1 School of Dental Medicine, University of Connecticut Health Center, Farmington, CT 06030-1605, USA
2 School of Dental Medicine, University at Buffalo, Buffalo, NY, USA
Correspondence
Jason M. Tanzer
tanzer{at}nso.uchc.edu
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ABSTRACT |
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Present address: Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD 21201, USA.
Present address: School of Dentistry, Virginia Commonwealth University, Richmond, VA 23298, USA.
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INTRODUCTION |
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Only animals that secrete significant -amylase in their saliva are colonized by ABS, thus suggesting the importance of amylase-binding in oral colonization by these bacteria (Scannapieco et al., 1994
). Salivary amylase retains its enzymic activity while bound to ABS and may facilitate dietary bacterial starch utilization (Douglas, 1990
; Rogers et al., 2001
; Scannapieco et al., 1990
). Amylase binds to high affinity receptors on S. gordonii that cluster around surface cell division sites (Scannapieco et al., 1992
). Bacterial cells in exponential growth bind more amylase than stationary-phase ones (Rogers et al., 2001
; Scannapieco et al., 1992
). Amylase-binding components of 20 and 82 kDa are released into culture liquors of S. gordonii strains (Douglas, 1990
; Gwynn & Douglas, 1994
). These findings suggest a multi-component, cell-surface-associated amylase receptor that is released into the in vitro milieu as a culture matures or, it may be speculated, into the fluids of the oral cavity as growth progresses in situ in the dental plaque biofilm.
Recently, the genes (abpA and abpB) encoding these two amylase-binding proteins have been identified, cloned and sequenced (Rogers et al., 1998, 2001
; Brown et al., 1999
; Li et al., 2002
). They are recognized in the recent draft publication of the genome of S. gordonii (http://www.tigr.org). Except for preliminary reports (Tanzer et al., 2002a
, b
), no data have been published, however, on the role of the amylase-binding proteins in colonization in vivo or on caries. This paper clarifies the impact of single and double mutations of abpA and abpB on the colonization of rats' teeth by S. gordonii, on competition for the tooth surface and on dental caries.
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METHODS |
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Inocula were prepared by growth to exponential phase in TMM and adjusted to an OD600 (1 cm light path) of 2·0 in that medium. Each rat was orally inoculated with 200 µl culture (approximately 109 cells) at 22 days old, 1 day after weaning and provision of either of two test diets. One group on each test diet remained uninoculated. Diet 2000 contains 56 % confectioners' sugar (97 % powdered sucrose/3 % powdered cornstarch); diet 2000CS contains 56 % cornstarch in lieu of confectioners' sugar. These diets were consumed, as was sterile demineralized water, ad libitum, for the duration of the experiments.
A total of 11 experiments were performed. The experiments compared colonization and persistence of wild-type S. gordonii (FAS4-S or Challis-S) with their respective abpA mutants, termed FAS4-ST and Challis-ST (Rogers et al., 2001; Li et al., 2002
). An abpB mutant (Challis-SE) was constructed from the more transformable strain, Challis-S, as was a double mutant (Challis-STE) defective in both abpA and abpB (Li et al., 2002
). We shall detail those two experiments that are the most informative and complete with respect to the Challis-S mutants.
In one experiment (Fig. 1) 10 groups were studied simultaneously. Five groups of rats were fed diet 2000 while another five groups were fed diet 2000CS. All animals originated from several litters born on the same day. There were 10 randomly assigned rats per group. One group consuming each of the diets remained uninoculated. After 1 day of consumption of test diets and demineralized water, the other groups were inoculated with either Challis-S or one of the following of its mutants: abpA mutant Challis-ST, abpB mutant Challis-SE or abpA/abpB- double mutant Challis-STE.
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Recovery of inoculants.
