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1 School of Dentistry, University of California, Los Angeles, CA 90095, USA
2 Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA
3 Institute for Cell Mimetic Space Exploration, University of California, Los Angeles, CA 90095, USA
4 California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
5 Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA
Correspondence
Fengxia Qi
fqi{at}dent.ucla.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Biofilm formation is one of the well-recognized virulence factors of S. mutans, which involves a sucrose-independent initial attachment, cell–cell aggregation, a sucrose-dependent stabilization, and eventual biofilm maturation (Kuramitsu, 2000).
The mechanism of sucrose-dependent biofilm formation is well understood. In the presence of sucrose, S. mutans produces extracellular polysaccharides named glucans from the glucose moiety of sucrose, through the enzymic activity of three glucosyltransferases (GtfB, -C and -D). Glucan formation allows S. mutans to firmly stick to the smooth tooth surface (Kuramitsu, 1993; Yamashita et al., 1993). Another group of proteins called glucan-binding proteins (i.e. GbpA, -B, -C and -D) are thought to play important roles in subsequent cell–cell aggregation and biofilm development (Douglas & Russell, 1982; Sato et al., 2002b; Shah & Russell, 2004; Smith & Taubman, 1996).
Compared with sucrose-dependent biofilm formation, the mechanism of sucrose-independent surface attachment is less well understood. The surface-associated protein antigen I/II (Pac), which binds to the salivary glycoproteins, was shown to be required for the initial attachment of S. mutans to the saliva-coated tooth surface (Bowen et al., 1991). Recently, LytR, a homologue of a regulator of autolysin activity in Bacillus subtilis, was shown to play an important role in sucrose-independent attachment to polystyrene surfaces in S. mutans (Wen & Burne, 2002; Yoshida & Kuramitsu, 2002). Another surface-associated protein, wall-associated protein A (WapA), is a well-studied human vaccine candidate (Russell & Johnson, 1987; Russell et al., 1995); however, the function of this protein remains controversial. It has been reported that antibodies to this antigen do not interfere with aggregation induced by sucrose or dextran (Douglas & Russell, 1982), or with adherence to saliva-coated hydroxyapatite (Douglas & Russell, 1984). However, Qian & Dao (1993) reported that inactivation of wapA in S. mutans strain GS-5 results in a significant decrease in sucrose-dependent adherence to surfaces. Later, Harrington & Russell (1993) constructed the same mutant in a different strain and found no such effect. More recently, Levesque et al. (2005) reported that, in the absence of sucrose, a wapA knockout mutant derived from strain UA159 forms stable and reproducible biofilms with a reduced biomass of 24.1 % of that of the wild-type. To obtain a clear answer on the function of WapA, we used different analytical methods, such as confocal and atomic force microscopy (AFM; Binnig et al., 1986), to elucidate the structural and biological functions of WapA in S. mutans. Our results demonstrate that WapA is involved in sucrose-independent cell–cell aggregation and biofilm formation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and its derivative were grown in Todd–Hewitt (TH; Difco Laboratories) medium or on brain heart infusion (BHI; Difco Laboratories) agar plates. For selection of antibiotic-resistant colonies, BHI plates were supplemented with 800 µg spectinomycin ml–1 (Sigma). All S. mutans strains were grown anaerobically (80 % N2, 10 % CO2 and 10 % H2) at 37 °C. Escherichia coli cells were grown in Luria–Bertani (LB; Fisher) medium with aeration at 37 °C. E. coli strains carrying plasmids were grown in LB medium supplemented with 200 µg spectinomycin ml–1.
Gene inactivation of wapA.
