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. 2021 Feb 3;100(6):631–638. doi: 10.1177/0022034520979798

Dissecting the Role of VicK Phosphatase in Aggregation and Biofilm Formation of Streptococcus mutans

S Wang 1, L Long 1, X Yang 1, Y Qiu 1, T Tao 1, X Peng 1, Y Li 1, A Han 2, DB Senadheera 3, JS Downey 4, SD Goodman 5, X Zhou 1,✉, DG Cvitkovitch 6,✉
PMCID: PMC9290112  PMID: 33530836

Abstract

VicRK (WalRK or YycFG) is a conserved 2-component regulatory system (TCS) that regulates cell division, cell wall biosynthesis, and homeostasis in low-GC Gram-positive bacteria. VicRK is also associated with biofilm formation of Streptococcus mutans on the tooth surface as it directly regulates the extracellular polysaccharide (EPS) synthesis. Of the 2 components, VicK possesses both autokinase and phosphatase activities, which regulate the phosphorylation and dephosphorylation of the regulator VicR in response to environmental cues. However, the dual mechanism of VicK as the autokinase/phosphatase in regulating S. mutans’ responses is not well elucidated. Previously, it has been shown that the phosphatase activity depends on the PAS domain and residues in the DHp domain of VicK in S. mutans. Specifically, mutating proline at 222 in the PAS domain inhibits VicK phosphatase activity. We generated a VicKP222A mutant to determine the level of VicR-P in the cytoplasm by Phos-tag sodium dodecyl sulfate polyacrylamide gel electrophoresis. We show that in VicKP222A phosphatase, attenuation increased phosphorylated VicR (VicR-P) that downregulated glucosyltransferases, gtfBC, thereby reducing the synthesis of water-insoluble polysaccharides (WIS-EPS) in the biofilm. In addition, VicKP222A presented as long-rod cells, reduced growth, and displayed asymmetrical division. A major adhesin of S. mutans, SpaP was downregulated in VicKP222A, making it unable to agglutinate in saliva. In summary, we have confirmed that VicK phosphatase activity is critical to maintain optimal phosphorylation status of VicR in S. mutans, which is important for cell growth, cell division, EPS synthesis, and bacterial agglutination in saliva. Hence, VicK phosphatase activity may represent a promising target to modulate S. mutans’ pathogenicity.

Keywords: caries, histidine protein kinase, glucosyltransferase, polysaccharides, gene expression regulation, agglutinins

Introduction

VicRK (also referred as WalRK or YycFG) is a 2-component regulatory system (TCS) that maintains the cell integrity, homeostasis, and bacteriocin production in low-GC Gram-positive bacteria (Senadheera et al. 2005; Deng et al. 2007; Dubrac et al. 2008). VicRK TCS contains a membrane-bound histidine kinase (HK), VicK. VicK autophosphorylates upon activation and transfers phosphate groups to its cognate response regulator (RR), VicR. Activated VicR then interacts with promoters of target genes to regulate their expression (Wang et al. 2013; Mattos-Graner and Duncan 2017). The VicK cytoplasmic domains are highly conserved and include HAMP (linker), PAS (potential signal binding), DHp (dimerization and histidine phosphorylation), and CA (catalytic ATPase) domains (Gao and Stock 2009; Lei et al. 2019). VicK also exhibits phosphatase activity toward VicR~P, for which the VicK PAS domain is required (Gutu et al. 2010). In addition, VicK can transphosphorylate noncognate RRs under certain conditions (e.g., presence of Mn2+) (Downey et al. 2014). Furthermore, deletion of vicK in Streptococcus mutans is reported to cause aberrant cell division (Senadheera et al. 2005; Lei et al. 2019).

