INTRODUCTION

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The oral pathogen Streptococcus mutans colonizes the hard surfaces in the human oral cavity and is considered to be the principal etiologic agent of dental caries (16). Key to understanding how S. mutans colonizes the oral cavity is discerning how the various molecular components comprising the bacterial cell surface interact with the acquired dental pellicle. There is much evidence in the literature suggesting that a number of cell surface molecules play important roles in the adherence (and cohesion) process and that the presence or absence of dietary sugar determines which of these molecules is operative (23, 24). It is widely accepted that adherence is mediated by mechanisms involving bacterial extracellular polymers (e.g., glucans) synthesized from sucrose when that common sugar is present. In the absence of sucrose, a 185-kDa cell wall-associated adhesin belonging to a family of oral streptococcal polypeptides called antigen I/II (22) has been suggested to play a major role in colonization (8, 9). The gene for this adhesin in S. mutans was cloned by Lee et al. (13) and called spaP and then by Okahashi et al. (20), who called it pac. Subsequent restriction fragment length polymorphism (RFLP) analysis revealed only minor differences between these adhesin genes (3). To be consistent with our earlier reports, we refer to the gene as spaP and to its product as P1 in this paper.

Early experiments to test the hypothesis that the spaP gene product is involved in non-sucrose-mediated adherence involved the construction of P1-negative mutants by insertional inactivation of spaP (14). In vitro experiments with hydroxyapatite beads coated with either parotid gland saliva or salivary agglutinin demonstrated nonadherence by spaP mutant strain 834 (2, 14) as well as by naturally occurring non-P1-containing strains of S. mutans (5). Also, both native P1 and recombinant P1 competitively inhibited binding by whole cells in these in vitro assays (5). Likewise, Koga et al. (12) demonstrated a lack of adherence by two pac mutants to saliva-coated hydroxapatite and a lack of saliva-induced aggregation of these strains compared to the pac⁺ parent strain. Collectively, these findings suggested an important role for the spaP gene product in the primary colonization of teeth in the absence of glucan.

Previously, the virulence properties of P1 mutant strain 834 and its parent strain were tested in an in vivo system in which each strain was used to infect desalivated specific-pathogen-free rats fed a high (56%)-sucrose diet (2). Both parent and mutant strains caused similar levels of smooth-surface caries. These findings were confounded by our simultaneous discoveries that mutant 834, which retained an active promoter, expressed the N-terminal 612 amino acids of the 1,561-amino-acid P1 molecule (3) and that an alanine-rich salivary agglutinin-binding domain was associated with this retained fragment (6). In addition, because of the high-sucrose diet, the glucan-independent phase of colonization was likely obscured in this animal study. Therefore, to determine the role of P1 in virulence, we undertook a new study using germ-free rats fed a low (5%)-sucrose diet and infected with a newly constructed mutant of S. mutans NG8 engineered to be completely devoid of spaP.

(This work was presented in part at the 97th General Meeting of the American Society for Microbiology, Miami, Fla., 4 to 8 May 1997.)

	MATERIALS AND METHODS

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Bacterial strains, culture conditions, and plasmids. S. mutans NG8 and the NG8-derived spaP mutant strain, PC3370, were routinely cultured aerobically at 37°C in Todd-Hewitt broth (BBL, Cockeysville, Md.) supplemented with 0.3% yeast extract (THYE broth) or on THYE agar in a candle jar. THYE media were supplemented with tetracycline (15 µg/ml) as required. A comparison of the plating efficiencies of the parent and mutant strains was performed by inoculating 2 ml of THYE broth with single colonies of each strain, followed by growth for 16 h and enumeration of CFU by serial dilution and plating. Colonies on triplicate plates of each strain were counted, and the mean ± standard error of the mean (SEM) CFU per milliliter for each were determined.

