986). From initial colonization stages onward, S. mutans undergoes continuous dynamic challenges to which it must respond and adapt. Generally, in bacteria, specific responses are under the control of a dedicated two-component regulatory system (TCRS): signal transduction systems, which are usually composed of a membrane-bound sensor protein and a cytoplasmic response regulator. Through phosphorylation reactions, extracellular signals sensed by the sensor protein are transferred to the cognate response regulator protein, which mediates gene expression.
In S. mutans UA159, based on the genome sequence, 13 TCRSs can be identified (Ajdi We report here on our study of the role of the S. mutans VicRK system in response to oxidative stress, based on the homology of the VicK sensor that we found with several specific bacterial two-component sensors. By expression of a green fluorescent reporter-protein (GFP) under control of the autoregulated VicRK promoter, we followed activity of the promoter and, hence, signal transduction output by variation in fluorescence. Furthermore, the effect of H2O2 on the wild-type strain was compared with the effect on a VicK knockout strain.
Bacterial Strains, Plasmids, and Media Bacterial strains and plasmids are listed in the Table  . Escherichia coli strains were grown in liquid or on solid (1.5% agar) Luria-Bertani medium. Streptococcus mutans UA159 was grown in Todd-Hewitt (TH) broth or on 1.5% TH-agar containing 0.3% yeast extract (THY). Erythromycin (Em) was included where indicated at 200 µg/mL for E. coli and 10 µg/mL for S. mutans, ampicillin at 100 µg/mL for E. coli.
DNA Manipulation Plasmids were extracted and purified from E. coli with QIAprep Spin Miniprep Kits (Qiagen GMH, Hilden, Germany). DNA extracted from agarose gels (1%) was purified with QIAquick Gel Extraction Kits (Qiagen GMH, Hilden, Germany). DNA was transformed into heat-shock competent E. coli. Chromosomal DNA of S. mutans was isolated according to Hanna et al.(2001). PCR was performed with a Dyad DiscipleTM thermocycler (Bio-Rad, Veenendaal, Netherlands). Primers used are listed in the Table  .
Construction of Reporter Strains A synthetic constitutive reference promoter, CP25 (Hansen et al., 2001), with a synthetic ribosome binding site, was also fused to the gfpmut2 gene and inserted into pVA838, resulting in pDM15. Reporter strains of S. mutans containing these vectors were obtained by the natural transformation (Li et al., 2001) of strain UA159. All sequences of constructs were confirmed by nucleotide sequencing.
Construction of the S. mutans
Promoter Activity The general procedure for a stress-response test was as follows: A 1-mL sample was taken from an exponential-phase culture (OD600 = 0.2–0.4) before and 15, 30, 45, 60, 90, and 120 min after the application of the stresses. Subsequently, samples were immediately cooled to 4°C to stop further protein synthesis. A 50–µL quantity of the sample was diluted in 950 µL Isoton® (Beckman Coulter, Mijdrecht, Netherlands) and stored at 4°C overnight before flow cytometry. Where necessary, pH and OD600 (Microplate spectrophotometer, Molecular Devices, Sunnyvale, CA, USA) were measured. Stresses applied included H2O2 (0.0025%, 0.005%, 0.01%), oxygen, and chlorhexidine (0.000625%). When H2O2 and chlorhexidine were applied, concentrated solutions were diluted 10-fold in the culture. When oxygen was applied, cultures were placed in Erlenmeyer flasks and incubated in a shaker-incubator (250 rpm, 37°C). H2O2 and oxygen were chosen (instead of other oxidative stress agents) because these are specifically relevant for S. mutans toward defense responses of the host, and for oral ecology. Fluorescent signals were measured by Epics-XL-MCL flow cytometry (Beckman Coulter, Mijdrecht, Netherlands), with a 488-nm argon excitation-laser and fluorescence-detection through a 525/10 nm (FL1) band-pass filter. We also measured forward- and side-scatter to assay cell size distribution. We collected 10,000 ’events’ in each sample. All data were acquired and analyzed with the use of Expo32-ADC software (Beckman Coulter, Mijdrecht, Netherlands).
Susceptibility to H2O2
PAS Domain PAS is an acronym formed from the names of the proteins in which the domains were first recognized: The Drosophila period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded protein (SIM). The sequence of VicK protein of S. mutans was subjected to protein families database analyses (Bateman et al., 2004), which revealed 4 segments: (i) an N-terminal transmembrane segment between residues 10 and 32, (ii) a PAS domain (Wagner et al., 2002) between positions 94 and 198, (iii) a histidine-kinase domain (207–273), and (iv) a histidine-kinase-like ATPase domain (323–434). A subsequent BLAST search of the PAS domain revealed, as the first hit with experimental back-up, the arcB redox-sensing PAS domain of Lactococcus lactis (E-value = 3e-23).
Functionality of the GFP Reporter Strains
GFP Marker Validation To validate GFP as a marker for gene-expression/signal transduction in S. mutans, we assayed variations in fluorescent behavior of the positive control strain (containing pDM15) under various conditions. During growth, fluorescence was reduced. However, if the pH of the media, which had dropped to 5.5, was re-adjusted, the fluorescence intensity was constant (Fig. 2 ). The pH dependency of GFP fluorescence has been reported previously (Hansen et al., 2001). Hence, in subsequent experiments, samples were diluted in Isoton (pH 7.4) and stored overnight (4°C) before measurements were taken.
