Sequencing and characterization of the 185 kDa cell surface antigen of Streptococcus mutans

ArchsoralBid. Vol. 35, Suppl., pp. 33%38S, 1990

0003-9969/90 $3.00 + 0.00 Copyright c 1990 Pergamon Press plc

Printed in Great Britain. All rights reserved

SEQUENCING AND CHARACTERIZATION OF THE 185 kDa CELL SURFACE ANTIGEN OF STREPTOCOCCUS MUTANS C. KELLY,’ P. EVANS,’ J. K-C. MA,’ L. A. BERGMEIER,’W. TAYLOR.*L. J. BRADY,’ S. F. LEE.?

A. S. BLEIWEI? and T. LEHNER’

’ Department of Immunology, United Medical and Dental Schools of Guy’s and St Thomas’s Hospitals. London Bridge, London SE1 9RT, *Division of Mathematical Biology, National Institute for Medical Research, Mill Hill, London NW7 IAA, England and ‘Department of Oral Biology, University of Florida, Gainesville, FL 32610 U.S.A. gene spa P (formerly designated as spa PI) encoding the M, 185,000 surface antigen I/II of Srrepptococcus mutnns, serotype c (strain NG5) has been sequenced. The deduced amino acid sequence of antigen I/II (1561 residues) includes a putative signal peptide (residues l-38) as well as a transmembrane region (residues 1537-1556). The N-terminal part of the protein (residues 39-550) is particularly rich in alanine and includes three tandem repeats of a sequence of 82 residues. This region is predicted to be a-helical, adopting a coiled-coil formation, and may account for the cell surface hydrophobicity associated with expression of antigen I/II. In contrast the C-terminal region (residues 800-1549) is proline-rich, favouring an extended conformation. Comparison with the sequence determined from Sfrep. mu$ans strain MT8148 showed that antigen I/II is highly conserved with the exception of a short central region (residues 750-805). N-terminal sequencing of purified antigens I and II components indicated that antigen I extends from the amino-terminus of the intact M, 185,000 surface antigen while antigen II extends from residue 996. Summary-The

Key words: antigen I/II, streptococcal surface antigen, Spa P, base sequence, Streptococcusmutans.

INTRODUCTION Antigen I/II is an adhesin that mediates the sucrose-independent attachment of Strep. mutans to the tooth surface. McBride et al. (1984) demonstrated that variants of Strep. mutans that had decreased cell surface hydrophobicity and decreased binding to saliva-coated hydroxyapatite were deficient in a number of cell wall-associated proteins of high M,, including antigen I/II. Furthermore, antigen I/II-deficient mutants of Strep. mutans, which were produced by insertional inactivation of the gene encoding antigen I/II, termed spa P (Lee et al., 1989; Okahashi et al., 1989a), showed both decreased surface hydrophobicity and decreased binding to salivary agglutinin-coated hydroxyapatite. In addition, Russell and Mansson-Rahemtulla (1989) have shown that antigen I/II binds directly, albeit with low affinity, to two proline-rich salivary proteins of A4,28,000 and 38,000. The precise nature of the adhesin-receptor interaction, however, remains unclear. The gene spa P from Strep. mutans strain NG5 (serotype c) has been cloned (Lee, Progulske-Fox and Bleiweis, 1988) and sequenced (Kelly et al., 1989). The identical gene from a further Strep. mutans strain, MT8148, was also cloned (Okahashi et al., 1989a) and sequenced (Okahashi er al., 1989b). The sequences indicate that antigen I/II is a membranebound protein with an extended structure, in which the N-terminal region may adopt an cc-helical coiledcoil conformation. The antigen appears to be highly conserved between different strains and serotypes of Strep. mutans. We have now further investigated this cell surface antigen of Strep. mutans NG5.