PCRs. PCRs were performed with primers having the sequences 5'-CCGGATCCGTGTCAGGTACTATTGTCA-3' and 5'-GGCTGCAGACGCCTTCGCCTTGTTTAG-3' to amplify a 480-bp DNA sequence (bp 31 to 511 of the spaP sequence with GenBank accession no. X17390) upstream of the putative spaP promoter (underlined bases indicate engineered restriction sites for BamHI and PstI, respectively) and primers having the sequences 5'-GGAAGCTTTGACAGCATAGACATTACA-3' and 5'-CGGGATCCAAGGCAGTGCGAAGTACCT-3' to amplify a 275-bp DNA sequence downstream (including the translational stop codon) of spaP (bp 4895 to 5170 of pac [20], a gene homologous to spaP and encoding Pac [P1] in S. mutans MT8148 [GenBank accession no. X14490]) (underlined bases indicate engineered restriction sites for HindIII and BamHI, respectively). PCRs were performed with a Biometra (Tampa, Fla.) UNO Thermoblock thermocycler, S. mutans NG5 chromosomal DNA as the template, and Taq polymerase (Promega, Madison, Wis.) for 30 cycles under the following conditions: denaturation at 96°C for 30 s, annealing at 56°C for 1 min, and extension at 72°C for 2 min. A final extension cycle was carried out for 5 min at 72°C.

DNA preparations and sequencing. Plasmid DNA from E. coli recombinant strains and chromosomal DNA from S. mutans were prepared as described previously (7). All vector constructions were verified by restriction analysis and/or DNA sequencing, which was performed at the DNA Sequencing Core Laboratory of the University of Florida Interdisciplinary Center for Biotechnology Research. Primers were prepared at the DNA Synthesis Core Laboratory of the Interdisciplinary Center for Biotechnology Research.

Construction of the spaP mutant. spaP mutant strain PC3370 was constructed as outlined in Fig. 1. PCR-amplified streptococcal DNA fragments (described above) into which appropriate restriction enzyme recognition sequences had been engineered for cloning were ligated separately into the cloning vector pCR1000 to yield pCR52 and pCR330. Each cloned fragment was excised from the host vector so that, upon ligation with each other, the host plasmid was reconstructed and the two amplified DNA fragments were oriented in tandem to produce pCR3352. E. coli INVF' was used to propagate each plasmid construct. The PCR-amplified products were excised from the constructs as single fragments by use of PstI and ligated into the PstI site of pVA981 (25). The resulting recombinant plasmid, pVA3352, was propagated in E. coli JM109, restricted with BamHI to linearize it at a unique site engineered to lie between the amplified sequences, and used to naturally transform (21) S. mutans NG8. The spaP mutants of NG8 generated via allelic exchange were selected on THYE agar containing 10% sucrose and 15 µg of tetracycline per ml. Mutant PC3370 was selected for further evaluation as described below.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the construction of spaP mutant strain PC3370. fl, flanking.

Analysis of the spaP mutant. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), Western immunoblotting, radioimmunoassay (RIA), adherence to agglutinin-coated hydroxyapatite beads, and aggregation analyses of parent and mutant strains were all performed as described previously (4, 5). The API 20 Strep (bioMérieux Vitek, Inc., Hazelwood, Mo.) test was performed in accordance with the manufacturer's instructions. Antibodies used for Western and RIA analyses were mouse anti-P1 monoclonal antibodies 4-10A_8c and 1-6F_6b, respectively (1). Southern analysis was performed on chromosomal DNA from parent and mutant strains blotted onto nylon membranes by use of the Photogene nonradioactive detection system (Gibco BRL, Bethesda, Md.) in accordance with the manufacturer's instructions. Blots were probed with biotinylated pVA981 and a 763-bp internal fragment from the 3' end of spaP (bp 4019 to 4782 of the spaP gene sequence [10]).

Infection and analysis of germ-free rats. For caries assessments, groups of five 19-day-old weanling germ-free Fischer rats [CDF(344) GN/Crl BR] fed a low (5%)-sucrose diet (Diet 305 [18]) were inoculated orally for 3 consecutive days with saturated swabs of parent strain NG8 or spaP mutant strain PC3370 diluted to 3 × 10⁸ CFU/ml or a mixture of both strains (1 × 10⁸ CFU/ml each). After 6 weeks, whole mandibles were removed from each rat, sonicated as described above for bacterial enumeration, and stored in 95% ethanol for 24 h. Then, excess tissue was manually cleaned from around the teeth, which were stained overnight in 0.4% murexide for caries scoring (11).