S. mutans have higher capacity to form biofilms than other Streptococci in dental plaque. S. mutans can adhere to dental surfaces by both sucrose-dependent and sucrose-independent mechanisms. In the presence of sucrose, glucosyltransferases (GtfB, -C, -D) promotes adhesion. GtfC has a high affinity to bind to the acquired pellicle formed of salivary proteins (Bowen and Koo 2011) and uses dietary sucrose to produce both water-soluble polysaccharides (WS-EPS) and water-insoluble polysaccharides (WIS-EPS). WIS-EPS facilitates initial S. mutans adhesion by binding to surface glucan-binding proteins (Gbps) (Rainey et al. 2019). GtfB assists biofilm assembly by allowing more S. mutans to attach on the tooth surface, while GtfD regulates WS-EPS synthesis (Duque et al. 2011). Previous work demonstrated that VicRK regulates the expressions of gtfBC, ftf (fructosyltransferase), and gbpB by directly binding to their promoters. Regulation of genes involved in biofilm formation or extracellular polysaccharide (EPS) in VicK and vicRKX operon overexpression mutants have also been previously reported (Senadheera et al. 2005; Ayala et al. 2014).

In the absence of sucrose, S. mutans initiate adherence by expressing surface adhesins such as SpaP or WapA that interact with salivary glycoproteins such as agglutinin, collagen, and fibronectin (Nakano et al. 2006; Yang et al. 2018; Yang et al. 2019). However, when salivary agglutinin is not adherent and is in liquid phase, aggregation of S. mutans via interactions between SpaP and salivary agglutinin results in clearance ofS. mutans from the oral cavity (Robinette et al. 2014).

In this study, we investigated the importance of VicR phosphorylation via VicK in S. mutans’ physiology by constructing 2 point mutations in the VicK PAS domain, thus individually blocking VicK autophosphorylation and phosphatase activity. Wild-type (WT), UASpec, and ΔVicK strains were used as controls. We assessed various phenotypes and biological indices related to bacterial colonization and cell division. To determine the level of intracellular VicR-P Phos-tag, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used. Our results demonstrate essentiality of VicK autophosphorylation and phosphatase activities in maintaining normal cell growth, division, and 3-dimensional (3D) biofilm structure. Furthermore, we show that intracellular VicR-P accumulation severely suppressed gtfBC expression, abolished WIS-EPS synthesis, and reduced S. mutans aggregation in saliva.

Materials and Methods

Bacterial Strains, Plasmids, and Media

Bacterial strains, plasmids, and amplicons used in this study are listed in Appendix Table 1. S. mutans UA159 WT strain and its derivatives were routinely maintained on Todd-Hewitt yeast extract (THYE) media (BBL Becton Dickinson), while Escherichia coli DH5α strains were maintained on LB media, both containing appropriate antibiotics as described in the Appendix. All S. mutans cultures were routinely grown as standing cultures at 37°C in a 5% CO2–95% air mixture. Site-directed vicK mutants were generated using an allele replacement strategy (details in Appendix), and primers used are listed in Appendix Table 2. Genome-harboring differentially mutated vicK genes are listed in Appendix Table 1, and all mutations were confirmed by sequencing.

S. mutans Biofilm Formation

Biofilm formation experiments were performed using semidefined minimal media as described elsewhere (Li et al. 2002; Senadheera et al. 2005). Briefly, S. mutans biofilms were grown in 12-well polystyrene microtiter plates containing 1/4 THYE media supplemented with 10 mM sucrose or, when specified, with 20 mM glucose (Senadheera et al. 2005). All wells were inoculated with 1/100 dilution of overnight bacterial suspension and incubated for 24 h at 37°C. Thereafter, broth was removed and adherent cells were stained with crystal violet and photographed directly.

Scanning Electron Microscopy

Scanning electron microscopy (SEM; FEI) was performed as described previously (Viszwapriya et al. 2017). Briefly, mid-log phase bacterial cells were harvested, resuspended in phosphate-buffered saline (PBS), spotted on coverslip, and air dried to pellicles. Pellicles and 24-h biofilms from each strain were fixed with 2.5% glutaraldehyde for 12 h, serially dehydrated in ethanol, and sputter-coated with gold. Specimens were examined at specified magnifications.

Biofilm Analysis and Structural Imaging

S. mutans biofilms were generated as described above. Cells were labeled with SYTO9 and biofilm matrix was labeled by integrating Alexa Fluor 647–labeled dextran conjugate (Molecular Probes) (Zheng et al. 2015). Biofilm images were captured with a Leica DMIRE2 confocal laser scanning microscope equipped with a 60× oil immersion objective lens. Imaging gates were set to 495 to 515 nm for SYTO9 and 655 to 690 nm for Alexa Fluor 647. Each biofilm was scanned at 5 random positions, and optical sections were used for 3D reconstruction of bacteria/WIS-EPS biofilms with Imaris 7.0.0 (Bitplane) and quantified for bacteria/EPS biomass of the biofilms.

