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Novel Sucrose-dependent Adhesion Co-factors in Streptococcus mutans
L. Tao1,* and
J.M. Tanzer2
1 Department of Oral Biology, College of Dentistry, University of Illinois at Chicago, Chicago, IL 60612; and
2 Department of Oral Diagnosis, School of Dental Medicine, University of Connecticut Health Center, Farmington, CT 06030;
Correspondence: * corresponding author, ltao{at}uic.edu
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ABSTRACT
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Streptococcus mutans glucosyltransferases form extracellular glucans from sucrose to promote adhesion to the teeth. We tested whether additional factors are involved in S. mutans sucrose-dependent adhesion. By screening a pVA891-insertion mutant library of S. mutans LT11, we isolated four clones deficient in adhesion to glass in the presence of sucrose, but normal in glucosyltransferase activities. The genetic loci flanking the insertion sites were retrieved and identified. They encode glycerol-3-phosphate dehydrogenase, an ABC transporter, a multidrug-efflux pump, and either the ribulose monophosphate operon or ascorbate metabolism operon. The four mutants were analyzed for their phenotypic expression and in vivo colonization in rats. The multidrug efflux pump mutant failed to colonize the rats. Three other mutants colonized the rats by reverting to the wild type. Therefore, these four factors may contribute to S. mutans sucrose-dependent adhesion.
Key Words: Streptococcus mutans • sucrose-dependent adhesion • sucrose • virulence • glucosyltransferases • multidrug-efflux pump
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INTRODUCTION
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Streptococcus mutans is a major pathogen of dental caries (Hamada and Slade, 1980; Tanzeret al., 2001) its cariogenicity is largely determined by its ability to adhere to the tooth and make metabolic acids.S. mutans produces three extracellular glucosyltransferases (Gtfs) that convert sucrose into glucans (Kuramitsu, 1993). two genes,gtfB and gtfC, encode water-insoluble glucan synthesis, and one,gtfD, encodes water-soluble glucan synthesis. animal studies (Yamashitaet al., 1993) suggested that the expression of all threeS. mutans gtfgenes is required for maximal virulence.S. mutans interacts with glucanvia three glucan-binding proteins (Gbps): Gbp-A (74 kd) (Banaset al., 1997), Gbp-B (59 kd) (Smithet al., 2001), and Gbp-C (60 kd) (Satoet al., 1997).
Besides Gtfs and Gbps, two lines of evidence suggest that other factors also play a role in S. mutans' sucrose-dependent adhesion. First, inactivation of S. mutans wall-associated protein A (wapA) decreases sucrose-dependent adhesion without affecting Gtf activities (Qian and Dao, 1993). Second, inactivation of the S. mutans serotype-specific rhamnosyl (rml) polysaccharide synthesis diminishes sucrose-dependent adhesion due to its sensitivity to glucan (Yamashita et al., 1999). rml mutation is lethal in medium containing sucrose unless a deletion occurs between the two adjacent Gtf genes, gtfB and gtfC, reducing S. mutans' production of insoluble glucan. Thus, two types of sucrose-dependent adhesion co-factors may exist in S. mutans. One is independent of glucan synthesis, while the other affects glucan synthesis. In the present study, we identified four genetic loci encoding factors contributing to S. mutans sucrose-dependent adhesion that are independent of glucan synthesis.
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MATERIALS & METHODS
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Bacterial Strains and Culture Media
Highly transformable S. mutans strain LT11 (Tao et al., 1993a) was grown in Todd-Hewitt broth (Difco, Detroit, MI, USA) or modified Jordan medium (Tanzer et al., 1973) and appropriate sugars. The mitis-salivarius agar (Difco) and trypticase soy sheep blood agar (BBL, Becton Dickinson, Franklin Lakes, NJ, USA) were used for recovering S. mutans and total flora from rats, respectively. Erythromycin was added to Todd-Hewitt broth at 15µg/mL for the selection of S. mutans strains transformed with the streptococcal integration plasmid pVA891 (Macrina et al., 1983). Chloramphenicol was added to LB (Luria-Bertani) medium at 15µg/mL as selective medium for Escherichia coli transformed with pVA891.