Cell surface antigens of Streptococcus mutans have attracted considerable interest because of their potential use as sub-unit vaccines against dental caries. Russell and Lehner (1978) identified four cell wallassociated antigens-I, II, III and I/II. Antigen I/II, also termed antigen B (Russell, 1980) and Pl (Forester, Hunter and Knox, 1983) is a major surface protein of M, 185,000 and is associated with the fuzzy coat present on the outer surface of the cell wall (Moro and Russell, 1983). Immunization with antigen I/II prevents colonization with Strep. mutans and the development of dental caries in non-human primates (Lehner ef al., 1981; Russell, Beighton and Cohen, 1982). Antigens I and II can also induce protection against dental caries (Lehner et al., 1981) and are both components of the 185,000 M, surface protein. Antigen I was purified from culture supernatants of Strep. mutans as a polypeptide of M, 150,000 (Russell et al., 1980b). Antigen II represents a protease-resistant core, of approx. M, 50,000, which was purified following pronase digestion of antigen I/II (Russell et al., 1980a). The two components do not cross-react serologically and monoclonal antibodies that are specific for each have been produced (Smith, Lehner and Beverley, 1984).

Abbreriuiions: GPI, glycosyl-phosphatidylinositol; polymerase chain reaction.

PCR, 33s

34s

C.

KELLY et al.

MATERIALS AND METHODS

Nucleotide sequencing

Restriction fragments from the recombinant clone pSM2949 (Lee et al., 1988) were subcloned into M13mp18 or M13mp19. The sequence of spa P was determined on both strands using the dideoxy chain termination procedure @anger, Nicklen and Coulson, 1977). Oligonucleotide primers (17-20 nucleotides), based on the sequence determined, were used to extend the sequences of fragments >400 bp. Double-stranded PCR products (see below) were sequenced directly using a modified procedure (Winship, 1989). Sequences were assembled and analysed using the Staden plus programme (Amersham International, Amersham, England). Pol.ymerase chain reaction

Chromosomal DNA was prepared from Strep. mutans strain NG5 as described by Lee et al., (1988). Using the primers CAAATGGGACAAACAGGC, nucleotides 39854002 from the NG5 sequence, and AAGGCAGTGCGAAGTACC, complementary to nucleotides 5 153-S 170 from the MT8148 sequence (Okahashi et al., 1989b), a 1 kb fragment that included the 3’ region of spa P was amplified by the PCR (Saiki et al., 1988). The reaction (30 cycles: 94°C for 2 min, 55C for 1 min, 72°C for 2 min) was made with an automated thermal cycler (Bioexcellence; Techne Ltd, Cambridge). Chromosomal DNA was prepared from a further 9 strains of Strep. mutans. Using the primers GCTTCTGCTTATACAGG (nucleotides 1965-198 1 from the coding sequence of NG5) and GGAAGATTAACCGCACG (complementary to nucleotides 24542470) a 0.5 kb DNA fragment that included the coding region for amino acid residues 750-810 was amplified from each strain as above. Protein pwfication

and sequence analysis

Antigens I/II and I were prepared from the culture supernatant of Strep. mutans serotype c (Guy’s strain) by chromatography on DEAEcellulose and Sepharose 6B (Russell et al., 1980a, 1980b). Antigen II was prepared from antigen I/II by treatment with pronase, followed by gel filtration on Ultragel AcA34 (Russell et al., 1980a). A peptide of IV, 4 K was isolated from a subtilisin digest of antigen I/II by reversed-phase high-pressure liquid chromatography (Mitchell and Lehner, 1989). A recombinant antigen of M, 155,000 was prepared by gel filtration of the periplasmic fraction, released following osmotic shock, from Escherichia coli LC 137 transformed with pSM2949 (Lee et al., 1988). N-terminal amino acid sequence analyses were performed on the purified polypeptides (lOO200 pmol) using the 470 A gas-phase sequencer with on-line 120 A PTH analyser (Applied Biosystems, Foster City, CA, U.S.A.). RESULTS