Growth Curves

Growth of test strains was monitored using a Bioscreen plate reader (Bioscreen C Labsystems). Overnight bacterial cultures were inoculated at 1/100 dilution in fresh THYE media. Uninoculated THYE was used as control. Biolink software (Labsystems) was used to monitor OD600 at 37°C every 20 min for 24 h, with 25-s high shaking before each measurement. OD600 measurements were plotted against time to generate growth curves.

RNA Extraction

Overnight cultures were diluted into fresh THYE broth as described above. Mid-log phase (0.4–0.5 OD600) cells were pelleted and resuspended in Trizol reagent (Invitrogen) prior to RNA isolation using the Fast Prep system (Bio 101 Savant). Total RNA was digested with DNase (RNAse-free kit; Promega) at 37°C for 1 h and reverse transcribed using first-strand complementary DNA (cDNA) synthesis kit (Fermentas). Expression was normalized to the 16S ribosomal RNA (rRNA) gene, and statistical analyses were performed using Student’s t test (P < 0.05).

Phos-tag Gel and Western-Blot

The Phos-tag SDS-PAGE method was based on Wayne et al. (2012) with slight modifications. Briefly, a Phos-tag acrylamide resolving gel was prepared as recommended. Prior to experiment, gels and running buffer were maintained at 4°C. Mid-log phase cells were pelleted and lysed at 4°C. Sample was then diluted in sample buffer and loaded on the gel. For dephosphorylated controls, sample was heated at 95°C for 10 min and then chilled prior to loading. Gels were soaked in chilled transfer buffer with EDTA to remove Mn2+ and then in chilled transfer buffer to remove EDTA. Gels were then processed for Western blotting. Anti-VicR polyclonal antibody (Abmart) was used for primary binding and horseradish peroxidase (HRP)–conjugated anti-rabbit antibody (Abcam) was used as secondary antibody at manufacturer-recommended dilutions. Luminescent signal was detected and quantitated using a ChemiDoc XRS+ system (Bio-Rad).

Aggregation Assay

Unstimulated whole saliva was collected from human volunteers with good oral health via a spitting method (Silverman et al. 2010). S. mutans overnight cultures were diluted in prewarmed medium and grown until OD600 reached ~0.8, harvested, washed with potassium phosphate buffer (KPBS buffer), and resuspended in KPBS to an OD600 = 1.0. Bacterial suspension and fresh unstimulated whole saliva were mixed in equal ratios and transferred to cuvettes and incubated at room temperature. OD600 of samples was recorded at 15-min intervals for 120 min. Ethical approval for human saliva was obtained from the institutional review board of West China Hospital of Stomatology, Sichuan University.

Results

Critical Roles of VicK as Autokinase and Phosphatase in S. mutans

We targeted VicK autokinase and phosphatase activity in S. mutans with an in-frame deletion, VicKH217A and VicKP222A containing substitution of histidine at 217 to alanine and proline at 222 to Alanine respectively. UASpec had a spectinomycin cassette inserted and served as a control. VicKH217A overnight culture aggregated to the bottom of glass tube similar to ΔVicK as described in a previous study (Senadheera et al. 2005). We observed that VicKP222A grew as a uniform turbid suspension similar to UASpec, but the growth was much slower (Fig. 1A, B). SEM analysis revealed that UASpec presented as individual short-rod cells with a middle septum, whereas ΔVicK and VicKH217A had intact septae but failed to separate. Interestingly, VicKP222A presented as long-rod cells with asymmetrical division (Fig. 1C).

Figure 1.

Figure 1.

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Biological characteristics, growth kinetic assay, and scanning electron microscopic analysis of cell morphology (40,000×). (A) Overnight cultures (top), untreated and crystal violet stained biofilms (middle) and biofilm mass analysis (bottom) of UASpec, ΔVicK,VicKH217A, and VicKP222A. (B) Growth kinetics of UASpec and VicKP222A were monitored for 12 h in OD600. Each data point is the average of 3 independent OD values per sample. The results shown are representative of 2 other independent experiments conducted with UASpec and VicKP222A. (C) UASpec, ΔVicK, VicKH217A, and VicKP222A cell morphologies were monitored at exponential growth phase. **P < 0.01; ****P < 0.001.