The inactivation of wapA was achieved via single crossover homologous recombination. A 330 bp fragment of SMU.987 (wapA) was amplified from chromosomal DNA of strain UA140 with primers WapA-F (5'- CTATTACTTTCCCAGATGAAG-3'), corresponding to +202 to +223 of the coding region, and WapA-R (5'-GTTAACATCTGGACTTATTGG-3'), corresponding to +527 to +548 of the coding region (GenBank accession number, AE014133; wapA locus tag, SMU.987). The PCR product was cloned into the pCR 2.1 TOPO TA vector (Invitrogen), and the correct fragment insertion was confirmed by sequencing. To construct the isogenic mutant in wapA, the wapA fragment was excised from pCR 2.1 TOPO with SalI and HindIII (New England Biolabs), following DNA extraction from agarose gel (1 %, w/v) with the Qiagen QIAquick Gel Extraction kit. The backbone vector for the single-crossover inactivation was pFW5, which contains a spectinomycin-resistance marker (gene aad9) that works in both Gram-negative and Gram-positive bacteria (Podbielski et al., 1996). pFW5 was also digested with SalI and HindIII, and ligated with the wapA fragment to generate pFW5 : : wapA, using a standard ligation procedure. The resulting plasmid containing the wapA gene fragment was confirmed by PCR and sequencing. The plasmid was transformed into S. mutans UA140 and transformants were selected on TH plates with 800 µg spectinomycin ml–1. Insertion of the plasmid into the S. mutans chromosome via single-crossover integration was further confirmed by PCR.
Confocal laser scanning microscopy (CLSM).
S. mutans cells were cultured in TH medium at 37 °C under anaerobic conditions. For biofilm formation, cells were grown anaerobically in TH medium overnight, and diluted 1 : 100 into TH medium with 0, 0.01 and 0.5 % sucrose in the Lab-TekII Chamber Slide System (Nalge Nunc International) for confocal imaging. Biofilm cells were stained with CellTracker Orange CMTMR [5-(and-6)-(((4-chloromethyl)benzoyl) amino)tetramethylrhodamine] according to the manufacturer's instructions (Molecular Probes). After 24 h incubation, CLSM was performed using an LSM 5 PASCAL with LSM 5 PASCAL software (Carl Zeiss). Images were obtained with a 10x0.3 Plan-Neofluar and a 40x1.4 Plan-Neofluar oil objective. Images were further processed using CorelDraw 10.
Immobilization of S. mutans cells for AFM analysis
(i) Dry immobilization.
A volume (0.5 ml) of each overnight culture was washed twice with nanopure water and resuspended in the same volume. Serial dilutions in water (10–1, 10–2 and 10–3) were prepared and 8 µl was transferred onto a 10-well microscope slide (Erie Scientific). The slide was air-dried and imaged directly with AFM.
(ii) Wet immobilization.
Two millilitres of an overnight culture in TH medium (5x108 bacteria ml–1) were filtered through an isopore polycarbonate membrane (Millipore) with a pore size of 0.6 µm (i.e. slightly smaller than the diameter of streptococcal cells) to immobilize the bacteria through mechanical trapping, a very common technique used to gather AFM images and force measurements of molecular interactions at microbial surfaces (Pelling et al., 2004; van der Aa et al., 2002; Kasas & Ikai, 1995). After filtering, the filter was carefully removed from the filter device and fixed with double-sided sticky tape onto a small Petri dish. Five millilitres of 20 % (v/v) BHI broth was added to the sample prior to AFM imaging.
AFM methodology.
All imaging in air and fluid was conducted using a Nanoscope IV Bioscope (Veeco Digital Instruments). AFM images were collected in contact mode using sharpened silicon nitride cantilevers with experimentally determined spring constants of 0.02 N m–1 and a tip radius of <20 nm (Levy & Maaloum, 2002). Fluid imaging and mechanical measurements were obtained at room temperature (
20 °C), with force measurements recorded at a pulling rate of 1 Hz. Height and deflection images were simultaneously acquired, and are both of importance with regard to microbial cell surface characterization as they yield complementary information. Deflection-mode images consistently reveal cellular ultrastructure with higher sensitivity, although these images are not representative of the true sample surface height variations; however, height-mode images provide quantitative height measurements, yielding accurate surface-roughness and surface-feature measurements (van der Aa et al., 2002; Paige et al., 1998; Pelling et al., 2005). The images presented in this study are deflection-mode images; however, all quantitative cellular measurements were taken from the corresponding sample-height data.
Roughness analysis with AFM.
