Restriction Fragment Length Polymorphisms and Sequence Variation ...

Vol. 59, No. 5

INFECTION AND IMMUNITY, May 1991, p. 1803-1810 0019-9567/91/051803-08$02.00/0

Copyright C 1991, American Society for Microbiology

Restriction Fragment Length Polymorphisms and Sequence Variation within the spaP Gene of Streptococcus mutans Serotype c Isolates L. J. BRADY,' P. J. CROWLEY,' J. K.-C. MA,2 C. KELLY,2 S. F. LEE,' T. LEHNER,2 AND A. S. BLEIWEISl* Department of Oral Biology, University of Florida, Gainesville, Florida 32610,1 and Department of Immunology, United

Medical and Dental Schools

of Guy's and St. Thomas's Hospitals, London Bridge, London SE] 9RT, England2 Received 31 October 1990/Accepted 8 February 1991

A restriction fragment length polymorphism study was undertaken to determine the extent and location of heterogeneity within spaP encoding the Mr 185,000 cell surface protein P1 (antigen I/II) of Streptococcus mutans serotype c isolates. The gene was found to be highly conserved except for a central variable (V) region predicted to encode less than 150 amino acids. Sequence analysis identified two V-region variants. These differences were independent of the geographic source of the isolates. Southern analysis using synthetic oligonucleotide probes indicated that nonretention of P1 (I/II) by some isolates is not due to a deletion of the 3'-terminal DNA necessary to encode an intact carboxy terminus.

(residues 219 to 464 of P1, with residues 186 to 218 representing a fourth partial degenerate repeat), a proline-rich (P) region located in the central portion of the molecule comprising three 39-residue tandem repeats (residues 847 to 963 of P1) preceded by a 7-residue partial repeat sequence, a C-terminal region rich in proline residues believed to span the cell wall, and a potential transmembrane domain consisting primarily of hydrophobic amino acids followed by a charged cytoplasmic tail. The C-terminal molecular architecture is similar to that observed for other streptococcal wall proteins, most notably group A streptococcal M protein (9). The alanine-rich repeats of the A region demonstrate a 7-residue periodicity predicted to result in an a-helical coiled coil structure (22), a feature also observed with M protein (9) and pneumococcal PspA (44a). A schematic diagram of spaP is shown in Fig. 1. The spaP and pac genes are highly conserved, with most nucleotide substitutions resulting in alternative codon usage or conservative amino acid substitutions (21). There are a total of 36 predicted single amino acid substitutions between P1 (I/Il) and PAc; however, 20 of these substitutions are clustered within a variable segment (V region) of less than 150 amino acids [residues 679 to 823 of P1 (I/II) and 679 to 827 of PAc] immediately upstream of the P-region repeats (Fig. 1). A single residue deletion in PAc [corresponding to residue 752 of P1 (I/Il)] and the replacement of 9 P1 (I/Il) residues (796 to 804) with 14 nonhomologous PAc residues (795 to 808) occur within the V region as well. However, only two amino acid substitutions occur in the A region and only one occurs in the P region. To examine the extent and location of heterogeneity within the spaP gene at the molecular level among various isolates of serotype c S. mutans, a restriction fragment length polymorphism (RFLP) study of the entire gene was undertaken. Specific probes were generated by using cloned spaP DNA. Of particular interest was the V region, in which most of the differences between spaP and pac are clustered. Sequence analysis of this part of the gene from several S. mutans isolates was performed to determine whether they were spaP-like or pac-like or possessed unique V-region sequences. In addition, oligonucleotide probes corresponding to the 3' terminus of the gene were used to determine