VicKH217A and VicKP222A Formed Aberrant Biofilms

Biofilms of various VicK mutants were analyzed with crystal violet staining. UASpec formed smooth biofilm, but ΔVicK and VicKH217A biofilms appeared to be rough. Although VicKP222A formed uniform biofilms, they were easily detachable and appeared thinner compared with UASpec (Fig. 1A).

We next employed double staining as described (Materials and Methods) to examine biofilm matrix structure (Fig. 2). Interestingly, disrupting VicK in ΔVicK or in VicKH217A severely disrupted biofilm matrix. Furthermore, due to inseparable cell clusters, accumulated cells with less WIS-EPS showed uneven but thicker localized matrix ΔVicK and VicKH217A (data not shown). VicKP222A displayed fragile biofilm matrix, with distributed bacterial cells and limited WIS-EPS production. Biofilm thickness in VicKP222A was also significantly reduced compared with UASpec.

Figure 2.

Figure 2.

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Phenotypic characteristics of Streptococcus mutans biofilm. (A) Bacterial cells and water-insoluble polysaccharides (WIS-EPS) of 24-h S. mutans biofilms were labeled with SYTO9 (Molecular Probes) and Alexa Fluor 647–labeled dextran conjugate (Molecular Probes). Green, bacteria (SYTO 9); red, EPS (Alexa Fluor 647). Images were taken at 60× magnification. Three-dimensional reconstruction of the biofilms of bacteria/extracellular polysaccharide (EPS) was performed with Imaris 7.0.0. (B) Quantification of bacteria/EPS biomass was performed with Imaris 7.0.0. Results are the average of 5 randomly selected positions of each sample and are presented as mean ± SD. P < 0.05. ****P < 0.001.

VicKP222A Affected gtfBCD and ftf Expression and Cellular VicR~P Levels

Since VicKP222A formed thinner biofilms that easily detached and exhibited less WIS-EPS in the matrix, even in the presence of sucrose, we hypothesized that VicKP222A modulated the expression of gtfBCD and ftf. To test this, we performed transcriptional analysis of gtfBCD and ftf in UASpec, ΔVicK, VicKH217A, and VicKP222A. As shown in Figure 3B, expression of gtfD and ftf in VicKH217A was downregulated, and the expression of gtfB was significantly lower in both VicKH217A and VicKP222A than in UASpec. Expression of gtfC remained unchanged in VicKH217A compared with UASpec. Notably, in ΔVicK, gtfB and gtfC were upregulated, whereas gtfD and ftf were downregulated compared with UASpec. In addition, in VicKP222A, gtfD and ftf expression increased by 19.2- and 41.7-fold, respectively. These findings suggested a severe repression of WIS-EPS and a significant induction of WS-EPS in VicKP222A.

Figure 3.

Figure 3.

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VicRK regulon expression in different vicK mutant strains and extracellular proteins of Streptococcus mutans. (A) Concentrated protein profiles of culture supernatant. Culture supernatant proteins were extracted from the stained gel and subjected to mass spectrometric analysis. M: Marker; 1: UASpec; 2: VicKH217A; 3: VicKP222A. (B) Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis showed the gene transcripts of 4 types strains (UASpec, ΔVicK, VicKH217A, and VicKP222A). Values represent means of data obtained from 3 different experiments with standard deviations. ****P < 0.001, of Student’s t test using Prism 7.0 (GraphPad Software). Error bars indicate standard deviations.

Given that Gtfs and Ftf are important for biofilm matrix formation, we performed mass spectrometry on concentrated supernatants from overnight cultures, which confirmed abundance of the Ftf band slightly above 100 KD in VicKP222A and reduced in VicKH217A relative to UASpec. Two other bands between 130 KD and 170 KD in VicKP222A were verified as 2 GtfD forms. Two bands below 170 KD from UASpec were GtfB and GtfC. Of note, corresponding bands in the VicKH217A lane were significantly weaker (Fig. 3A).

