molecular oral microbiology molecular oral microbiology

Contribution of glucan-binding protein A to firm and stable biofilm formation by Streptococcus mutans Y. Matsumi, K. Fujita, Y. Takashima, K. Yanagida, Y. Morikawa and M. Matsumoto-Nakano Department of Pediatric Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan

Correspondence: Michiyo Matsumoto-Nakano, Department of Pediatric Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan Tel.: + 81 86 235 6715; fax: + 81 86 235 6719; E-mail: [email protected] Keywords: biofilm; glucan-binding protein; Streptococcus mutans Accepted 21 September 2014 DOI: 10.1111/omi.12085

SUMMARY Glucan-binding proteins (Gbps) of Streptococcus mutans, a major pathogen of dental caries, mediate the binding of glucans synthesized from sucrose by the action of glucosyltransferases (GTFs) encoded by gtfB, gtfC, and gtfD. Several stress proteins, including DnaK and GroEL encoded by dnaK and groEL, are related to environmental stress tolerance. The contribution of Gbp expression to biofilm formation was analyzed by focusing on the expression levels of genes encoding GTFs and stress proteins. Biofilm-forming assays were performed using GbpA-, GbpB-, and GbpC-deficient mutant strains and the parental strain MT8148. The expression levels of gtfB, gtfC, gtfD, dnaK, and groEL were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, the structure of biofilms formed by these Gbp-deficient mutant strains was observed using confocal laser scanning microscopy (CLSM). Biofilm-forming assay findings demonstrated that the amount formed by the GbpA-deficient mutant strain (AD1) was nearly the same as that by the parental strain, while the GbpB- and GbpC-deficient mutant strains produced lower amounts than MT8148. Furthermore, RT-qPCR assay results showed that the expressions of gtfB, dnaK, and groEL in AD1

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 217–226

were elevated compared with MT8148. CLSM also revealed that the structure of biofilm formed by AD1 was prominently different compared with that formed by the parental strain. These results suggest that a defect in GbpA influences the expression of genes controlling biofilm formation, indicating its importance as a protein for firm and stable biofilm formation.

INTRODUCTION Streptococcus mutans has been implicated as a major causative agent of dental caries in humans. Cell surface proteins, such as glucosyltransferases (GTFs; GTFB, GTFC, and GTFD), and protein antigen c, as well as glucan-binding proteins (Gbps; GbpA, GbpB, GbpC, and GbpD) are considered to be components associated with the adhesion phase of caries development (Hamada et al., 1984; Banas & Vickerman, 2003). The major function of Gbps is binding of glucans synthesized from sucrose by the action of GTFs (Kuramitsu, 2003). Gbps are also associated with biofilm structure and its accumulation (Banas & Vickerman, 2003). GbpA, the first designated glucan-binding protein reported, contains C-terminal repeats similar to the glucan-binding

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domain of GTF enzymes (Banas et al., 1990; Shah & Russell, 2004). This protein is involved in cellular adherence to the tooth surface, and has been shown to contribute to the cariogenicity of S. mutans both in vitro and in vivo (Matsumoto-Nakano et al., 2007). GbpA contributes to the development of optimal plaque biofilm, which minimizes stress on the bacterial population (Banas et al., 2007). A defect of GbpA causes changes in biofilm structure, including reduced microcolony height and spreading over the substratum, as well as changes in localized pH compared with the non-defective parent strain (Hazlett et al., 1999). Such alteration in structure causes harbored bacteria to be exposed to acid, therefore making them susceptible to gene introduction, with the stress-response protein RecA possibly related to this response, though the detailed mechanism remains unclear (Banas et al., 2007). GroEL and DnaK are stress-response proteins that have important roles for inducing generation or alteration of new proteins (Lindquist & Craig, 1988; Craig et al., 1993; Jayaraman & Burne, 1995). In the present study, we analyzed the contribution of Gbp expression to biofilm formation using Gbpdeficient mutant strains. We found that GbpA has a strong relationship with biofilm structure, so we also investigated the expression levels of genes encoding GTF and stress proteins to elucidate the effects of GbpA on those genes in biofilm formation.