Streptococcus mutans has been implicated as a causative agent of human dental caries (14). Serotype c organisms express a protein of approximate Mr 185,000, first identified as antigen I/MI (36, 45) and also referred to as antigen B (37, 38), IF (18), PAc (33, 34, 43), MSL-1 (7), and P1 (11). Analogous proteins have also been described for Streptococcus sobrinus serotype d and g strains (1, 16, 32), S. mutans serotype f strains (2), and Streptococcus sanguis strains (6). These proteins are designated SpaA (or PAg), SR, and SSP-5, respectively. The cell surface protein P1 (I/II) is associated with a fuzzy surface layer surrounding the cell wall in strains designated "retainers" or released into the culture supernatant in strains designated "nonretainers" (3). P1 has been shown to mediate the binding of retainer strains of S. mutans to salivary agglutinin-coated hydroxylapatite beads (25, 27) and to increase the hydrophobicity of such strains (24, 25, 27, 31). At present, P1 (I/II) is believed to function as an adhesin enabling S. mutans to bind to the salivary pellicle of tooth surfaces or to other plaque microorganisms (8, 12, 27, 29). The gene encoding P1 (I/II) has been cloned (26, 33) and sequenced independently by two research groups (21, 34), and they designated it spaP and pac, respectively. Comparison of the sequences derived from the two strains of S. mutans serotype c, NG5 and MT8148, reveals a high degree of homology, although some differences can be noted. Amino acid sequences deduced from spaP and pac indicate that these genes encode polypeptides of 1,561 [P1 (I/II)] and 1,565 (PAc) amino acids with predicted Mrs (following the cleavage of signal peptides) of 166,159 and 166,817, respectively. Differences between predicted Mrs and those measured by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis have been attributed to an abundance of proline residues. This has been proposed to cause aberrant gel migration of group A streptococcal M6 protein (15) and group G streptococcal protein G (13). P1 (1/11) and PAc share the following features: a 38-residue N-terminal signal peptide, an alanine-rich (A) region in the amino-terminal third of the molecule comprising three 82-residue tandem repeats * Corresponding author. 1803

1804

INFECT. IMMUN.

BRADY ET AL.

5'

3'

(1)

1 6mmJ%

SIGNAL SEQUENCE

PROMOTER REGION

2

3

.% /o. %1 Fo/Rw J % %,%-%L' % %I% %

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\A - REGION/ (Alanine-rich) (655-1491) (a.a.186-464)

HINDIII (1538) 1

HINDIII

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1 2 3 rT.-:.-}-.-.T-.7-.-3 i

b-REGI N V-REGIO1 (Variable) (Proline-rich) (2134-2512) (a.a.679-823)

(2617-2988) (a.a.840-963)

1(4019)

(4782K -XXI-I

MEMBRANE SPANNING REGION (Proline-nch) (4705-4766) (4555-4704) (a.a. 1536-1556) WALL SPANNING REGION

(a.a.1486-1535)

CYrOPLASMIC

TAIL (4767-4782) (a.a.1557-1561)

FIG. 1. Schematic representation of spaP based on the sequence derived from S. mutans serotype c isolate NG5 (21). Bold numbers in parentheses denote nucleic acid positions, whereas numbers following "a.a." denote amino acid residues.