In all experiments samples were spiral plated onto the following non-selective and selective agars: Trypticase soy sheep blood agar (BA, for total recoverable flora); mitis salivarius agar (MS, for total streptococci, including possible cross/extraneous contaminants and revertants); MS supplemented with 200 µg streptomycin sulfate ml-1 (MSS, for FAS4-S and Challis-S, as well as FAS4-ST and Challis-ST); MS supplemented with both 200 µg streptomycin sulfate ml-1 and 10 µg tetracycline hydrochloride ml-1 (MSST, for FAS4-ST and Challis-ST); MS with 10 µg erythromycin ml-1 (MSE, for Challis-SE); and MS supplemented with all three of the antibiotics (MSSTE, for Challis-STE). Recoveries were expressed as both absolute c.f.u. values and as relative values, the percentage of total recoverable c.f.u. on BA that was the inoculant, on extracted teeth. Data could be expressed only in relative terms from the swab samples of the teeth, due to the variable size of such samples. In doubly inoculated animals, Challis-S c.f.u. recoveries were computed as equal to (CFUMSS-CFUMSST).
Carious lesion scoring.
Forty-two or 41 days after inoculation, in the two experiments, animals were euthanized by anaesthetic overdose. Specimens were prepared, randomly coded and blindly scored (Tanzer, 1979; Tanzer et al., 2001a
). Only after scoring was completed and raw data entered into the laboratory computer were the random number codes broken.
Statistical analyses.
Bacteriological as well as caries score data were statistically analysed as described previously (Tanzer, 1979; Tanzer et al., 2001a
) by ANOVA. Statistically significant differences among the group means were isolated using the Fisher LSD procedure (Snedecor & Cochran, 1967
; SPSS version 10.1, Chicago, IL, USA). Bacterial recoveries, expressed as percentages, were arcsine-transformed to improve normalcy of distribution before parametric analysis. c.f.u. numbers and caries scores were analysed without transformation. To compute growth rates of cultures and possible differences among them, equations were fitted, slopes computed with standard errors and 95 % confidence intervals determined (NONMEM, version V, GloboMax LLC).
Glucosyltransferase activity of wild-type and mutants.
GtfG activity of wild-type and abpA, abpB and abpA/abpB mutants was initially explored. Relative amounts of Gtf enzyme activity were determined by measuring enzyme activity in polyacrylamide gels as described by Vickerman & Minick (2002). Briefly, the wild-type and mutant strains were grown in broth to late exponential phase, as determined by optical density. Equal amounts of cell-free supernatants were run on SDS-PAGE gels. After electrophoresis, gels were incubated overnight in 3 % sucrose and the resulting glucan bands were visualized by staining with periodic acid and pararosanaline. The intensity of the stained bands was densitometrically assessed and reflected the relative activities of the Gtf protein.
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RESULTS |
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Colonization and competition studies
A total of 11 in vivo experiments were done. In no case was an uninoculated animal observed to harbour one of the inoculants and in no case was evidence of reversion of mutant to wild-type phenotype noted. In no experiment were the weight gains of the diet 2000-fed rats different from those of rats eating diet 2000CS, within that experiment. This was as expected, because the diets are isocaloric.
In all experiments S. gordonii strains colonized rats fed a high sucrose/low cornstarch diet better than rats fed the diet containing only cornstarch. At the termination of the experiments, in sonicates of the molar teeth of one hemi-mandible, abpA mutants, abpA/abpB mutants and their respective wild-types colonized the rats' teeth and were generally recovered in 5·06·5-fold higher numbers when the animals ate the sucrose/starch diet than when they ate the starch-only diet (Fig. 3 and Fig. 4
). This directional difference was also observed intra-experimentally in swab samples of the dentition (data not shown).