METHODS Bacteria strains We used S. mutans MT8148 and its GbpA- (AD1), GbpB- (BD1), and GbpC-deficient (CD1) mutant strains (Matsumoto-Nakano et al., 2007), as well as strains NG8, GS5, and UA159 (Matsumoto-Nakano & Kuramitsu, 2006) in the present study (Table 1). All were grown in brain–heart infusion broth (Becton Dickinson, Franklin Lakes, NJ), Todd Hewitt (TH) broth (Becton Dickinson), and mitis-salivarius agar (Becton Dickinson), as required, with the appropriate antibiotics (erythromycin 10 lg ml1, kanamycin 500 lg ml1, and spectinomycin 1 mg ml1) used for selection. Construction of GbpA-defective mutants GbpA-defective mutant strains were constructed using a method previously described (MatsumotoNakano et al., 2007). Briefly, after being digested to become linear at a unique restriction site, the plasmid pMMN26 containing an erythromycin-resistance cassette was introduced into S. mutans NG8, GS5, and UA159 by transformation to allow allelic exchange, using the method described by Tobian & Macrina (1982) (Table 1). Transformants were screened on mitis-salivarius agar plates containing the appropriate

Table 1 Strains and plasmids used in this study Strains or plasmids Streptococcus mutans MT8148 UA159 NG8 GS5 AD1 UA159 AD1 NG8 AD1 GS5 AD1 BD1 CD1 Escherichia coli DH5a Plasmids pGEM-T pMMN26

Relevant charateristics

Source or reference

Wild type Wild type Wild type Wild type MT8148::ΔgbpA::Ermr UA159:: ΔgbpA::Ermr NG8:: ΔgbpA::Ermr GS5:: ΔgbpA::Ermr MT8148:: ΔgbpB::Spr MT8148:: ΔgbpC::Kmr General cloning

Ooshima et al. (1983) University of Alabama University of Florida Perry et al. (1985) Matsumoto-Nakano et al. (2007) This study This study This study Matsumoto-Nakano et al. (2007) Matsumoto-Nakano et al. (2007) Invitrogen

Cloning vector; Ampr pGEM-T harboring an inactivated gbpA gene with an Emr cassette; Ampr, Emr

Promega Matsumoto-Nakano et al. (2007)

Ampr, Ampicillin resistance; Emr, Erythromicin resistance; Spr, Spectinomycin resistance; Kmr, Kanamycin resistance.

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antibiotics. Appropriate insertional inactivation of the mutants was confirmed by determining the nucleotide sequences of erm by polymerase chain reaction (PCR) as well as Western blotting of whole cells from the mutant strains with GbpA-specific rabbit antiserum (Matsumoto-Nakano et al., 2007). Evaluation of GTF expression The tested organisms were grown in brain–heart infusion broth at 37°C to an optical density at 550 nm (OD550) value of 1.0. Bacterial cells were then re-suspended with phosphate-buffered saline (PBS) for a final dilution in 1 9 sodium dodecyl sulfate (SDS) gelloading buffer, while the supernatants were concentrated by 50% ammonium sulfate precipitation and dissolved in the same buffer. An equal amount of each protein was separated by 7.5% SDS–polyacrylamide gel electrophoresis (PAGE) and then transferred onto polyvinylidene difluoride membranes (Immobilon; Millipore, Bedford, MA). Transferred protein bands were exposed to anti-rabbit antibodies against cell-associated GTF (CA-GTF), and simultaneously reacted with GTFC and GTFD, kindly provided by Professor Shigeyuki Hamada (Hamada et al., 1989). In addition, an antibody against the 190kDa cell-surface protein antigen c (PAc) was also used as an internal control for Western blotting analysis. Next, bands were visualized using the alkaline phosphatase-conjugated anti-rabbit immunoglobulin G antibody (New England Biolabs, Beverly, MA) and 5-bromo-4-chloro-3-indolylphosphate-nitro-blue tetrazolium substrate (Moss Inc., Pasadena, MD). The