Isolation of Sucrose-dependent Adhesion Negative Mutants
LT11 was mutagenized by integration with the pVA891-S. mutans DNA fragment library (Tao et al., 1993b). Because our purpose was to select mutants with defects in S. mutans surface molecules other than Gtfs, sucrose was not added to the agar medium. LT11 has raised, rough-edged colonies on Jordan-plus-glucose agar (Tao et al., 1993a). We hypothesize that mutation in cell-surface structure may result in a change in this morphology. To isolate such mutants, we screened erythromycinR transformants for colonial morphological changes on Jordan-plus-glucose agar. Smooth, flattened colonies, which differed from the wild type, were selected as sought-for candidates. They were tested for adherence to the bottoms of glass test tubes and a 96-well polystyrene microtiter plate (Fisher Scientific, Pittsburgh, PA, USA) in the presence of sucrose.
Cloning and Sequence Determination of the Interrupted Gene Loci
Chromosomal DNA was isolated from the four presumptive sucrose-dependent adhesion-defective mutants. The DNA was sheared by vigorous vortex for 20 sec and used to transform LT11 to establish a linkage between insertion locus and the mutant phenotype and to rule out the possibility of insertion by multiple plasmids in the chromosome. DNA flanking the vector was recovered by marker rescue (Tao et al., 1993b). One plasmid was recovered from each mutant clone. The retrieved DNA fragments in the four plasmids were sequenced with the use of a reverse primer (5'-GCCACGATGCGTCCGGCTAGA-3') derived near the BamHI site in pACYC184 that, in turn, is a part of pVA891. Their sequences were determined by the Automated Sequencing Facility at the University of Missouri-Kansas City. Nucleotide sequence data were analyzed by the BLAST program (Altschul et al., 1990) and by searching the University of Oklahoma S. mutans genomic database (http://www.genome.ou.edu/smutans.html). The four gene loci were assigned GenBank accession numbers from AF397165 to AF397168.
Extracellular Protein Profile and Polysaccharide Synthesis Activity
S. mutans LT11 and the four adhesion-negative mutants were grown for 16 hrs in 20 mL of Jordan-plus-glucose broth. Culture liquors were separated by centrifugation at 27,000 x g, filtered through a Whatman 40 paper, and concentrated 20-fold with an Ultrafree-15 centrifugal filter device (Millipore, Bedford, MA, USA). The cell pellets were washed and re-suspended in 0.5 mL (pH 7.0) phosphate-buffered saline. Cell suspensions were sonified on ice by 6 strikes, 5 sec each, at maximal output (Micro Ultrasonic cell disruptor, KT50, Kontes, Vineland, NJ, USA). Cells were separated with a microfuge at maximal speed for 15 min. Supernatants were assayed for sonication-released surface proteins, while surface proteins not released by sonication in the pellets were eluted for 5 min at room temperature by the sample buffer (2% SDS/5% 2-mercaptoethanol, pH 6.8). The samples were subjected to SDS-10% PAGE in duplicate without being boiled. One gel was stained for protein with Coomassie blue, while the other was stained for polysaccharide by periodic acid-Schiff's after incubation in phosphate-buffered saline with 3% sucrose for 16 hrs as described by Qian and Dao (1993).
Growth Rates and Tolerance to Environmental Stress
Growth and tolerance to environmental stress of the four mutants and LT11 were analyzed. An overnight culture (200 µL) was diluted 20-fold in 37°C Todd-Hewitt broth, and optical density was measured spectrophotometrically (Genesys TM, Model 4001, Spectronic Instruments, Inc., Rochester, NY, USA) at OD550. Tolerance of the bacteria to environmental stresses including low pH, high temperature, and osmotic pressure (0.3-0.6 M NaCl) was evaluated by the growth criteria of Yamashitaet al.(1998).
Survival Rates of Wild-type and Mutants Grown in the Presence of Sucrose
Because mutation in rml affects S. mutans tolerance to sucrose (Yamashita et al., 1999), survival rates of the mutants and LT11 were analyzed in the presence of sucrose. The bacterial strains were grown in 10 mL of Todd-Hewitt broth in 18 x 150 mm test tubes for 16 hrs. The KT50 sonifier was used to disrupt streptococcal chains as described by Freedman and Tanzer (1974). After dilution, sonicates were spread on Jordan agar supplemented with either 1% sucrose or glucose as described by Yamashitaet al.(1999).