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It was unexpected that while the wild-type strains colonized the teeth very well, ranging from 20 to 70 % of the total recoverable flora in the series of experiments, when comparisons were made with their abpA mutants, the latter colonized even better, if the animals ate the sucrose-rich diet (Fig. 3). This difference was statistically significant in 3 of 11 experiments, despite the ability of the wild-type cells to colonize the teeth very well, both on a c.f.u. basis (P=0·002) and as a percentage of the total recoverable flora (P=0·002), for the data in this figure. [For the other two trials in which significance was detected using this experimental model, the significance values on a relative recovery basis were P<0·001 and P=0·002, and on an absolute c.f.u. basis were P=0·220 and P=0·002, respectively.] If the animals ate the starch diet, this advantage of the abpA-defective cells could sometimes be statistically significant (on a percentage basis, P=0·002; on an absolute c.f.u. basis, P=0·330; Fig. 3
).
In the two experiments in which it was tested, double mutant abpA/abpB (Challis-STE) colonized as well or better than its wild-type progenitor (Challis-S) and better than the abpB mutant (Challis-SE), independent of dietary carbohydrate (Fig. 3).
We sought further evidence of the advantage of abpA mutant Challis-ST versus its wild-type Challis-S using a competition paradigm. We felt that the abpB mutant would not be useful for such experiments, because it could not even compete well with the indigenous flora of the rats. For this study, with rats eating either the sucrose-starch-supplemented diet or the starch-only supplemented diet, weanlings were inoculated with either the wild-type, abpA or, simultaneously and equally, with both strains (Fig. 2). In the cornstarch-only group, the absolute count recoveries of the wild-type and the abpA mutant cells were not different, but the abpA mutant constituted a higher proportion of the total recoverable flora than its wild-type (P=0·028) (Fig. 4
). For the sucrose-fed rats, the absolute recoveries of the wild-type and abpA mutant were not different, but approached significant difference as a percentage of the total recoverable flora (P=0·122). For the doubly infected rats, there was no difference in the colonization abilities of these two strains, if the rats ate the starch-only diet. By contrast, if animals ate the high sucrose diet, the abpA mutant and wild-type strains not only colonized at much higher levels than in the starch-only group (P<0·001), but the abpA mutant also competed better for colonization of the teeth than its wild-type on an absolute count basis (P<0·001) and constituted 85 % of all bacteria, compared to 8 % for the wild-type (P<0·001). In aggregate, the wild-type and the AbpA mutant accounted for
94 % of all bacteria on the teeth.
Within any experiment there were either no differences of caries scores between uninoculated rat groups and those inoculated by S. gordonii wild-types FAS4-S or Challis-S or slight ones. If there were statistically significant increases in caries scores, they were biologically unimpressive. However, caries scores were sometimes higher (Fig. 5) in rats inoculated with the abpA mutant Challis-ST than those that were either uninoculated or inoculated with the abpB or abpA/abpB mutants (P=0·001, 0·008 and 0·019, respectively). The contrast of the abpA-mutant-infected group with the wild-type-infected group approached statistical significance (P=0·067). As always, rats eating the sucrose-containing diet had much higher caries scores than those eating the starch-only diet (P<0·001). No contrasts among the low scores of the starch-fed rats were significant statistically.
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DISCUSSION |
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The ecological consequences of amylase binding to S. gordonii are clearly more complex than initially envisioned. For example, while AbpA-deficient mutants adhere less well to amylase-coated hydroxyapatite and are defective in biofilm formation in vitro (Rogers et al., 2001), they colonize the teeth of rats to a greater extent than their parental strains, especially when the animals are fed a sucrose-rich diet. Although this suggests that AbpA fosters net clearance of S. gordonii in vivo, previous in vitro data did not reveal amylase agglutination of any ABS (Scannapieco et al., 1995
). It is possible that other important colonization factors are unmasked or redistributed on the cell surface in AbpA-deficient strains in vivo and may serve to augment bacterial adhesion to the tooth. Other surface components that might be redistributed or up-regulated in the abpA mutant to enhance oral colonization include SspA and SspB (Demuth et al., 1996
), the sialic-acid-binding protein Hsa (Takahashi et al., 2002
) and CshA (McNab et al., 1996
).