intensity of positive bands for each strain was determined using the National Institutes of Health IMAGE software application (version 1.43; Macintosh computer application, Scion, MD). Quantification of GTF activity GTF protein enzyme activities were quantified using polyacrylamide gels as previously described (Inagaki et al., 2009). Briefly, the strains were grown to OD550 = 1.0, and cells were collected and washed with PBS buffer (pH 7.4), then resuspended in PBS buffer and adjusted again to OD550 = 1.0. Next, 15 ll of each cell suspension was run on 7.5% SDS–PAGE gels. After electrophoresis, the gels were incubated overnight at 37°C in 3% sucrose, 0.5% Triton X-100, and 10 mg ml1 dextran T10 in 10 mM sodium phosphate (pH 6.8) at 37°C, and the resulting glucan bands were treated with periodic acid and pararosaniline. The intensities of positive bands of each strain were determined using the National Institutes of Health IMAGE software package (version 1.43; Macintosh computer application) and used to show the activities of the GTF proteins. Analysis of expression of genes encoding GTF and stress-response proteins Primers for 16S rRNA were designed as internal controls. All primers used in this study are shown in Table 2. Total RNA was isolated from 15 ml of midlog-phase cell cultures. After centrifugation, the cells were suspended in 0.3 ml of diethylpyrocarbonatetreated water. Samples were then transferred to

Table 2 Primers used for real-time quantitative polymerase chain reactions in the present study Gene 16S rRNA gtfB gtfC gtfD dnaK groEL

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

Sequence (50 ?30 )

References

CAG CGC AGC TGA TAG CTG TTT GT CT CTG CTG GCA AAT TCG CTT ACT TG GAT GGG TGA CAG TAT CTG TTGC GAG CTA CTG ATT GTC GTT ACTG GAT GCT TCT GGG TTC CAA GCT CGA TTA CGA ACT TCA TTT CCGG GTT TGA TTA CCT TGG GCA CCA CAA CAT TGG ACG TTT GCC TGA CTT TGG GTC TGC GTT TGT GGT ACA ACA AAC TCA GCA GTT GCA GTT CTT CCC CAT CTT AGA TTT GAT GGA AAG AAT TGT GCA GAT GCA AGA AGT ATG GTG CGT GGT AGA AGC AAC CTC TGA AAC TAG TTT AGC TCC

Matsumoto-Nakano & Kuramitsu (2006)

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Inagaki et al. (2009) Inagaki et al. (2009) Inagaki et al. (2009) This study This study

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FastRNA tubes with blue caps (Qbiogene, Inc., Carlsbad, CA) and 0.9 ml of TRIzol reagent (Invitrogen, Carlsbad, CA) was added. Cells were broken using a FastPREP FP120 homogenizer (Qbiogene) at a speed setting of 0.6 for 25 s. After the samples were placed on ice for 2 min, 0.2 ml of chloroform was added and the tubes were vortexed and centrifuged again, as described above. RNA was finally precipitated from the aqueous phase with isopropanol, and the resulting pellets were dried and resuspended in 20 ml of diethylpyrocarbonate-treated water. For reverse transcription-quantitative PCR (RT-qPCR) analysis, RNA samples were treated for 15 min at 37°C with 1.0 U ml1 of RNase-free DNase (Promega, Madison, WI) to remove contaminating DNA. Reverse transcription was carried out with SuperScript III (Invitrogen), according to the directions of the supplier. Real-time RT-qPCR was performed using cDNA samples with either 16S rRNA or specific primers using IQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) in an iCycler thermal cycler, according to the manufacturer’s recommendations (Bio-Rad). Relative expression levels of the target gene transcripts were then calculated by normalizing the levels of specific RNA of each target gene to the level of 16S rRNA. By normalizing the Ct values for the target genes to the total amount of 16S rRNA, all samples were compared and relative fold changes were calculated using the DDCt method described for the MyIQ real-time PCR detection system (Bio-Rad). Biofilm analysis using confocal laser scanning microscopy Quantification of biofilm formation Biofilm formation was assayed using a method previously described (Matsumoto-Nakano & Kuramitsu, 2006), with some modifications. Briefly, 96-well polystyrene microtiter plates were prepared by adding 1 ll of a pregrown cell suspension to 100 ll of TH broth including sucrose (0.5%) in individual wells. The plates were then incubated at 37°C with 5% CO2 for 48 h, after which liquid medium was removed and the wells were rinsed six times with sterile distilled water. The plates were then air-dried and stained with 1% crystal violet for 15 min. After staining, the plates were rinsed with sterile distilled water to remove excess dye and air-dried. Stained biofilms were 220