In vivo Colonization
Colonization studies were done essentially as described by Tanzer et al. (1985, 2001a). Rats free of mutans streptococci were used to test implantation and persistent colonization by LT11 and its four mutants. Fifty 21-day-olds were pooled, fed high-sucrose diet 2000 and, a day later, randomly assigned, two per cage, to 5 groups of 10 animals. Each rat was inoculated orally with approximately 109 cells of either LT11 or one of its mutants. To avoid suppression of the rats' indigenous flora, erythromycin was not added to either the ad libitum-provided food or sterile demineralized water, so that each strain had to compete successfully with the indigenous flora. Twenty-three days after inoculation, swabs of all animals' mouths were placed in buffered yeast extract, vortexed, and spiral-plated within 30 min onto mitis-salivarius agar and blood agar. After incubation, colonies on mitis-salivarius agar (which contains 5% sucrose) with morphology typical of mutans streptococci and total colony-forming units on blood agar were enumerated. Data were expressed as a percentage of S. mutans colonies among total recoverable flora in each swab sample. Statistical comparisons among the groups were done by ANOVA after arc-sine transformation of the percentage normalized data. Differences among groups were isolated by Fisher's least-significant difference test. Selected mutans streptococcal morphotypes recovered from mitis-salivarius agar were subcultured to erythromycin-supplemented media to check for revertants.
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RESULTS
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Isolation of Adhesion-defective Mutants
Approximately 20,000 erythromycinR transformants were screened on Jordan-plus-glucose agar for colonial variants. Thirty-two smooth colonies were isolated and tested for their ability to adhere to glass test tubes or polystyrene microtiter plates in Jordan medium with 3% sucrose. Four clones were defective in adhesion (Fig. 1A ). They were designated LT41, LT42, LT43, and LT44.
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Figure 1. Phenotypic analysis of S. mutans sucrose-dependent adhesion mutants. (A) Comparison of S. mutans wild-type and mutant adhesion to microtiter plate wells. Well number: 1, LT11 (wt); 2, LT41; 3, LT42; 4, LT43; and 4, LT44. Bacteria were grown at 37°C in 0.2 mL of Jordan medium supplemented with 3% sucrose in a sterile polystyrene microtiter plate. After 48 hrs, culture liquor was removed, and wells were washed three times with de-ionized water. The adherent bacteria were stained with 1% crystal violet for 1 min, rinsed with de-ionized water, and photographed. (B) Coomassie Blue stain of SDS-PAGE of S. mutans extracellular proteins. Lanes: M, molecular-weight standards; 1 (LT11); 2 (LT41); 3 (LT42); 4 (LT43); and 5 (LT44). (a) Surface proteins in culture liquor (20 X concentrated); (b) surface proteins released by sonication; and (c) surface proteins not released by sonication. Little surface protein was released from LT41 by sonication (lane b2). The sample buffer elution (lane c5) released a band of  160 kD (arrows) from LT44 that was weakly evident in the culture liquor (lane a5), and not evident after sonication (lane b5). (C) Periodic acid-Schiff's stain of SDS-PAGE of the same S. mutans samples. Two polysaccharide bands at the 150-kD and 90-kD levels, corresponding to Gtf and fructosyltransferase (Ftf) activities, respectively, are similar between LT11 and four mutants in the cultural liquor fraction. There was no noticeable Gtf or Ftf activity in the sonication-released fraction for LT41. Although little protein was released (Panel B) from LT43 by either sonication (b4) or sample buffer (c4), its free Gtf and Ftf activities (Panel C, a4) appeared strong.
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Recovery of the Mutated Loci and Nucleotide Sequence Analysis
The transformation assay with sheared chromosomal DNA isolated from the four mutants showed that all transformants changed colonial morphology to the mutant phenotype (smooth). This suggested that only one plasmid was inserted into the relevant chromosomal locus in each mutant. With the marker rescue strategy, we recovered DNA flanking the integration vector by cloning the inserted plasmid into E. coli JM109. This resulted in four plasmids: pLT41, pLT42, pLT43, and pLT44. In each cloned DNA fragment, a sequence of 500 to 700 bases was determined and used to search the S. mutans genomic database to identify their genetic loci. The structural organizations of these loci suggest strong homologies with glycerol-3-phosphate dehydrogenase, an ABC transporter, a multidrug-efflux pump, and the ribulose monophosphate operon or ascorbate metabolism operon (Fig. 2).