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	RESULTS AND Discussion

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Characterization of the spaP mutant. Transformation of parent strain S. mutans NG8 with 1 µg of BamHI-linearized pVA3352 yielded 65 Tet^r colonies. All transformants were initially screened for the lack of surface expression of P1 by an RIA with an anti-P1 monoclonal antibody. All but one transformant showed no reactivity with the antibody (data not shown). One P1-negative transformant, PC3370, was chosen for further analysis. The generation time for parent and mutant strains in THYE broth was 80 min. Biochemical and fermentation profiles for parent and mutant strains were identical, as determined with the API 20 Strep test (data not shown). Southern analysis of BamHI-restricted chromosomal DNA from mutant strain PC3370 (Fig. 2, lane 1) showed no hybridization with a labeled internal fragment of spaP, while the parent strain hybridized with the two predicted fragments (Fig. 2, lane 2). SDS-PAGE (Fig. 3A) analysis of SDS extracts of mutant and parent cells demonstrated nearly identical protein profiles, except that bands corresponding to P1 were not present in the mutant. Western immunoblotting with an anti-P1 monoclonal antibody confirmed the loss of P1-reactive polypeptides in the mutant (Fig. 3B). Additionally, there was virtually no in vitro adherence (3.2% ± 2.1%) of radiolabeled mutant PC3370 cells to agglutinin-coated hydroxyapatite, whereas 55.8% ± 2.0% of NG8 cells adhered. Taken together, these data provide ample evidence that spaP was deleted from the genome of mutant PC3370, that P1 expression was eliminated, and that in vitro adherence properties were dramatically affected.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Southern analysis of BamHI-restricted chromosomal DNA from spaP mutant strain PC3370 (lane 1) and parent strain NG8 (lane 2). The membrane was probed with a biotinylated internal fragment of spaP (see Materials and Methods). Lane std contains biotinylated HindIII-digested lambda DNA markers.

View larger version (87K): [in this window] [in a new window]   FIG. 3.   SDS-PAGE and Western immunoblot analysis of whole cells of parent strain NG8 (lanes 1) and spaP mutant strain PC3370 (lanes 2) boiled in nonreducing SDS sample buffer. After electrophoresis, proteins were stained with Coomassie brilliant blue R-250 (A) or transferred to nitrocellulose membranes and reacted with a 1:1,000 dilution of anti-P1 monoclonal antibody 4-10A8c (B).

Virulence in gnotobiotic rats. Caries scores from four separate experiments in which rats fed the low (5%)-sucrose diet were infected with either parent strain NG8 or spaP mutant strain PC3370 or coinfected with both strains are compiled in Table 1. The data clearly indicate a role for the spaP gene product in the virulence of S. mutans. There were significantly fewer enamel and dentinal lesions on all molar surfaces in rats infected with mutant PC3370 than in those infected with NG8 (P  0.009), except for both moderate and deep proximal dentinal lesions, for which there were either no or too few lesions to compare. The number of streptococci recovered from week-6 plaque samples was not significantly different (P = 0.3471) in animals infected with NG8 (26.9 × 10⁶ ± 8.8 × 10⁶ CFU/ml) and those infected with PC3370 (21.4 × 10⁶ ± 6.8 × 10⁶ CFU/ml), demonstrating that the mutant strain clearly was able to colonize the oral cavity of the rats to the same extent as the parent strain. Therefore, P1 may function as an adhesin but is not a necessary requirement for adherence leading to colonization in this model. Other mechanisms that contribute to colonization, such as glucan-mediated adherence, must be considered here, since sucrose was provided in the rat diet, albeit at a low concentration. In this regard, it has been shown that as little as 0.1% dietary sucrose is a sufficient substrate for the accumulation of S. mutans plaque in rats infected with a single strain (19). Therefore, it is possible that the reduction in caries scores seen in mutant-infected animals might have been greater in the absence of sucrose, since glucan might have contributed to the adherence of these organisms and therefore likely obscured the actual ability of the mutant to colonize the oral cavities of the rats in a sucrose-independent manner. Unfortunately, some sucrose in the diet is necessary for colonization by wild-type S. mutans to occur in this particular animal model.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Time course of colonization. (A) Groups of 20 rats were infected with parent strain NG8 () or mutant strain PC3370 () or coinfected with both strains (), and the number of cells recovered from mandibles of three animals at each time point after infection was enumerated. (B) The percentage of tetracycline-sensitive cells (NG8) recovered from mandibles of coinfected animals was determined as described in Materials and Methods. Error bars indicate standard errors of the means.