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quantified by measuring absorbance at 570 nm with an enzyme-linked immunosorbent assay microplate reader (model 3550; Bio-Rad). Each assay was performed in triplicate and wells without biofilm were used as blank controls after crystal violet staining. Staining to show cell aggregation Determination of viable and non-viable cells was performed using LIVE/DEAD BacLight viability staining (Molecular Probesâ, Life Technologies Corp., Carlsbad, CA) according to the manufacturer’s instructions (Inagaki et al., 2009). Biofilms were formed by MT8148 and Gbp-deficent mutant strains using the method described above. Cells were stained and kept in the dark for 15 min, then examined with a confocal laser scanning microscope (CLSM; LSM 510, version 4.2; Carl Zeiss MicroImaging Co., Ltd., Jena, Germany) with a reflected laser at 405 nm and 488 nm, while obtained images were digitally reconstructed using IMAGE J 1.42q software (National Institutes of Health, Bethesda, MD). Each biofilm was scanned at three randomly selected positions. Quantitative and structural analysis of biofilms Quantitative and structural analyses of biofilms were performed using a CLSM and subsequent image analysis (Kuboniwa et al., 2009). Tested strains were cultured at 37°C for 16 h in TH broth and stained with hexidium iodide (15 lg ml1; Molecular Probesâ). After washing with PBS, the cells were adjusted to OD600 = 0.1 with Chemically defined medium including sucrose (0.5%) (sCDM) (Bouvet et al., 1981). The biofilms were developed in individual chambers with sCDM in a Lab-Tekâ Chambered #1.0 Borosilicate Coverglass System 8 (Numc, Rochester, NY) for 24 h. They were observed using a CLSM (LSM 780, Axio Observer.Z1, Carl Zeiss) with a reflected laser at 568 nm, whereas obtained images were digitally reconstructed using ZEN LITE 2012 64bit version (Black edition; Carl Zeiss). Each biofilm was scanned at three randomly selected positions. Sonic disruption assay Sonic disruption assays were performed as previously reported by Kuboniwa et al. (2009), with some modification. The tested strains were inoculated into 5-ml portions of one-quarter strength TH broth in six-well polystyrene microtiter plates and allowed to form © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 217–226

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biofilms. After 18 h of incubation, the resulting biofilms were sonicated for 1 or 2 min at an output level of 7 (output power 25 W, oscillating frequency: 28 kHz, tip diameter: 2.5 mm) using a Handy ultrasonic disruptor (UR-20P; Tomy Seiko, Tokyo, Japan). Immediately after sonication, supernatants containing floating cells were removed by aspiration and the remaining biofilms were gently washed with PBS. Attached cells were removed with a cell scraper and suspended in PBS, then inoculated onto trypticase soy agar plates at 37°C for 48 h and counted. The rates of the remaining cells following sonication were calculated as the percentage relative to the number of total biofilm cells for each strain. Statistical analysis Intergroup differences of various factors were estimated using statistical analysis of variance for factorial models. Fisher’s protected least-significant difference test was used to compare individual groups. Statistical computations were performed with STAT-VIEW II (Macintosh computer application). RESULTS Biofilm formation The quantity of biofilm formed by AD1 was similar to that formed by MT8148, whereas that of those formed by the GbpB-deficient mutant strain BD1 and GbpCdeficient mutant strain CD1 was lower (Fig. 1A). Furthermore, biofilms formed by GbpA-defective strains generated from NG8, UA159, and GS5 were the same as those of their respective parental strain (Fig. 1B). Viability staining also demonstrated that biofilms formed by AD1 had a higher density compared with biofilms by other strains (Fig. 1C). In biofilms formed by BD1 and CD1, the rate of bacterial growth was lower and aggregation intensity was reduced compared with MT8148 biofilm. The growth rates of all strains were not significantly different (data not shown). Expressions of GTFs and stress protein genes The expression levels of gtfB and gtfC in AD1 were increased by approximately three-fold compared with those in MT8148 (Table 3). On the other hand, gtfB © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 217–226