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Figure 2. Structural organization of the four loci encoding sucrose-dependent adhesion co-factors of S. mutans. The triangle indicates the insertion site of pVA891. (A) The locus in LT41, a putative glycerol-3-phosphate dehydrogenase (gpdA). Downstream of gpdA, separated by one base, is gluA (UDP-glucose-1-phosphate uridylyltransferase; Yamashita et al., 1998). Upstream gpdA locate two divergent putative promoters and two ORFs on the opposite strand. The first ORF encodes a putative dUTPase (dutA). (B) The locus in LT42, a putative ABC transporter located between two other ABCs. On the left locates an operon of five genes for amino acid transport and metabolism (atm). On the right is a protein-secreting ABC (psa) transporter of three genes (Ross et al., 1993). The function of the central (mutated) ABC is unknown. Six genes had homologies to known genes: cnhA (carbon-nitrogen hydrolase), gidB (glucose-inhibited division protein), htpX (heat-shock protease), lemA (lemA-like protein), ylbN (YibN-like protein), and gcrR (regulator of Gbp-C; Sato et al., 2000). (C) The locus in LT43, a putative multidrug efflux pump (mepA). A transcriptional regulator was found upstream, while downstream mepA is gbpA separated by a small ORF on a separate strand. (D) The locus in LT44, the putative ribulose monophosphate operon. It encodes three PTS enzyme II components (rmpABC), a probable hexulose-6-P synthase (rmpD), a hexulose-6-P isomerase (rmpE), and a ribulose-5-P-4 epimerase (rmpF).
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Extracellular Protein Profile and Polysaccharide Synthesis Activity
The secreted and surface-bound protein profiles and polysaccharide synthesis activities are shown in Figs. 1B and 1C . The result showed that Gtf and Ftf activities were similar among the four mutants and the wild type.
Growth Rates, Environmental Stress Tolerance, and Sensitivity to Sucrose
Under normal growth conditions, three of the four mutants (LT41, LT42, and LT43) had 10% longer doubling time than the wild-type strain LT11 (Table). Low pH did not differentially affect the growth of these mutants. Growth of LT41 was more affected by high temperature than that of the other strains, and mutant LT41 was least tolerant to NaCl. The number of colonies, colonial sizes, and morphologies on the sucrose-supplemented agar plates were not noticeably different among the five tested strains.
In vivo Colonization Study
Wild-type LT11 and its mutants LT41, LT42, and LT44 colonized all rats equally (7.1-13.9%) of recoverable oral flora, but mutant LT43 failed to colonize any rats detectably (P < 0.01). Subculture of S. mutans colonies growing on mitis-salivarius agar plates from LT41-, LT42-, and LT44-inoculated rats revealed that none was resistant to erythromycin, and thus these strains appeared to have reverted in vivo to wild-type phenotype.
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DISCUSSION
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We have identified four genetic loci encoding potentially novel S. mutans sucrose-dependent adhesion co-factors: glycerol-3-phosphate dehydrogenase (LT41), an ABC transporter (LT42), a putative multidrug efflux pump (LT43), and the ribulose monophosphate operon (LT44). We have demonstrated that the putative multidrug efflux pump plays an important role in sucrose-associated colonization in vivo. In the absence of any of these co-factors, S. mutans did not effectively adhere to smooth surfaces in vitro, even with the presence of sucrose and functional Gtfs.
Glycerol-3-phosphate dehydrogenase (gpdA) is involved in bacterial cell membrane synthesis (Morbidoni et al., 1995). Its gene is closely linked to gluA, which encodes glucose-1-phosphate uridylyltransferase responsible for UDP-glucose synthesis (Yamashita et al., 1998). Inactivation of gpdA in LT41 by an integration vector should also inactivate gluA. Therefore, the adhesion defect of LT41 may be caused by mutations in both genes.
In S. gordonii, inactivation of the oligopeptide permease gene hppA affects its adhesion (McNab and Jenkinson, 1998), while inactivation of the transmembrane protein gene (htpX) affects its cell-surface topology (Vickerman et al., 1997). In Pseudomonas aeruginosa, an ABC transporter exports cell-surface lipopolysaccharides (Rocchetta and Lam, 1997). A multidrug efflex pump in the bacterium Pseudomonas putida is related to its adhesion to corn seeds (Espinosa-Urgel et al., 2000). This suggests that an ABC transporter, a transmembrane protein, and/or a multidrug efflux pump may also facilitate S. mutans adhesion.