The present seemingly disparate observations could be explained if one or both of the Abps of S. gordonii not only interact with amylase but with other, as yet undefined, salivary or streptococcal surface proteins to either enhance or inhibit oral colonization. Because S. gordonii releases both AbpA and AbpB into its in vitro milieu, AbpA may serve to block adhesive interactions in the in vivo plaque biofilm and promote net clearance of these bacteria from the oral cavity. Because AbpB is required for S. gordonii colonization of teeth in rats fed a starch-only diet, it may augment adhesive interactions and retention in the in vivo plaque biofilm. In the presence of sucrose, however, the effect of absence of AbpB is masked, perhaps, by the glucosyltransferase activity of these cells that is implicated as a positive colonization factor (Sulavik et al., 1992; Vickerman & Jones, 1995
; Vickerman & Minick 2002
).
BLAST searching of the AbpB protein sequence suggests its relationship to bacterial di-peptidases (Li et al., 2002). The absence of this enzyme activity in the abpB mutant strain may block an important nutritional pathway in vivo that severely compromises the bacterium's ability to compete with other oral flora in a salivary environment.
The observation that GtfG activity may be greater in AbpA- and AbpA/AbpB-defective cells than in their wild-type or in the corresponding AbpB-defective strain may help to explain the behaviour in vivo of AbpA-defective mutants, but this explanation requires more detailed study and must be regarded as speculative at present.
In view of the two seemingly counter-effects of AbpA and AbpB vis-à-vis tooth surface colonization, and the additional putative pro-colonization effect of GtfG, it is not surprising that a double mutant, abpA/abpB, colonizes at least as well as its wild-type in vivo. Perhaps the anti-colonization effect of AbpA and the pro-colonization effect of Gtf in sucrose-fed rats, effectively mitigate the positive colonization effect of AbpB. While this double mutant binds no amylase in vitro, and there is no evidence of another, previously cryptic amylase-binding cell-surface determinant, one cannot presently exclude the possible unmasking or upregulating of other colonization factors in S. gordonii by the double deletion of abpA and abpB.
Consistent with previous data (Tanzer et al., 2001a), S. gordonii appears to be either a non-cariogen or, at most, a feeble cariogen, despite its striking ability to prominently colonize the surface of teeth of our rats or humans (Scannapieco et al., 1993
; Tanzer et al., 2001b
). The present data suggest that deletion of its abpA slightly increases its virulence, and that this cariogenicity increase is associated with its augmented ability to colonize the surface of teeth in the in vivo plaque biofilm. The increased cariogenicity observed for the AbpA-deficient mutant may be related to dis-inhibition of GtfG activity; increased GtfG activity may contribute to the observed subtly augmented cariogenicity by this strain as compared to its wild-type. Of course, it is only from sucrose that Gtf of oral streptococci synthesize extracellular or cell-surface glucan homopolymers, and abundant evidence associates this sucrose-dependent phenomenon with cariogenicity in experimental animals and humans (Tanzer et al., 1974
, 2001b
; Tanzer 1979
). The association of higher, albeit relatively weak by comparison with the mutans streptococci, cariogenicity of S. gordonii in rats has been reported recently (Tanzer et al., 2001a
).
In summary, this study demonstrated that strains of S. gordonii defective in AbpB could not colonize starch-only eating rats, but could colonize rats if sucrose was added to the diet. By contrast, strains of S. gordonii defective in AbpA colonized better than their wild-type, already good colonizers. A double mutant lacking both AbpA and AbpB colonized like its wild-type. Studies are now in progress to determine the molecular bases for these findings. The strains of S. gordonii studied are, at most, feeble cariogens in the presence of sucrose. The results illustrate that the complex nature of the in vivo plaque biofilm may not be adequately characterized from in vitro model data.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 26 September 2002;
revised 19 May 2003;
accepted 3 June 2003.
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