Stress Responses in S. mutans Vic-promoter expression is affected by 2 types of oxidative/redox stresses (Fig. 3A ). The highest induction was achieved upon the addition of 0.005% of H2O2. Within an average of 100 min, fluorescence intensity increased from 235 AU to 300 AU. O2 also served as an inducing signal for the Vic-promoter. When cells were agitated (i.e., incubated in a flask on a rotary shaker), the average fluoresence intensity increased from 235 AU to 270 AU. A marginal drop in fluorescence was observed in the "no-induction" experiment. This small drop in expression (after 120 min) from the Vic-promoter was routinely observed when the cells were going into stationary phase (data not shown). No induction was observed when chlorhexidine was added to the cells. Fluorescence intensity changes under H2O2 and agitation treatments were significantly higher than in the controls. The concentration of chlorhexidine (0.000625%) was chosen to parallel the inhibitory effect of 0.005% H2O2. These concentrations, which reduced growth rate to approx. 70%, were determined in pilot experiments (data not shown), where growth inhibition of S. mutans was titrated (with various concentrations of H2O2 and chlorhexidine). Therefore, the experiments show that the response from the vic-promoter was neither a "general" stress response, nor a response to changes in growth rate (Fig. 3A). No change in average fluorescence intensity (either upon aeration, H2O2, or chlorhexidine addition) could be seen in the negative control strain (which remained at autofluorescence levels), or in the positive control strain (which remained at a value of approx. 440 AU), ruling out side-effects of the inhibitors.
To examine the effects of various H2O2 concentrations on Vic-expression, we also examined induction after the addition of 0.0025% and 0.01% H2O2. The 0.005% H2O2 gave the maximal response (Fig. 3B ). The system was also significantly induced at 0.0025% H2O2, but not at the higher concentration of 0.01%.
Characteristics of the vicK Mutant Strain
Evidence presented in this report shows that the VicK two-component sensor in S. mutans is involved in sensing oxidative stress. By conducting a computational analysis on the VicK protein, we found a PAS domain, which predicted the involvement of the VicRK signal transduction system in sensing redox changes. This prediction was subsequently supported by the experimental data presented. We found increased fluorescent intensities in a vic promoter GFP-fusion strain when oxidative stress was applied. We also found that a  vicK mutant was more sensitive to H2O2 treatment than the wild-type. Obviously, bacterial growth was notably inhibited under H2O2-stress and aerobic, oxidative stress. One could argue that, rather than the stress itself, a change in growth rate is the inducing signal toward vicRK induction. To exclude this possibility, we tested the responses of vicRK and the constitutive promoter to chlorhexidine. The concentration of chlorhexidine was chosen to parallel the inhibitory growth effect of 0.005% H2O2, i.e., resulted in a similar reduction in growth rate. Fluorescence intensity changes, in response to the stress, remained absent in both reporter and control strains, indicating that VicK responds specifically to oxidative stress, and not to a reduction in growth rate or alternative effects of chlorhexidine. Similar results were obtained when the growth rate was decreased by heat stress (50°C, data not shown). The constant fluorescence in the control strain excluded other side-effects of these stresses. Several aspects of the vic system in S. mutans have been studied earlier. It has been shown that the system is involved in regulation of gtfBCD, gbpB, and ftf expression, biofilm formation, and genetic competence development (Senadheera et al., 2005). The results from the current study suggest that oxidative stress is one of the triggers for the regulation of these important virulence parameters. While this manuscript was being reviewed, a new study by Senadheera et al.(2007) also indicated that the Vic-system is involved in the response to oxidative stress. There are differences, however, in the behavior of the knock-out in that study and the one described here, possibly due to differences in growth and/or experimental conditions. There is much evidence suggesting a relation between oxidative stress and biofilm formation (Loo et al., 2004; Sampathkumar et al., 2006), e.g., oxidative stress response genes were shown to be up-regulated during biofilm formation in Staphylococcus aureus (Resch et al., 2005). Clearly, all gene products that are currently known to be under control of the S. mutans VicRK system are important traits in the cariogenicity of the organism. This identifies the TCRS as a potential target for prevention or therapy. Indeed, TCRSs have long been considered interesting targets for inhibiting pathogenic micro-organisms (e.g., Stephenson and Hoch, 2004). Specifically, from an oral ecological/oral healthcare point of view, it might be effective to gather as much fundamental insight as possible on the regulatory mechanisms that translate environmental/ecological signals toward cariogenicity, and, subsequently, to use this knowledge to avoid or tackle these signals.
We thank the ACTA Research Institute for financial support. Received for publication August 29, 2006. Revision received February 28, 2007. Accepted for publication March 5, 2007.
Journal of Dental Research, Vol. 86, No. 7,
606-610 (2007) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
et al., 2002
), agitation (
), or 0.000625% chlorhexidine (*), or were without any additions (
). In (B), cells in exponential phase were subjected to various concentrations of H2O2: 0 (
). The results were averaged from 3 independent experiments and were presented as the means with standard deviations. We used a two-way analysis of variance to compare the increase of fluorescence intensity in time among stresses or concentrations (*p < 0.05).