The complete nucleotide sequence of the gene (4683 bp) encoding antigen I/II was determined and the deduced amino acid sequence (1561 residues) is shown in Fig. 1. The reading frame was confirmed by the results of analysing the N-terminal amino acid

sequence of antigen I/II, the recombinant gene product and the polypeptide fragments derived from antigen I/II. In each case the amino acid sequence matched that predicted from the nucleotide sequence. The N-terminal sequence of purified antigen I/II corresponded to the predicted sequence from Asp 39 to Thr 43. Similarly, the N-terminal sequence of the recombinant antigen I/II corresponded to the sequence Asp 39 to Gln 67. Furthermore, the amino acid sequence around Asp 39 satisfies the requirements for cleavage by signal peptidase (von Heijne, 1983). The observations suggest that the N-terminal 38 residues form a signal peptide. In addition to the signal sequence, the predicted amino acid sequence of antigen I/II includes a potential transmembrane region located at the C-terminal (Ala 1537-Leu 1556), suggesting that the antigen is an integral membrane protein. Immediately preceding the transmembrane region is a sequence rich in Pro and otherwise consisting almost entirely of polar residues (Thr 1486Asn 1536). Similar sequences in other streptococcal cell surface proteins are believed to span the cell wall (Hollingshead, Fischetti and Scott, 1986). Two series of tandem repeats are evident with residues 191464 forming 3.3 repeats of an 82 residue Ala-rich sequence and residues 847-963 representing 3 repeats of a Pro-rich 39 residue sequence. Nucleotide sequencing of pSM 2949 indicated that the recombinant plasmid did not contain the entire spa P gene. Comparison with the sequence of Strep. mutans strain MT8148 (Okahashi et al., 1989b) indicated that the complete reading frame comprised a further 800 bases beyond the 3’ Hind111 site of pSM 2949 (Lee et al., 1988). A 1 kb DNA fragment that overlapped with pSM 2949 and that included this region was generated from Strep. mutans NG5 DNA by means of the PCR, using primers based on the sequences of strains NG5 and MT8148. Sequencing of the PCR product confirmed that it coded for residues 1308-l 561. The antigen I/II sequences from strains NG5 and MT8148 showed a considerable degree of conservation with only 36 single amino acid substitutions most of which were conservative (Kelly et al., 1989). In addition, a sequence of 9 residues (796804) in strain NG5 is replaced by a non-homologous sequence of 14 residues in strain MT8148. The substitutions are not distributed evenly throughout the protein sequence but most occur in the central region (residues 750-805) shown boxed in Fig. 1. The extent of variation in amino acid sequence between different strains of Strep. mutans (serotype c) was investigated further. By means of the PCR, the fragment of spa P encoding residues 656-823 was amplified from 9 unrelated strains. The deduced amino acid sequences of 5 of these strains were identical to that of strain MT8148, while those of the remaining four were identical to strain NG5 (L. J. Brady et al., unpublished data). The positions of antigen I and antigen II within the intact 185 K M, antigen I/II were determined from N-terminal sequence analyses of the purified components. The N-terminus of antigen I is identical to that of antigen I/II (residue 3943). The peptide of M, 4 K, generated by digestion with subtilisin, is known to react with antisera raised against antigen I

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Fig. 2. Principal features of the sequence of antigen I/II from Strep. mutans. Regions of the sequence identified in Fig. 1 are shown diagrammatically. The leader sequence (residues l-38) is followed by three tandem repeats of an Ala-rich sequence (residues 219464), which are preceded by a possible degenerate further repeat q . The region where most amino acid substitutions occur (residues 750-805 is shown 69 followed by the tandem repeats of a Pro-rich sequence (residues 846-963). At the C-terminal, wallspanning (residues 148&1536) and transmembrane (residues 1537-1556) regions are indicated. The N-terminal residue of antigen I is Asp 39 and of antigen II is Gln 996. The C-terminal of antigen I is located around residue 1400 (see text), while that of antigen II is uncertain. and Lehner, 1989). As the N-terminus of this peptide corresponded to the sequence starting at residue 1362 it must be derived from, or overlap with, the C-terminal of antigen I, which therefore represents a large N-terminal fragment. Antigen II extends from residue 996 and thus represents a C-terminal fragment. The main features of the sequence of antigen I/II are summarized diagrammatically in Fig. 2. (Mitchell