In E. coli, the ribulose monophosphate (rmp) pathway may transport and metabolize pentose, pentulose, or pentitol (Reizer et al., 1997). But in Mycobacterium gastri (Mitsui et al., 2000) and Bacillus subtilis (Yasueda et al., 1999), it is involved in formaldehyde fixation. Recently, Yew and Gerlt (2002) reported that this operon is involved in L-ascorbate conversion to xylulose and subsequently, ribulose in E. coli. Although S. mutans transports xylitol (a pentitol) and forms xylitol-5-phosphate by a fructose PTS (phosphoenolpyruvate phosphotransferase system) (Trahan et al., 1985), it does not further metabolize the pentitol. Moreover, S. mutans does not metabolize any other pentose, pentulose, or pentitol, nor does it fix formaldehyde. The S. mutans msm operon is homologous to the E. coli arabinose operon, but it encodes for the metabolism of raffinose, melibiose, sucrose, and isomaltose, all of which contain hexose residues (Tao et al., 1993c). The msm encodes transport and metabolism of sugars with hexose residues that S. mutans can metabolize and may generate a precursor for surface polysaccharides related to adhesion (Tao et al., 1993c). For example, the synthesis of S. mutans serotype antigen-specific polysaccharide requires glucose-1-phosphate (Yamashita et al., 1998). Mutation of gtfA, which encodes an enzyme producing glucose 1-phosphate from sucrose (Tao et al., 1993c), reduces S. mutans adhesion and virulence (Yamashita et al., 1993).
Hazlett et al. (1998) and Yamashita et al. (1999) reported that inactivation of gbpA or rml causes deletion between gtfB and gtfC, and, consequently, reduces S. mutans sucrose-dependent adhesion. The rml mutants are sensitive to sucrose and high salt concentrations. Although their tolerance to environmental stress varied, all four mutants in the present study were normal in sucrose tolerance and glucan synthesis. This suggests that the defects in sucrose-dependent adhesion in the four mutants were probably caused by a change in cell-surface topology, like the wapA mutant (Qian and Dao, 1993), not in glucan synthesis. In fact, no change in glucan synthesis was noted.
One of the mutants (LT43) did not colonize rats, consistent with the initial in vitro adhesion assay. However, the other three mutants apparently reverted to the wild type in rats. Reversion of mutants constructed by insertion-duplication usually occurs at a low frequency, about 10-6 cells/generation in bacteria, due to spontaneous segregation by a single crossover recombination (Leenhouts et al., 1991). Because erythromycin was not added to the animal diet, in vivo ecological pressures of the rat might have selectively enriched for the revertants adherent to the animals' teeth. No conclusion can be drawn from this study in rats for the three mutants that clearly reverted. Further animal studies using gene-deletion mutants will be needed to avoid genetic reversions. We do not know why reversion did not occur in LT43. We speculate that the location of the integration vector might be a "cold spot" for DNA recombination. Such a "cold spot" locus may allow the co-existence of two homologous genes, such as gtfB and gtfC, in the bacterial chromosome. Alternatively, the inactivation of the mepA gene might retard the recombination event, in contrast to the gbpA mutation, which promotes recombination between gtfB and gtfC (Hazlett et al., 1998).
The discovery of these adhesion co-factors suggests that the synthesis of water-insoluble glucan, although necessary, may not be sufficient for S. mutans' sucrose-dependent adhesion. Further characterization of these co-factors may help us understand the molecular mechanisms of S. mutans' sucrose-dependent adhesion processes and explore new strategies for caries prevention.
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ACKNOWLEDGMENTS
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We acknowledge the Streptococcal mutans Genome Sequencing Project funded by NIH/NIDCR grant and B.A. Roe, R.Y. Tian, H.G. Jia, Y.D. Qian, S.P. Linn, L. Song, R.E. McLaughlin, M. McShan, D. Ajdic, and J. Ferretti of the University of Oklahoma. This work was supported by NIH/NIDCR grant R29 DE11400.
Received for publication July 11, 2001.
Revision received April 12, 2002.
Accepted for publication April 18, 2002.
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Journal of Dental Research, Vol. 81, No. 7,
505-510 (2002)
DOI: 10.1177/154405910208100715
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ABSTRACT
INTRODUCTION