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gene expression in BD1 and CD1 was decreased to half of that in MT8148. As for gtfD expression, no significant change was observed in AD1 and BD1, whereas its expression in CD1 was decreased compared with MT8148. When comparing gtfB and gtfC expression between BD1 and CD1, there were no significant differences found. Western blotting analysis showed that the amounts of GTFB and GTFC proteins in AD1 strains were significantly increased compared with those of their parental strains (Fig. 2A). Furthermore, activity staining demonstrated increases in each AD1 strain as compared with their parental strains (Fig. 2B). Furthermore, the expressions of the dnaK and groEL genes in the AD1 strain were significantly increased compared with the parental strain (Fig. 3). Biofilm architecture Assays of biofilm quantity using microtiter plates to measure the optimal density did not clarify thickness. Therefore, confocal microscopic analysis was performed to elucidate the structure of the formed biofilms. Figure 4 presents a representative three-dimensional image of bacteria and glucan in biofilms formed by AD1 and each parental strain. As for vertical distribution, biofilms formed by AD1 were markedly thicker than those by each parental strain. Furthermore, biofilms formed by the parental strain MT8148 were found to be rigid and uniform, whereas those formed by AD1 were relatively patchy and sparse. Cross-sectional images of biofilms formed by AD1 showed that they were thicker than those formed by each parental strain. To analyze the influence of molecules on biofilm vulnerability, the physical strength of the biofilms against brief ultrasonication was compared (Fig. 5), which revealed that the AD1formed biofilms were quite fragile compared with those formed by the other strains. DISCUSSION Although Gbps do not possess enzymatic activities (Shah & Russell, 2004), they are known to be involved in bacterial adhesion and accumulation (Banas et al., 1990). Synthesized glucan binds to specific Gbps expressed on the surface of S. mutans cells (Lynch et al., 2007). Furthermore, Gbps may provide mechanical stability by tightly and stably 221

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1.6

OD570

1.2

*

0.8

*

0.4

0.0

B

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BD1

CD1

0.4

OD570 Values

0.3

0.2

0.1

10 μm

binding bacterial cells together and to tooth surfaces, so providing a supporting frame for developing biofilm (Koo et al., 2010). Our results regarding the amount of biofilm produced suggest that GbpA-deficiency does not influence that formation compared with GbpB and GbpC deficiency. On the other hand, GbpA was shown to be strongly related to cariogenicity in our previous studies (Matsumoto-Nakano et al., 2007). In addition, the role of GbpA may be linked to glucan molecules, more or less independent of individual bacteria, as its loss does not adversely affect biomass (Lynch et al., 2007). A previous study reported that S. mutans that are defective for GbpA 222

1 D S5 G

AD1

10 μm

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G

C MT8148

S5

1 D

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8A G N

8 G

D 9A 15 A U

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D A

15 A U

M

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14

8

0 Figure 1 Biofilm formation by Streptococcus mutans strains. (A) Amounts of formed biofilms. (B) Amounts of biofilm formed by MT8148 and glucan-binding protein A (GbpA)-deficient mutants constructed from various laboratory strains. There were statistically significant differences between MT8148 and the GbpA-deficient mutant strains (*P < 0.001). (C) Confocal microscopic images of MT8148 and AD1 following LIVE/DEAD staining. Viable cells were stained green by SYTO9, while cells with damaged membranes were stained red by propidium iodine.

show a strong trend toward covering more of the substratum, and that loss led to both macroscopic and microscopic changes in biofilm structure (Hazlett et al., 1999). Therefore, we investigated the structure of biofilms formed by AD1. In the present study, the structure of biofilm formed by AD1 was drastically altered and its biomass was increased compared with that by the parental strain. These results support previously reported findings. Furthermore, LIVE/DEAD staining showed extensive changes with regard to the mass of cells formed in the biofilms formed by AD1 compared with the parental strain MT8148, which we considered was due to © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 217–226