whether P1 (I/II)-nonretaining isolates of S. mutans lack the DNA necessary to encode an intact carboxy terminus. MATERIALS AND METHODS Bacterial strains and culture conditions. S. mutans serotype c isolates used in this study were as follows: Ingbritt 175, Ingbritt 162, NG8, NG7, and NG5 from K. Knox, Institute of Dental Research, Sydney, Australia; CCY1, CCY2, Ingbritt, Guy's c, and K2 from T. Lehner, United Medical and Dental Schools of Guy's and St. Thomas's Hospitals, London, England; GS5 from R. Gibbons, Forsyth Dental Center, Boston, Massachusetts; and DP5 and DP6, fresh isolates from our laboratory. All streptococcal isolates were grown aerobically for 16 h at 37°C in Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) supplemented with 0.3% yeast extract. Escherichia coli JM109 (International Biotechnologies, Inc., New Haven, Conn.) and recombinant strains from our laboratory were grown aerobically at 37°C with vigorous shaking in L13 medium (1% tryptone, 0.5% yeast extract, and 1%o [wt/vol] NaCl, pH 7.0). Medium for the growth of recombinant strains was supplemented with 20 ,ug of ampicillin per ml (wt/vol) (Sigma Chemical Co., St. Louis, Mo.). Isolation of chromosomal DNA. Streptococcal cells from stationary-phase cultures (1 liter) were harvested by centrifugation at 10,000 x g (10 min, 4°C) and washed twice with 30 ml of 10 mM Tris-HCl-1 mM EDTA buffer, pH 7.5 (TE). Cell pellets (-1.5 g wet weight) were suspended in 5 ml of TE and incubated at 65°C for 20 min. Five milliliters of TE containing 40 mg of lysozyme per ml (wt/vol) and 200 U of mutanolysin (Sigma) were added to each suspension (1 h, 37°C), followed by the addition of 0.5 ml of TE containing 10 mg of predigested pronase E per ml (wt/vol) (Sigma) (1 h, 37°C). Suspensions were diluted to 15 ml with TE, and 5 ml of 20% (wt/vol) SDS (Swartz/Mann Biotech, Cleveland, Ohio) was added to lyse the cells. Cesium chloride (Sigma) was added to each sample to a density of 1.55 ± 0.03 g/ml (-30 g), and samples were incubated (15 min, 65°C) to dissolve precipitated SDS. Samples were centrifuged in 38.5-ml Beckman ultraclear Quick Seal tubes in a VTi 50 vertical tube rotor (175,000 x g, 24 h, room temperature). The DNA band collected from each gradient was dialyzed once against 200 volumes of sterile 10 mM Tris-HCl-5 mM EDTA buffer, pH 8.0 (16 h, room temperature), and twice against 10 mM Tris-HCl, pH 8.0 (4 h, 4°C and 16 h, 4°C), treated with 100 jtg of boiled RNase per ml (wt/vol) (Sigma)

for 1 h at 37°C and then with 100 p.g of proteinase K per ml (Sigma) for 2 h at 37°C, brought to 15 ml with TE, extracted once with an equal volume of phenol-chloroform and once with an equal volume of chloroform, and precipitated with 2 volumes of ice-cold 95% ethanol containing 2.5% (wt/vol) potassium acetate (30 min, -85°C). Precipitated DNA was spooled onto sterile glass rods, rinsed with ice-cold 70% ethanol, and air dried. The DNA from each isolate was dissolved in 1 ml of TE and quantitated by reading absorbance at 260 and 280 nm. Isolation of plasmid DNA. Plasmids were isolated by the method of Ish-Horowitz and Burke (19). Subcloning. The gene encoding P1, spaP, was cloned from HindIII partially digested NG5 chromosomal DNA and ligated into pUC18 as previously described (26), resulting in the generation of the recombinant plasmid pSM2949. A subclone containing recombinant plasmid p26L10 was generated by partially digesting pSM2949 with HindIII. A 5.3-kb DNA fragment containing the 2.7-kb pUC18 vector and the 2.6-kb 3' half of the original spaP insert was isolated from an agarose gel by electroelution (28) and purified by using an Elutip-d column (Schleicher and Schuell, Inc., Keene, N.H.). The DNA was ligated with T4 ligase (International Biotechnologies, Inc., New Haven, Conn.) and transformed into E. coli JM109 as described previously (26). A second subclone containing the recombinant plasmid p26R3, corresponding to the 5' half of the original spaP insert, was generated by digesting pSM2949 to completion with HindIl, PstI, and Scal. The 2.6-kb fragment was isolated and purified as described above and cloned into E. coli JM109 by using pUC18. Oligonucleotide synthesis. Oligonucleotides were synthesized with a DNA synthesizer (model 380B; Applied Biosystems, Foster City, Calif.). Restriction enzymes. The following restriction enzymes were used in this study: AlwNI, BspHI, HgiAI and DraIII (New England Biolabs, Beverly, Mass.); NheI, DraI, and EcoRV (Promega Corp., Madison, Wis.); HaeII, Ncil, ScaI, and PvuII (Bethesda Research Laboratories [I3RL], Gaithersburg, Md.); EaeI, BsmI, XMnI, and HaeII (Stratagene, La Jolla, Calif.); Eco47III (United States Biochemical Corp., Cleveland, Ohio); XbaI (Pharmacia-LKB Corp., Piscataway, N.J.); HindIII and PstI (International Biotechnologies, Inc.); and Nsp(7524)I (Amersham Corp., Arlington Heights, Ill.). Each enzyme was used according to the manufacturer's instructions.