Surface-roughness values (Pelling et al., 2005) for the extracellular ultrastructure of the cells were calculated from high-resolution AFM height-mode images using Nanoscope software (Veeco Digital Instruments). For both the wild-type UA140 and the wapA mutant, the mean Rrms value was calculated by collecting 100 Rrms values over areas of 250 nm2 on 10–20 cells for each strain (giving a mean 5–10 measurements per cell), and calculating the mean (Pelling et al., 2005). For a particular scan line over the cell surface, the calculated roughness, R, was defined as the SD (where rrms noise is the SD for large datasets) in the ith height value, hi, from the mean height, <h> (Pelling et al., 2005). The mean rms height divergence (<Rrms>) was calculated from high-resolution AFM height-mode images according to the following equation:
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RNA extraction and real-time RT-PCR.
Strain UA140 cultures were grown in 100 ml TH medium with 0, 0.01 and 0.5 % sucrose. Cells were harvested at OD600 0.3 by centrifugation (4700 r.p.m. for 15 min at 4 °C), washed three times with PBS, pelleted and stored at –80 °C. For RNA extraction, frozen cell pellets were ground three times with liquid nitrogen to break up the cells. Subsequently, 0.5 ml chloroform, 2 ml TRIzol (Invitrogen) and 2 ml Tris/EDTA buffer were added to the cell pellets for extraction. RNA was precipitated with one-tenth vol. 3 M sodium acetate (pH 5.2) and 0.7 vols. 2-propanol (100 %). Total RNA (3 µg) was used for cDNA synthesis using Stratascript RT (Stratagene), according to the manufacturer's protocol. For real-time RT-PCR, primers were designed according to sequence data provided by GenBank (accession no. AE014133), and SYBR green (Bio-Rad) was used for fluorescence detection with the iCycler (Bio-Rad) real-time PCR system, according to the manufacturer's protocol. Real-time RT-PCR primer sequences were as follows: wapRTf 5'- TCAAACGAATGTTCCGACAA-3' and wapRTr 5'-CAAAAGCCCTGCTTGTTCAC-3'. Total cDNA abundance between test samples was normalized using the 16s rRNA gene as a housekeeping control.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Fischetti et al., 1990; Levesque et al., 2005). Previous studies have demonstrated a ubiquitous localization of WapA on the cell surface (Moro & Russell, 1983). To further study the role of WapA in cell surface structures and related functions, we constructed an isogenic mutant of wapA by insertional inactivation. The cell morphology of the wapA mutant was then examined by optical microscopy. As shown in Fig. 1(a), the cell morphology of the wapA mutant differed dramatically from that of the wild-type when grown in TH medium without sucrose. While the wild-type displayed long cell chains (mean 10.2±5.7 cells per chain), the wapA mutant showed only short cell chains (mean 2.1±1.2 cells per chain). Interestingly, this difference diminished as the sucrose concentration in the medium was increased. At 0.01 % sucrose, the mean chain length of wapA became a little longer (2.7±1.6 cells per chain), but much less cell–cell aggregation was observed at this sucrose concentration compared with the large cell aggregates formed in the wild-type culture (Fig. 1b). At 0.5 % sucrose, the morphology of wapA was nearly indistinguishable from that of the wild-type, either by chain length (wild-type 9.8±5.6 cells per chain, wapA 8.3±4.0 cells per chain) or by sucrose-dependent aggregation (Fig. 1c). These results suggest that WapA may play a role in cell–cell attachment between sister cells in the absence of sucrose.
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; Wen et al., 2005; Yoshida & Kuramitsu, 2002), but under our experimental conditions, strain UA140 did not form a biofilm in the absence of sucrose. The biofilms were first observed with a 10x0.3 Plan-Neofluar objective to get an overview of the entire field, and then a 40x1.4 Plan-Neofluar oil objective was used to take seven random snapshots from different positions in the confocal field of each chamber. A representative image of the biofilm of each strain at 0.01 and 0.5 % sucrose is shown in Fig. 2. It is apparent that, at 0.01 % sucrose, the wild-type cells formed a biofilm with large but sparsely distributed microcolonies and large areas occupied by unstructured or single-cell chains (Fig. 2a). This is typical of the biofilm morphology at 0.01 % sucrose displayed by strains UA140 and UA159 (Kreth et al., 2004). In contrast, the wapA mutant attached to the surface mostly as unstructured cell layers and formed a thin biofilm. It was conspicuous that these biofilms were quantitatively different. However, at 0.5 % sucrose, both the wild-type and wapA mutant formed thick, confluent biofilms consisting of dense microcolonies with small, water-channel-like void areas (Fig. 2b). These results are consistent with the morphological difference between the wild-type and mutant cells presented in Fig. 1
, suggesting that WapA also plays a role in determining S. mutans biofilm architecture at lower sucrose concentrations.