DISCUSSION The deduced sequence of antigen I/II is consistent with that of a cell surface protein anchored in the membrane by means of a C-terminal hydrophobic sequence. Recent findings, however, suggest that antigen I/II may be attached to the membrane not by the C-terminal hydrophobic sequence of 20 residues but by some form of membrane attachment complex, possibly an acyl group, added post-translationally. Thus the sequence of the homologous antigen from Strep. mutans serotype f (Ogier et al., 1990) did not contain a transmembrane region. Furthermore, Pancholi and Fischetti (1988) provided evidence that the membrane-bound M proteins from group A streptococci lack a C-terminal hydrophobic sequence, predicted to be present from the DNA sequence. Subsequently, an enzymic activity associated with the cell membrane and capable of releasing membrane-bound M protein was identified (Pancholi and Fischetti, 1989). It was suggested that this enzymic activity resembled that of a phospholipase and, by analogy, that M proteins may be attached to the membrane by means of a GPI complex. In support of this hypothesis, a common amino acid-sequence motif was identified (consensus LPXTG) near the C-terminus of 5 streptococcal membrane proteins, as well as at the predicted cleavage and attachment sites of three GPI-linked eukaryote proteins. Although there is no evidence for GPI-linked proteins in prokaryotes, the presence of the consensus sequence in streptococcal proteins that show no other homology is remarkable and may well provide a signal for attachment of some form of membrane anchor. The sequence is also present in antigen I/II from Strep. mutans serotype c (residues 1528-1532) and serotype $ The C-terminal region of antigen I/II (residues 800-1540) does not exhibit any obvious structural motif. The relative abundance of Pro suggests it may adopt a somewhat extended structure similar to the

stable polyPro conformations, i.e. helices of three residues per turn but less tightly packed than collagen. In contrast, the N-terminal region (residues 60-550), which includes the Ala-rich tandem repeats, is predicted to be a-helical (Chou and Fasman, 1974). Further evidence that this region is helical is provided by the observation that each long 82-residue) repeat comprises a series of 10 heptad repeats where three of the repeats are extended by 4 residues as shown in Fig. 3. Such repeats are characteristic of an a-helical coiled-coil conformation comprising either two chains, e.g. intermediate filaments (Steinert and Parry, 1985), or three chains, e.g. the influenza virus haemagglutinin (Wilson, Skehel and Wiley, 1981). Although there is no homology between antigen I/II and streptococcal M proteins, heptad repeats also occur in the latter for which an cc-helical coiled-coil structure has been proposed (Manjula et al., 1984). In view of the considerable overlap between the C-terminus of antigen I and the N-terminus of antigen II, the lack of serological cross-reaction between these two components is surprising. Release of antigen I may result in conformational changes such that epitopes present in the antigen II region are not available for antibody binding. Alternatively, immunodominant epitopes of antigen II may be located exclusively in the C-terminal region of the M, 50 K polypeptide. Release of antigen I is presumably the result of cleavage at the C-terminal by endogenous proteases, with likely cleavage sites being the clusters of Lys located between residues 1396 and 1440. The functional significance, if any, of release of antigen I Reoeat

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Cell surface antigen of Strepiococcus mutans