VOL. 59, 1991

Biotinylation. A 1.4-kb DraIII-HindIll fragment of p26R3 and the entire p26L10 plasmid were labeled with biotin-7dATP (BRL) by using a nick translation kit (BRL) according to the manufacturer's suggested protocol. Four 18-base oligonucleotides (spanning bases 3961 to 3979, 4029 to 4047, 4321 to 4339, and 4731 to 4749 of the 3' terminus of spaP) were end labeled with biotin-7-dATP as described previously (20). Southern hybridization. Complete DNA digests were electrophoresed at 15 V/cm of gel on 0.7% agarose in lx TAE buffer (0.04 M Tris-acetate-0.002 M EDTA, pH 8.6), depurinated in 0.25 M HCl (15 min), denatured in 1.5 M NaCl-0.5 M NaOH (twice for 20 min), and neutralized in 1.5 M NaCI-1.0 M Tris-HCl, pH 7.5 (20 min). Gels were washed in 0.1 x TAE, and the DNA was transferred onto PhotoGene nylon membranes (BRL) by electroblotting in 0.1 x TAE (1 h, 200 mA). Southern blots were prehybridized, hybridized with biotinylated probe DNA, and developed with the PhotoGene nonradioactive detection system (BRL) according to the manufacturer's suggested protocol. For Southern blots in which biotinylated oligonucleotides were used as probes, digested DNA was electroblotted onto nitrocellulose (Bio-Rad Laboratories, Richmond, Calif.) in 0.1 x TAE (1 h, 200 mA). Prehybridization was performed with 2x SSC (0.3 M NaCl-0.03 M sodium citrate, pH 7.0) containing 0.5% (wt/vol) Ficoll, 0.5% (wt/vol) polyvinylpyrrolidone, 0.5% (wt/vol) bovine serum albumin, 1% (wt/vol) SDS, and 0.5 mg of sheared denatured salmon sperm DNA per ml (2 h, 37°C). Hybridization was performed with the same solution containing approximately 2 ,ug of biotinylated oligonucleotide probe DNA per ml (16 h, 37°C). Posthybridization washes and development were performed with the BluGene nonradioactive detection system (BRL) according to the manufacturer's suggested protocol. PCR. Chromosomal DNA was prepared from S. mutans isolates as described above. Using the primers 5'-GCTTC CGCTTATACA-3' (nucleotides 2065 to 2079 of spaP) and 3'-TAGGAAGATTAACCGCACG-5' (complementary to nucleotides 2554 to 2572 of spaP), a 0.5-kb fragment which spanned most of the V region of spaP was amplified by the polymerase chain reaction (PCR) (39). The reaction was 30 cycles (94°C for 2 min, 55°C for 1 min, 72°C for 2 min) with a thermal cycler (Bioexcellence, Techne Ltd., Cambridge, England). Nucleotide sequencing. Double-stranded DNA from the PCR was sequenced directly by using a modified procedure (44) based on the dideoxy chain termination method (40). The oligonucleotide primers used for the PCR were also used to sequence the fragments. The sequences were assembled and analyzed by using the Staden Plus program (Amersham, England).