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. In general, the wild-type cells displayed distinct surface structures, most prominently, protrusive equatorial septa, as previously observed by Cross et al. (2006) (Fig. 3a). Moreover, single-cell surface cross-sections of wild-type cells showed the presence of protrusive septa in the nanoscale cell surface topology (data not shown). In contrast, the wapA mutant cells revealed a more amorphous cellular ultrastructure in both the AFM deflection-mode image (Fig. 3b) and the single-cell surface cross-section (data not shown).
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); both cells showed septum topology, as seen in the AFM deflection-mode images (Fig. 3c, d), and a more homogeneous overall cell surface morphology, as detected in the single-cell surface cross-sections (data not shown). This was probably due to the presence of glucan layers on the cell surface, which are produced during growth in the presence of sucrose.
To quantify the difference in nanoscale surface morphology between the wild-type and wapA mutant cells before and after sucrose treatment, a surface roughness analysis was conducted. The roughness of the cellular surfaces was calculated from one hundred 250 nm2 areas on AFM height images no larger than 4x4 µm in scan size (n=100 in each case; wild-type and wapA mutant before and after sucrose). Using the equation described in Methods, mean Rrms values were obtained (Pelling et al., 2005) for the wild-type and wapA mutant cells before and after sucrose addition (Fig. 4). The corresponding Rrms values for the wild-type and wapA mutant cells before sucrose were determined to be 7.20±1.31 nm and 5.43±0.74 nm, respectively. After the addition of sucrose, the mean surface roughness values were found to be 9.76±0.95 nm for the wild-type cells and 9.63±0.93 nm for the wapA mutant cells. A two-sample independent t test was conducted on the mean Rrms values before and after treatment with sucrose. At the 95 % confidence level, it was found that the population means before sucrose addition were significantly different between the wild-type and mutant cells (P<0.0005; Fig. 4a), whereas, after sucrose addition, the population means for the wild-type and mutant cells were not significantly different (Fig. 4b).
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The wild-type and wapA mutant cells were immobilized on a filter, as previously described by Dufrene et al. (1999). Force–displacement curves were then collected on the cells by positioning the AFM cantilever tip over a cell and pressing the tip against the cell surface, while recording cantilever deflection on approach and subsequent retraction of the tip from the cell. For these experiments, the applied force of the tip against the cell was 5 nN. During tip retraction, numerous rupture events occurred, forming sawtooth-like patterns (Fisher et al., 2000; Sen et al., 2005), and revealing the adhesive interactions between the tip and cell surface proteins. Most likely, the observed rupture events were due to breakage of multiple adhesive bonds formed between the tip and cell surface adhesive molecules, as suggested in similar studies involving surface-adherent substances in various other cell systems (Chen & Moy, 2000; Pelling et al., 2005; van der Aa et al., 2002). Force spectroscopy experiments involving tip–cell interactions are commonly used to quantitatively probe the mechanical properties of macromolecules at the microbial surface (van der Aa et al., 2001). The transient and local interactions between the cell surface and the AFM tip are a reasonable model for probing adhesive properties at the surface of microbial cells (Pelling et al., 2005; van der Aa et al., 2001). Moreover, force curves taken on the bare substrate, before and after those performed on the cell, revealed force curves lacking rupture events, with little to no variation between the approach and subsequent retraction curves, thus indicating null adhesion.