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an increase in gtf expression. Real-time RT-qPCR assay findings confirmed our theory, as they showed that the expression of gtfB was significantly increased in AD1 compared with MT8148 and the other Gbpdeficient strains. In addition, the expression and activity of GTFB in AD1 were increased compared with the parental strain. These phenomena may have been caused by environmental changes resulting from GbpA deficiency. Observations using CLSM revealed that AD1 formed biofilms that were more expansive compared with those formed by the parental strain. However, they were quite fragile, suggesting a structure of loosely connected microcolonies. GbpA seems to

Table 3 Ratios of gene expression for the parental strain MT8148 and Gbp-deficient mutant strains Gene expression ratio1 Strains

gtfB2

gtfC

gtfD

AD1 BD1 CD1

3.15  0.64*** 0.43  0.13 0.49  0.06

3.23  1.29* 1.34  0.36 1.09  0.25

1.1  0.02 1.17  0.03* 0.47  0.05***

1

Relative to that of MT8148. gtf gene expression in each Gbp-deficient mutant strain. Total cDNA abundance in the test samples was normalized using the 16s rRNA gene as a control. There were statistically significant differences between S. mutans MT8148 and the Gbp-deficient mutant strains, as shown by Fisher’ s PLSD analysis (*P < 0.05, ***P < 0.001). 2

A

MT8148 AD1

UA159 UA159AD1 NG8 NG8AD1 GS5

GS5AD1

200 kDa 150 kDa 180

** *

140

Gray value

**

*

160

120 100 80 60 40 20 0

MT8148

UA159 UA159AD1 NG8

MT8148 AD1 UA159 UA159AD1 NG8

B

NG8AD1

GS5

NG8AD1 GS5

GS5AD1 GS5AD1

200 kDa 150 kDa

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450

*

400 *

350 300

Area

Figure 2 Glucosyltransferase B (GTFB) expression and enzyme activity of MT8148 and glucan-binding protein A (GbpA) deficient mutants constructed from various clinical strains. Results are shown for (A) Western blot analysis and (B) activity staining. The intensities of positive bands for each strain were determined using the National Institutes of Health IMAGE software package (version 1.43; Macintosh computer application) and used to show the activities of the GTF proteins. Three independent experiments were performed in duplicate with each strain. There were statistically significant differences between the AD1 strains and their parental strains, as shown by Fisher’s protected least-significant difference analysis (*P < 0.05, **P < 0.01).

AD1

*

250

*

200 150 100 50 0

8148

AD1

UA159 UA159 AD1

NG8

NG8 AD1

GS5

GS5 AD1

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coordinate the integrity of biofilm in both the developing and maturation phases (Lynch et al., 2007), so its specific role may be regulation of biofilm formation with regard to dispersion, de-concentration, and/or

Gene expression related to that of MT8148

12

***

***

MT8148 AD1 BD1 CD1

8

***

4

0

dnaK

groEL

Figure 3 Expression levels of dnaK and groEL genes. The expressions are expressed as values relative to the parental strains. Total cDNA abundance in the test samples was normalized using the 16s rRNA gene as a control. There were statistically significant differences between MT8148 and the mutant strains, as shown by Fisher’s protected least-significant difference analysis (***P < 0.001).

8148

8148 AD1

NG8

NG8 AD1

UA159

UA159 AD1

GS5

GS5 AD1

224

detachment of microcolonies. An earlier study noted that GbpA contributes to a strong biofilm structure and plays an important role in linking glucan molecules, more or less independent of individual bacteria, while its loss does not adversely affect biomass (Lynch et al., 2007). Together, these results suggest that loss of the GbpA results in weak bacterial binding, resulting in alterations of biofilm structure. The bacterial composition of human dental biofilm remains relatively stable with minor environmental changes (Marsh, 2009). On the other hand, the structure of biofilm is clearly influenced by a number of biological factors such as twitching motility, growth rate, cell signaling, and extracellular polymeric substance (EPS) production, while the physical growth environment may also play a significant role in determination of that structure (Stoodley et al., 2002). In the present study, biofilm structure was drastically altered, as a deficiency of GbpA is the result of the physical growth environment. Our results suggest that

Figure 4 Biofilm analysis with confocal laser scanning microscopy. Representative threedimensional images of biofilms formed by the AD1 strains and their parental strains in the presence of 0.5% sucrose. Shown are x-y section and z-projection of biofilms formed by those strains created as digital images using ZEN LITE 2012, 64 bit version.