RESULTS A complete restriction map of spaP based on the sequence determined for S. mutans NG5 was generated (21). Those restriction endonucleases that demonstrated a single recognition site within the region of interest (e.g., the A or V region) or a single recognition site within the entire gene were chosen for use in this study (see Fig. 2). To ensure that any RFLPs detected were the result of alteration of the restriction site within the region of interest and not of changes elsewhere in the coding or flanking sequences, chromosomal DNA was predigested with "boundary" enzymes, which demonstrated conserved sites among the isolates tested. These sites corresponded as closely as pos-

spaP GENE OF S. MUTANS SEROTYPE c ISOLATES

1805

sible to the 5' and 3' ends of each probe. Analysis of strain NG5 served as a positive control. V-region analysis. Biotin-labeled p26L10 (see Materials and Methods) was used as the probe for V-region analysis (Fig. 2). The insert in this recombinant plasmid included that portion of spaP spanning the HindlIl sites from positions 1538 to 4019. The 5' and 3' boundary enzymes for the V-region experiments were EcoRV (position 1767) and HindIII (position 4019), respectively. The restriction endonucleases EaeI, HgiAI, NheI, NciI, and Nsp(7524)I were used to analyze polymorphisms within the V region. The V-region Southern analysis using HgiAI as the third enzyme is shown in Fig. 3A. Fragment sizes predicted on the basis of the spaP sequence were 1.68, 0.56, and 0.23 kb and were observed for isolates NG5, NG7, Ingbritt 175, Ingbritt 162, and GS5. Isolates NG8, DP5, and DP6 displayed a different pattern, indicating that they lacked the HgiAI site at position 2331. The summary of the results of all V-region experiments is shown in Table 1. RFLPs observed in this region paralleled the differences observed by comparison of spaP and pac gene sequences. On the basis of RFLPs within the V region, there appeared to be two families of isolates. Isolates NG8, DP5, and DP6 lacked those restriction sites present in the spaP sequence which were also absent from the pac sequence (34). These three isolates demonstrated different EaeI, HgiAI, NheI, Ncl, and Nsp(7524)I sites than the ones predicted for NG5; however, the exact locations of these sites could not be determined by RFLP analysis since corresponding partial recognition sequences were not located in the NG5 sequence. All other isolates possessed the spaP restriction sites predicted from the NG5 sequence with one exception; GS5 lacked the EaeI site at position 2216. A fragment of the gene (nucleotides 2065 to 2572) encoding the V region was amplified with the PCR from isolates GS5, NG8, DP5, and DP6. Five additional isolates, CCY1, CCW1, Ingbritt, Guy's c, and K2, were also included in this part of the study. Nucleotide sequencing of the PCR products demonstrated that there were only two major variant forms of this region, since all the isolates showed virtual identity with either spaP or pac. Occasional nucleotide substitutions were detected that were indicative of strain variation (Table 2). A-region analysis. The DNA probe encompassing the A region (Fig. 2) was generated by cleavage of p26R3 (see Materials and Methods) at the DraIllI site at position 154, to remove non-spaP 5'-flanking DNA, and at the HindIll site at position 1538. AlwNI was used as the 5' boundary enzyme in this series of Southern hybridizations since its recognition site at position 187 was conserved among all isolates tested. EcoRV, which demonstrated a conserved site at position 1767, was used as the 3' boundary enzyme (Fig. 2) since the HindIII site at position 1538 was not present in isolates NG8, DP5, and DP6. Chromosomal DNA from eight isolates of S. mutans serotype c was digested with AlwNI, EcoRV, and a third enzyme with a single recognition site within the spaP A region. The Southern analysis using BsmI as the third enzyme is shown in Fig. 3B. Fragment sizes predicted on the basis of the spaP sequence were 1.00 and 0.58 kb. All strains tested demonstrated these fragments. Fainter higher-molecular-weight bands represent hybridization to partially digested DNA. The restriction endonucleases EaeI, HgiAI, Eco47III, and HaeII also were used to investigate RFLPs within the A region (Table 1). Only one polymorphism was detected within the alanine-rich repeats of the A region. Isolates NG7, Ingbritt 175, and Ingbritt 162 demonstrated a Southern hybridization pattern that indicated the presence of

1806

BRADY ET AL.

INFECT. IMMUN.