37s REFERENCES

is not clear, although similar release of a N-terminal

fragment was reported for the wall-associated antigen III (A) (Ferretti, Russell and Dao, 1989). Regions of the protein that are directly involved in binding to the tooth surface have not been identified. The cell surface hydrophobicity that is associated with the expression of antigen I/II (McBride et aI., 1984; Okahashi et al., 1989a; Lee et al., 1989) and that is believed to contribute to binding may be a consequence of the abundance of Ala (approx. 25% of residues) in the N-terminal region. In the a-helical coiled-coil conformation predicted for this region, the Ala residues are arranged in such a way that they would be clustered to form continuous hydrophobic patches winding around the surfaces of the helices. The hydrophobic interactions may be secondary to a more specific adhesin-receptor binding. Ogier et al. (1985) identified a fragment of IV, 74 K from Strep. mutans serotypef, that binds to saliva-coated hydroxyapatite and appears to represent a C-terminal fragment of antigen I/II (Ogier et al., 1990), although it is likely to extend into the central region. On the assumption that the most conserved part of the sequence is likely to be functionally important, the central region may form the adhesin-binding site. Thus, Takahashi et al. (1989) have shown that the highest degree of homology between the genes for

Ayakawa G. Y., Bleiweis A. S., Crowley P. .I. and Cunningham M. W. (1988) Heart cross-reactive antigens of mutans streptococci share epitopes with group A streptococci and myosin. J. Immun. 140, 253-257. Bergmeier L. A. and Lehner T. (1983) Lack of antibodies to human heart tissue in sera of rhesus monkeys immunised with Streptococcus mutans antigens and comparative study with rabbit antisera. Infect.-Immun. 40, IOi%1082. Chou P. Y. and Fasman G. D. (19741 Prediction of orotein conformation. Biochemistry i3, 222-245. ’

antigen

A., Harris A. C., Aitken A., Bleiweis A. S. and Lehner T. (1989) Sequence analysis of the cloned streptococcai ceil surface antigen I/II. FEBS Lerrs 258, 127-132. Koga T., Okahashi N., Takahashi I., Kanamoto T., Asakawa H. and Iwaki M. (1990) Surface hydrophobicity, adherence, and aggregation of cell surface protein antigen mutants of Streptococcus mutans serotype c. Infect. Immun. 58, 289-296. Lee S. F., Progulske-Fox A. and Bleiweis A. S. (1988) Molecular cloning and expression of a Streptococcus mutans major surface antigen, Pl (I/II), in Escherichia

Strep.

I/II and the analogous

antigen

(PAg) from

sobrinus

occurs in the region encoding residues 612-919. Similarly, Southern blot analyses using a series of probes spanning the entire coding region of the gene confirmed that the central region was the most conserved sequence between the mutans groups of streptococci (J. Ma er al. unpublished data). Paradoxically, this region includes the sequence that shows most variation between the Strep. mutnns strains NG5 and MT8148, i.e. residues 750-805. The extent of variation is, however, limited because only two different sequences were found in the 9 strains of Strep. mutans (serotype c) analysed. Cell surface hydrophobicity and the binding to salivary agglutinin-coated tooth surfaces may therefore be associated with different regions of antigen I/II. In view of the potential use of antigen I/II as a sub-unit vaccine or as a target for topically administered monoclonal antibody (Lehner, Caldwell and Smith. 1985; Ma, Smith and Lehner, 1987), the demonstration that there is limited strain variation is of some importance. Although it has been suggested that the observed immunological cross-reactivity between Strep. mutnns and heart tissue is associated with antigen I/II expression (Hughes et al.. 1980; Russell et al., 1982; Forester et al., 1983), later studies have demonstrated that this is unlikely. Thus, immunization of non-human primates with antigen I/II failed to elicit cross-reactive antibodies for heart tissue (Bergmeier and Lehner, 1983). Furthermore, mutants of Strep. mutans that do not express antigen I/II yield on immunization heart cross-reactive antibody titres similar to those elicited by I/II-expressing strains (Lee et al., 1989; Koga et al., 1990). The heart

cross-reactivity is likely to be associated with other membrane components and a membrane protein of M, 62 K that has myosin-like epitopes has been identified (Ayakawa et al., 1988).

Ferretti J. J., Russell R. R. B. and Dao M. L. (1989) Sequence analysis of the wall-associated protein precursor of Streptococcus mutans antigen A. Molec Microbial. 3, 469-478.

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