Next, we wanted to determine the ratio of phosphorylated VicR to unphosphorylated VicR in these mutants using Phos-tag SDS-PAGE. Foremost, we observed that the total amount of VicR was constant in each strain tested (data not shown). During exponential growth, 8.9%, 8.1%, and 7.7% VicR were phosphorylated in UASpec, ΔVicK, and VicKH217A, respectively. Comparatively, both phosphorylated VicR~P (90.7%) and unphosphorylated VicR levels were higher in VicKP222A. As a control, heating samples from VicKP222A mutant converted all VicR~P to VicR, consistent with heat lability of aspartyl-phosphate bonds (Fig. 4A).

Figure 4.

Figure 4.

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Cellular amounts of VicR~P and transcriptional analysis of VicRK regulon. (A) Representative Western blots of Phos-tag gels containing extracts of VicK mutant strains grown exponentially in Todd-Hewitt yeast extract (THYE) broth. The positions of unphosphorylated VicR, VicR~P, and a very faint contaminant band are indicated. Heated sample of VicKP222A after the dotted line served as a control. (B) Expression profile of genes involved in biofilm formation in control strain UApIB, VicKP222A, and 2 vicR overexpression mutants (named VicR-O1 and VicR-O2). Data are presented as mean ± SD. (C) Representative Western blots of Phos-tag gels containing extracts of control and mutant strains grown exponentially in THYE broth. The positions of unphosphorylated VicR and VicR~P are indicated. *P < 0.05; **P < 0.01; ****P < 0.001.

Overexpression of vicR Increased Cellular VicR Levels but Did Not Affect Its Phosphorylation

We next questioned if increased expression of VicR or its phosphorylated form accounted for changes in gtfs and ftf expression in VicKP222A. For this, we constructed and randomly picked 2 mutant strains differentially overexpressing vicR. VicR expression and phosphorylation were confirmed by Phos-tag SDS-PAGE (Fig. 4C). Transcriptional analysis of gtfB and gtfC showed that their expression was relatively stable in vicR-overexpressing strains and less affected compared to VicKP222A (Figs. 3B, 4B). Interestingly, VicR~P levels were significantly decreased in both VicR overexpression strains (Fig. 4C), thus suggesting that it is the phosphorylated form that regulates gtfs and ftf expression.

VicKP222A Decelerated Agglutination in Saliva of S. mutans

To assess the sucrose-independent adhesiveness, UASpec and VicKP222A were tested for their abilities to aggregate in clarified whole saliva. Initially, UASpec aggregated faster than VicKP222A up to 45 min, followed by a gradual decrease (Fig. 5A). Interestingly, spaP was upregulated in ΔVicK and VicKH217A but down in VicKP222A (Fig. 5B). Furthermore, SpaP protein was less abundant in VicKP222A compared to UASpec, suggesting the transcriptional downregulation of the gene due to high VicR~P levels (Fig. 5C).

Figure 5.

Figure 5.

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Aggregation of UASpec and VicKP222A. (A) Aggregation of UASpec and VicKP222A in the presence of fresh unstimulated whole saliva (WS). Points are means of triplicates from a representative experiment repeated 3 times. *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001 of Student’s t test using Prism 7.0 (GraphPad Software). Error bars indicate standard deviations. (B) Transcriptional analysis of spaP in derivative strains of Streptococcus mutans. Data are presented as mean ± SD (****P < 0.001 of Student’s t test using Prism 7.0). (C) Representative Western blot analysis of SpaP levels in culture supernatant in mutant strains. The whole-cell lysates of each strain are shown below the Western blotting results as the control. M: Marker; 1: UASpec; 2: VicKP222A; 3: UASpec-His, the mutant whose SpaP protein bound with His-tag in UASpec strain; 4: VicKP222A-His, the mutant whose SpaP protein bound with His-tag in VicKP222A strain.