P and V-region probe

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a second HgiAI site. Examination of the NG5 spaP sequence revealed that five of six bases of the HgiAI recognition sequence were present at the analogous position of the

preceding A-region repeat as the conserved HgiAI site at base 1304. Fragment sizes were consistent with the second

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12 345678 FIG. 3. (A) Southern blot analysis of S. mutans serotype c chromosomal DNA digested with HindIII, EcoRV, and HgiAI and probed with biotinylated p26L10. Fragment sizes predicted on the basis of the NG5 spaP sequence were 1.68, 0.56, and 0.23 kb (arrows). Lanes: 1, NG5; 2, NG7; 3, NG8; 4, Ingbritt 175; 5, Ingbritt 162; 6, GS5; 7, DP5; 8, DP6. (B) Southern blot analysis of the same isolates as above (lanes 1 to 8) digested with AIwNI, EcoRV, and BsmI and probed with biotinylated A-region probe. Predicted fragment sizes based on the NG5 spaP sequence were 1.00 and 0.58 kb (arrows).

HgiAI site corresponding to a position at base 1059. The nucleotide substitution which resulted in this additional HgiAI site would not change the amino acid sequence. P-region analysis. Biotin-labeled p26L10 (see Materials and Methods) was also used as the probe for P-region analysis (Fig. 2). The only restriction endonuclease which could be used for P-region analysis was AlwNI. As a consequence of the highly repetitive nature of this section of the gene, other enzymes predicted to restrict within the P region showed multiple recognition sequences. AlwNI was predicted to have two restriction sites within the P region (at positions 2779 and 2896), the only two AlwNI sites between positions 1767 and 4019 demarcated by boundary enzymes EcoRV and Hindlll. All isolates except NG8, DP5, and DP6 showed a digestion pattern identical to that of the positive control, NG5. These three isolates displayed the AlwNI site at position 2896 as well as an additional AlwNI site, the location of which was consistent with a potential AlwNI site (eight of nine bases of the recognition sequence) corresponding to position 2669 of NG5 (Table 1). It is likely that these isolates also retain the AlwNI site at position 2779; however, the complex restriction pattern generated by three cleavage sites within close proximity of one another makes the definitive identification of that particular site impossible by using this technique. Analysis of non-A-, non-V-, and non-P-region DNA. Restriction enzymes with limited recognition sites in regions flanking the A, V, and P regions were also used to provide information regarding sequence conservation outside these specific areas of interest. The enzymes included AlwNI, BspHI, HindIII, XbaI, EcoRV, PstI, DraI, and XmnI. The results of these Southern hybridizations are summarized in

spaP GENE OF S. MUTANS SEROTYPE c ISOLATES

VOL. 59, 1991

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Table 1. With the exception of the HindIII site at position 1538 (missing from isolates NG8, DP5, and DP6), there were no RFLPs detected with these enzymes. Southern analysis of nonretaining isolates with 3' oligonucleotides. To determine whether S. mutans isolates which do not retain P1 (I/II) on their cell surfaces but which release the molecule into culture supernatants have a deletion at the 3' end of the spaP gene (where the C-terminal membrane and wall anchor sequences are located), Southern hybridizations were performed with four end-labeled 3' oligonucleotide probes. The positions of these 18-residue oligonucleotides within the spaP sequence derived from NG5 are shown in Fig. 2. All four oligonucleotide probes hybridized with HindIII-restricted DNA from all Pl-nonretaining isolates, including GS5, which produces a truncated Mr 155,000 protein instead of a full-length Mr 185,000 product (23). A representative Southern blot is shown in Fig. 4. Since all isolates were shown to have a conserved HindIII site at position 4019, differences in fragment sizes reflect polymorphisms in flanking DNA downstream of the 3' terminus of spaP.