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70 *

The rate of remained cells (%)

60 50 40 30

*

20

**

10 0

8148

AD1

BD1

CD1

Figure 5 Tenacity of biofilms formed by MT8148 and glucan-binding protein (Gbp) -deficient mutant strains. The rate of remaining cells after sonic disruption was calculated as a percentage relative to the number of total biofilm cells for each strain. Three independent experiments were performed in duplicate with each strain. There were statistically significant differences between MT8148 and the other strains, as shown by Fisher’s protected least-significant difference analysis (*P < 0.01, **P < 0.001).

GbpA is an important protein to determine biofilm structure, though its mechanism remains to be elucidated. Future studies should focus on stressresponse proteins and the tenacity of biofilm formed by GbpA-defective mutant organisms. Expressions of the dnaK and groEL genes, known to be stress-response proteins, were found to be increased in AD1 compared with MT8148. We speculated that when the AD1 strain is exposed to stress, such as low pH or acid, stress proteins, such as the molecular chaperones GroEL and DnaK, are increased, as a result of its deficiency of GbpA and lack of binding to the EPS matrix. The molecular chaperone (GroEL) was previously shown to be upregulated in S. mutans within a mixed-species biofilm community, as well as increases in genes associated with glucan synthesis and remodeling (Klein et al., 2012). Furthermore, the protein profile obtained by two-dimensional gel analysis indicated that many other proteins in AD1 had altered expressions (data not shown), suggesting that GbpA may exert, directly or indirectly, pleiotropic effects. In addition, a deficiency of GbpA produces a condition of stress for S. mutans and the signal transduction system may react against this stress to change protein expression. Biofilm is composed of communities of bacteria embedded in a matrix of extracellular polysaccharide (Xiao et al., 2012). The universal characteristics of biofilm structure, such as copious extracellular polysaccharide production and decreased antibiotic susceptibility, have also been well documented, and © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 217–226

analysis of specific gene products in biofilm formation has been reported (Kolter & Losick, 1998). Specific proteins associated with exopolysaccharide matrix assembly, and metabolic and stress adaptation processes were reported to be highly abundant following sucrose introduction as biofilm transited from earlier to later developmental stages (Klein et al., 2012). The structure of biofilm primarily consists of an EPS matrix or glycocalyx in which bacterial cells are embedded (Costerton et al., 1995) and under normal conditions forms a uniform structure according to the EPS matrix and cells become connected in a regular manner. Biofilm that is relatively stiff is resistant to such stress factors as acid, antibiotics, and low pH. However, a deficiency of GbpA results in loose binding to the EPS matrix, and a weak and non-uniform biofilm is formed. In addition, that deficiency may change the synthesis of glucan and protein expressions to adapt to environmental changes. Therefore, GbpA has an important role in binding proteins and exopolysaccharides to construct biofilm and maintain a balanced environment, while its lack alters the structure of biofilm and its tolerance to various types of stress. In summary, GbpA deficiency was shown to cause changes in EPS production and protein gene expression, leading to a weak and unstable biofilm structure. On the other hand, detailed information about surface proteins and the stress response is scant, and additional studies are needed to clarify those factors. ACKNOWLEDGEMENTS This study was supported by a Grant-in Aid for Scientific Research (B) 2339047315 from the Japan Society for the Promotion of Science. REFERENCES Banas, J.A. and Vickerman, M.M. (2003) Glucan-binding proteins of the oral streptococci. Crit Rev Oral Biol Med 14: 89–99. Banas, J.A., Russell, R.R.B. and Ferretti, J.J. (1990) Sequence analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingbritt. Infect Immun 58: 667–673. Banas, J.A., Fountain, T.L., Mazurikiewicz, J.E., Sun, K. and Vickermann, M.M. (2007) Streptococcus mutans glucan-binding protein-A affects Streptococcus gordonii

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Contribution of glucan-binding protein A to firm and stable biofilm formation by Streptococcus mutans.

Glucan-binding proteins (Gbps) of Streptococcus mutans, a major pathogen of dental caries, mediate the binding of glucans synthesized from sucrose by ...
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