Discussion

VicK Autokinase Activity Is Crucial for Normal Growth of S. mutans

Similar to a ΔVicK mutant, the absence of VicK autokinase activity in vicKH217A severely affected cell division, morphology, and biofilm formation. These phenotypes contrast with the moderate growth defects reported recently for a VicKSpnH218A in Streptococcus pneumoniae, which is distantly related to S. mutans (Barendt et al. 2011; Wayne et al. 2012). The growth difference may reflect VicRK regulon composition in these 2 bacterial species. In S. pneumoniae, VicKSpn inactivation downregulated genes that mediate peptidoglycan (PG) hydrolysis, a similar effect to when a VicRKSpn regulon, pcsB, was underexpressed (Barendt et al. 2009). Similarly, vicK deletion in S. mutans downregulated glucan binding protein B, gbpB. However, ΔVicK and mutant strain with low GbpB showed different morphologies, the latter having formed chains of shorter cocci without change in chain formation as in ΔVicK (Duque et al. 2011).

UASpec presented even biofilms, whereas VicKH217A formed rough, discrete biofilms similar to those of ΔVicK (Senadheera et al. 2005). Previously, we reported that the vic operon influenced gtfBCD and ftf expression, and VicR bound to gtfB, gtfC, and ftf promoter regions. In this study, we report upregulation of gtfB and gtfC expression in ΔVicK and slight repression of gtfD and ftf. When VicK autokinase activity was abolished in VicKH217A, gtfB was downregulated and gtfC remained unchanged, while gtfD and ftf showed moderate repression. Since gtfB and gtfC encode enzymes involved in WIS-EPS synthesis, our results suggest that alterations in WIS-EPS–related gene expression did not account for the major phenotypic changes in biofilm formation by ΔVicK and VicKH217A.

Molecular and structural mechanism of VicK-mediated VicR phosphorylation has been reported (Wang et al. 2013). In this study, we report similar percentages of VicR~P in VicKH217A and ΔVicK to that in UASpec, which partially explained moderate alteration of vic regulon expression. Given the severities of phenotypes, our observations suggest additional biological functions of VicK. Likewise, VicR~P detected in VicKH217A and ΔVicK suggests a transphosphorylation by noncognate HKs. In S. pneumoniae, phosphorylated noncognate HKs PnpS~P, HK08~P, and CiaH~P can phospho-transfer VicRSpn only under long incubation times and are not kinetically favored compared with cognate VicKSpn~P (Laub and Goulian 2007; Laub et al. 2007). As reported elsewhere, in the absence of both VicKSpn and PnpR, VicRSpn~P increased and cell growth was similar to WT. However, this increase of VicRSpn~P was abrogated in the ΔvicKSpn ΔpnpRS triple mutant, indicating that VicRSpn phosphorylation occurs via PnpS in the ΔvicKSpn ΔpnpR double mutant (Wayne et al. 2012). Furthermore, DNaseI footprinting indicated that the gcrR promoter is protected by purified VicK and that VicK can transphosphorylate GcrR in vitro (Negrini et al. 2012; Downey et al. 2014).

VicK Phosphatase Activity Regulates S. mutans Morphology

Lack of VicK phosphatase activity in VicKP222A caused VicR~P accumulation, abnormal cell morphology and division, and reduced growth. This growth phenotype bore resemblance to the vicKT222A phosphatase-deficient mutant in S. pneumoniae D39, wherein growth was consistently lower than parent cells (Wayne et al. 2012). VicRK coordinates cell wall architecture with cell division in Bacillus subtilis, S. pneumoniae, and S. mutans (Senadheera et al. 2005; Fukushima et al. 2008; Gutu et al. 2010). PG hydrolase is important in cell division and cell morphology (Sham et al. 2011). Genes related to PG hydrolysis are regulated via VicRK TCS, such as pcsB and lytA in S. pneumoniae, yoeB and yjeA in B. subtilis, and gbpB in S. mutans. Moreover, depletion of these genes causes division and morphology defects (Fukushima et al. 2008). In S. pneumoniae, PcsB depletion caused growth arrest similar to ΔVicKSpn, whereas depletion of its ortholog, GbpB, affected cellular morphology but not chain formation in S. mutans (Ng et al. 2004; Duque et al. 2011). However, purified PcsB lacks detectable enzymatic activity, and its PG hydrolase relied on interaction of CHAP domain and cell division complex FtsEX (Ng et al. 2005). In B. subtilis, VicRK modulated ftsAZ operon, and VicKBsu overexpression produced short cocci chains (Fukuchi et al. 2000). Upon initiation, FtsZ protein self-organizes into a heterogeneous ring (Z-ring) at the division site and recruits other proteins involved in division (Sham et al. 2011), suggesting that correct location and appropriate FtsZ expression were critical for cell division. In VicKP222A, misshaped cells and asymmetric divisions were observed. Two daughter cells with different sizes were divided by a Z-ring, and 2 Z-rings appeared on 1 cell.