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Analysis of S. mutans serotype c isolates indicated that the spaP gene is highly conserved, with the exception of variable region differences first identified by comparing spaP and pac sequences. Analysis of the A region demonstrated complete conservation of five restriction sites, with an additional HgiAI site in three of eight isolates tested. Analysis of the P region was limited to the use of AlwNI. Three of eight isolates demonstrated an additional AlwNI site upstream from those predicted for NG5 spaP. Given the repetitive nature of the A and P regions, the identification of additional restriction sites in some isolates is not surprising. RFLP analysis of spaP outside the A, P, and V regions demonstrated no polymorphisms among eight restriction sites, with the exception of three of eight isolates which lacked the HindIII site at position 1538. RFLP analysis of the V regions of eight isolates revealed two patterns. One is spaP-like (21), and such isolates retain those V-region restriction sites predicted for spaP from the NG5 sequence. The second is pac-like (34), and these isolates lack the restriction sites for the five enzymes tested. Furthermore, the sequences encoding the V region from a number of S. mutans isolates were shown to be either spaPorpac-like. No other variants were found. Whether isolates were spaP- orpac-like was independent of their geographic source. Both spaP- and pac-like isolates were identified among isolates from New Guinea, the United States, and England. Thus, at least two types of S. mutans serotype c organisms appear to exist worldwide. Molecular heterogeneity among streptococcal proteins has been demonstrated. Huang et al. (17) reported RFLPs within the streptokinase gene of group A streptococci in strains of both the same and different serotypes. More recently, Johnston et al. (19a) used the PCR and sequence analysis to define two variable regions, Vl and V2, also within the streptokinase gene of group A streptococci. Their results suggested the existence of at least four different Vl sequences, two of which appear to be associated with strains implicated in the pathogenesis of poststreptococcal glomerulonephritis. The significance of the V-region polymorphisms within the spaP genes of serotype c S. mutans organisms is not yet known since it is still unclear how the variable region may impact on the structure and function of

1808

INFECT. IMMUN.

BRADY ET AL.

TABLE 2. V-region nucleotide substitutions Nucleotides

Amino acid

Isolate

sequenceda

Substitution'

Position'

spaP-like GS5 Ingbritt Guy's c

2246-2526 2231-2525 2122-2522

T/C T/C T/C C/A T/A T/C C/A T/A

2511 2511, 2514 2124

None None None

2175 21% 2124

None None None None None

CCY1

2359-2642 2390-2638 2401-2629 2424-2626

T/C T/C T/C C/T

CCW1

2386-2638

G/A None

2540 2540 2540 2425 2575

K2

pac-like NG8 DP5 DP6

2089-2553

2175

21%

changeb 23kb '*Ei 9A 4A

Trp/His Trp/His Trp/His None None

a For spaP-like isolates, comparisons were made with NG5 DNA sequence (21); for pac-like isolates, comparisons were made with MT8148 DNA sequence (34). b For spaP-like isolates, isolate/NG5; forpac-like isolates, isolate/MT8148. c Position is the number of the nucleotide pair.

1

23 34 5

6

FIG. 4. Southern blot analysis of S. mutans serotype c chromosomal DNA digested with Hindlll and probed with biotinylated synthetic oligonucleotide no. 1 (see Fig. 3). Lanes: 2, Ingbritt 162; 3, Ingbritt 175; 4, GS5; 5, NG5. Lanes 1 and 6, biotinylated DNA size markers.

P1. Work is currently under way in our laboratory to identify the functional domain(s) of the P1 molecule. The results of this RFLP study will aid in the interpretation of data regarding any phenotypic variants of S. mutans serotype c isolates which may be identified. In addition, a panel of anti-Pl monoclonal antibodies, all of which react with M, 185,000 protein solubilized from all isolates tested to date, show differential reactivity with intact whole bacterial cells (unpublished observations). Experiments are in progress to determine whether these observed antigenic differences parallel the restriction fragment length polymorphisms reported in this study, and they may shed light on how the V-region sequences may affect the configuration of the P1 molecule in its native form on the bacterial cell. Despite a high degree of immunological cross-reactivity and sequence homology between P1 (I/II) or PAc and related molecules (e.g., SR, SpaA [PAg], and SSP-5), functional differences have been recognized. For example, SSP-5 and PAc demonstrate approximately 60% homology at the amino acid level, with even greater homology observed through the alanine- and proline-rich repeat regions; however, the interaction of S. sanguis with salivary agglutinin is dependent on sialic acid residues of the agglutinin, while that of S. mutans serotype c is not (7). In addition, although extensive amino acid sequence homology (66%) has been demonstrated within the central regions of PAc and SpaA (43) and within the first 600 amino acids of SpaA and P1 (I/II) (42), SpaA from S. sobrinus serotype g has been reported to be involved in sucrose-induced aggregation and to affect dextranase activity (4), while pac-deficient mutants of S. mutans serotype c can still aggregate in the presence of sucrose (although activity is somewhat decreased) and the dextranase function of such mutants is unaffected (25). Common properties also have been shown. P1 (I/II) or PAc has been shown to mediate sucrose-independent adherence to saliva-coated hydroxylapatite (8, 25, 27, 41), as have the S. mutans serotype f antigen SR (2) and S. sanguis surface protein (10, 30). The binding of both S. sanguis SSP-5 (5, 6) and S. mutans serotype c P1 (I/II) (unpublished observations) to salivary