Adhesion forces for living S. mutans wild-type UA140 and wapA mutant cells were obtained, revealing mean rupture forces of 84±156 and 42±13 pN, respectively. Probed under analogous conditions, the wild-type cells exhibited considerably more adhesion events than the mutant cells. In particular, wild-type (Fig. 5b inset) cells revealed sawtooth-like patterns in the retraction traces of the force–displacement curves indicative of multiple tip–cell interactions, whereas wapA mutant cells (Fig. 5a inset), in general, only revealed single tip–cell adhesion events. As shown by the histogram in Fig. 5(a), the rupture forces observed due to tip–cell adhesion fell within a much narrower range of 20 to 80 pN for the wapA mutant cells, as compared to the wild-type cells, whose rupture events ranged from 20 to 330 pN (Fig. 5b). Moreover, the number of rupture events observed for the wild-type cells was significantly more than that exhibited by the mutant cells. Since these analyses were conducted for the wild-type and mutant cells under the same conditions, these results suggest that the wild-type cells are more sticky than the wapA mutant cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, these cellular ultrastructural changes might relate to the significantly reduced wall-associated lipoteichoic acid level in wapA mutants. Moreover, the mutant cell surface appeared to be less sticky when probed using in vivo force spectroscopy. Consequently, the wapA mutant formed less structured biofilms compared to the wild-type. Interestingly, all the observed phenotypes of wapA mutation disappeared when cells were grown in 0.5 % sucrose. Taken together, these results suggest that WapA is more likely to be involved in sucrose-independent cell–cell aggregation than in sucrose-dependent adherence. This result is consistent with the findings of Russell's group (Douglas & Russell, 1982, 1984; Harrington & Russell, 1993), but contradictory to those of Qian & Dao (1993). Recently, at least two mutations have been identified in strain GS-5, the strain studied by Qian & Dao. The two mutations are localized on two surface-protein genes, gbpC and pac (Murakami et al., 1997; Sato et al., 2002a). Based on our own experience, strain GS-5 has cell morphology that is very distinct from that of most S. mutans clinical isolates, and forms biofilms with architecture different from that of strains UA159 and UA140. The wapA phenotype reported by Qian & Dao (1993) may well be caused by multiple surface-protein gene mutations.
The optical and confocal microscopy studies showed shorter cell chains in the wapA mutant when grown in the absence of sucrose. This phenotype may be due to the distribution of WapA during cell division. It has been proposed that the chain formation is due to incomplete separation of peptidoglycan between sister cells, which permits an easy orientation of cell division sites at the contact points between cells (Cole & Hahn, 1962). Newly synthesized surface proteins first appeared at the contact sites between cells, and then extended slowly over the entire surface of the cell. This suggests that WapA may be required for maintaining cell–cell attachment during cell replication or may prevent complete separation of peptidoglycan between cells.
As a surface structural protein, WapA may also be involved in protecting the bacteria from some forms of environmental stress. To test this hypothesis, we compared the resistance of the wild-type and wapA mutant to antibiotics (ampicillin, kanamycin, tetracycline, chloramphenicol and ofloxacin), antimicrobial peptides, triclosan and chlorhexidine. The MIC data did not show any difference between these two strains. We also measured growth rates of the wild-type and mutant strains under different stress conditions, such as oxidative stress, low pH and high temperature, but no difference was observed between the two strains (data not shown). Therefore, we conclude that WapA is probably not involved in functions other than those associated with cell surface structure and properties.
Real-time RT-PCR demonstrated repression of wapA gene expression by sucrose. Why does S. mutans modulate wapA expression according to the sucrose concentration in the environment? From the point of view of the bacterial cell, we speculate that it is related to the bio-economics of the cell. Since both WapA and sucrose are involved in cell–cell aggregation (through cell–cell contact for the former, and glucan formation for the latter), and glucan-mediated attachment appears to be stronger than WapA-mediated cell–cell aggregation, the production of glucan in the presence of sucrose makes biosynthesis of WapA unnecessary. Thus, it makes more bio-economical sense to shut down wapA gene expression when sucrose is present. On the other hand, in the absence of sucrose, the production of WapA becomes important for maintaining cell–cell contact and aggregation, which is probably pivotal for successful colonization of S. mutans in the oral cavity.
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ACKNOWLEDGEMENTS |
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Received 31 January 2006;
revised 29 March 2006;
accepted 9 April 2006.
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