Role of VicK Phosphatase in EPS Biosynthesis and Biofilm Formation via Manipulation of VicR~P Levels

In VicKP222A, accumulation of VicR~P, asymmetrical cell division, abnormal morphology, and misstructured biofilm were observed. gtfBC were downregulated and caused severe reduction in WIS-EPS (Rainey et al. 2019). These changes were absent in vicR overexpression strains, which had only VicR accumulation but not VicR~P (Fig. 4), suggesting the aforementioned phenotypes were mainly attributed to VicR~P accumulation. Previous study also demonstrated that overexpression of the vic operon elevated both gtf and ftf expression (Senadheera et al. 2005; Chakraborty and Burne 2017).

Reduction of SpaP in VicKP222A Partially Led to Low Aggregation of S. mutans in Saliva

VicKP222A displayed low bacterial aggregation in saliva compared with UASpec. Usually, surface proteins contribute to this process. In S. mutans, a knockout mutant, ΔspaP, displayed reduced aggregation compared with WT in unstimulated whole saliva (Sato et al. 2011; Robinette et al. 2014; Yang et al. 2018). SpaP has been shown to adhere to various salivary components, including salivary agglutinin, also called gp340, promoting aggregation in fluid saliva (Jonasson et al. 2007; Purushotham and Deivanayagam 2014). We observed a significant reduction in spaP transcription and protein levels in VicKP222A compared with UASpec. However, the difference in rate of aggregation between UASpec and VicKP222A was not significant, probably due to other adhesins involved in this process (Igarashi et al. 2003).

In summary, S. mutans has developed multiple adhesion and virulence factors that enable it to colonize and compete with other bacteria in dental plaque. In this study, we report that abolishing VicK phosphatase activity increased the intracellular VicR~P to >90% and led to severe repression of WIS-EPS and SpaP, which are the main adherence factors of S. mutans. These findings suggest that VicK phosphatase activity could be a novel biological target to modulate S. mutans growth and colonization.

Author Contributions

S. Wang, contributed to conception, design, data acquisition, analysis, NS interpretation, drafted the manuscript; L. Long, X. Yang, contributed to data acquisition, analysis, and interpretation, drafted the manuscript; Y. Qiu, T. Tao, contributed to data acquisition, drafted the manuscript; X. Peng, Y. Li, contributed to conception, design, and data acquisition, critically revised the manuscript; A. Han, X. Zhou, D.G. Cvitkovitch, contributed to conception and design, critically revised the manuscript; D.B. Senadheera, J.S. Downey, S.D. Goodman, contributed to design, data analysis, and interpretation, drafted the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Supplemental Material

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Supplemental material, sj-pdf-1-jdr-10.1177_0022034520979798 for Dissecting the Role of VicK Phosphatase in Aggregation and Biofilm Formation of Streptococcus mutans by S. Wang, L. Long, X. Yang, Y. Qiu, T. Tao, X. Peng, Y. Li, A. Han, D.B. Senadheera, J.S. Downey, S.D. Goodman, X. Zhou and D.G. Cvitkovitch in Journal of Dental Research

Acknowledgments

We thank Professor Chaoliang Zhang, who performed the scanning electron microscope test on the specimens.

Footnotes

A supplemental appendix to this article is available online.

Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Youth Foundation of Sichuan University (grant 2014SCU11034).

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Supplementary Materials

sj-pdf-1-jdr-10.1177_0022034520979798 – Supplemental material for Dissecting the Role of VicK Phosphatase in Aggregation and Biofilm Formation of Streptococcus mutans
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Supplemental material, sj-pdf-1-jdr-10.1177_0022034520979798 for Dissecting the Role of VicK Phosphatase in Aggregation and Biofilm Formation of Streptococcus mutans by S. Wang, L. Long, X. Yang, Y. Qiu, T. Tao, X. Peng, Y. Li, A. Han, D.B. Senadheera, J.S. Downey, S.D. Goodman, X. Zhou and D.G. Cvitkovitch in Journal of Dental Research

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