agglutinin has been shown to be calcium dependent. Increased hydrophobicities have been reported for those strains of S. sanguis (10, 30) and S. mutans serotype c (24, 25, 27, 31) which express P1 (1/11)-like molecules on their surfaces. Nonretention of P1 (I/Il) on the cell surface does not appear to be due to a deletion of 3'-terminal spaP DNA. Synthetic oligonucleotide probes complementary to four short 18-residue stretches of DNA ranging from position 4019 to the 3' terminus of the gene (Fig. 2) hybridized with DNA from nonretaining isolates. These oligonucleotides spanned a region of spaP DNA including the hydrophobic membrane anchor domain and proline-rich wall-spanning region typical of streptococcal surface proteins. The 3' pac sequence from isolate MT8148, a retaining strain, is identical to that of spaP from NG5, a nonretaining isolate. It is unclear why P1 (I/Il) is retained by some strains and not by others. More than one mechanism may be involved. GSS produces a truncated protein of Mr 155,000, although hybridization of GSS DNA with the oligonucleotide probes indicates that the 3' region of the spaP gene is present. Proteolytic degradation of P1 (I/II) to the Mr 155,000 polypeptide is unlikely, as a gene encoding the Mr 185,000 protein has been inserted into 055 and the intact protein was expressed on the surface of the organism (25). A point mutation resulting in a premature termination codon is a possible explanation for the production of the truncated protein. Other nonretainer isolates such as NG5 and Ingbritt 162 release the Mr 185,000 protein into culture supernatants. This may involve a mechanism similar to that proposed by Pancholi and Fischetti (35) for the group A streptococcal M

protein, in which a membrane attachment complex that has been added posttranslationally is removed by a thiol-dependent membrane anchor cleaving enzyme. A potential signal for the posttranslational modification, with a consensus sequence of Leu-Pro-X-Thr-Gly-X, has been identified. This consensus sequence is present in both P1 (I/Il) and PAc. Such a multistep mechanism for the assembly and release of

VOL. 59, 1991

streptococcal surface proteins provides an explanation for the nonretention of P1 (MI/I) by isolates which appear to encode all the information necessary for cell-surface attachment. Nonretention of P1 may result from differences outside spaP, i.e., in the gene(s) encoding the membrane anchor cleaving enzyme activity or the putative membrane anchor complex. ACKNOWLEDGMENTS This work was supported by U.S. Public Health Service grant R37-DE-08007 to A.S.B. from the National Institute of Dental Research, NIH, Bethesda, Md., and by the Dickinson Trust (to T.L.). REFERENCES 1. Abiko, Y., M. Hayakawa, H. Aoki, S. Saito, and H. Takiguchi. 1989. Cloning of the gene for cell-surface protein antigen A from Streptococcus sobrinus (serotype d). Arch. Oral Biol. 34:571575. 2. Ackermans, F., J. P. Klein, J. A. Ogier, H. Bazin, F. Cormont, and R. M. Frank. 1985. Purification and characterization of a saliva-interacting cell wall protein from Streptococcus mutans

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