Vol. 58, No. 9

INFECTION AND IMMUNITY, Sept. 1990, p. 3064-3072

0019-9567/90/093064-09$02.00/0 Copyright C 1990, American Society for Microbiology

Characterization of Streptococcus gordonii (S. sanguis) PK488 Adhesin-Mediated Coaggregation with Actinomyces naeslundii PK606 PAUL E. KOLENBRANDER* AND ROXANNA N. ANDERSEN Laboratory of Microbial Ecology, National Institute of Dental Research, Bethesda, Maryland 20892

Received 9 March 1990/Accepted 27 June 1990 Intergeneric coaggregation of Streptococcus gordonii (S. sanguis) PK488 and Actinomyces naeslundii PK606 was studied by using coaggregation-defective (Cog-) mutants of both strains. A streptococcal protein of 38 kilodaltons was identified with anti-S. gordonii serum absorbed with Cog- cells of the streptococcus. Absorbed immunoglobulin G specifically blocked coaggregation of the streptococcus-actinomyces pair but did not affect the coaggregation of the streptococcus with other coaggregation partners. The 38-kilodalton protein was found in the supernatant of mild sonicated cell suspensions and was extracted from whole cells with sodium barbital or with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). An immunoreactive protein of the same size was found in sonicated cell supernatants of several other oral streptococci that also coaggregated with A. naeslundii PK606. Inhibition of S. gordonii PK488-A. naeslundii PK606 coaggregation was not observed with any of 16 different sugars tested. We propose that a functionally similar adhesin that mediates coaggregations with A. naeslundii PK606 is expressed by several species of the genus Streptococcus.

Most human oral bacteria can participate in intergeneric coaggregations, which involve cell surface recognition between complementary adhesin-carbohydrate molecules on the partner cells (6, 7). Of the 692 strains from 13 genera examined in this laboratory for potential coaggregation partnerships, most of the strains exhibit interactions that are inhibited by lactose and other galactosides. The lectin responsible for one of these has been identified on the surface of Bacteroides loeschei PK1295 (18-20) and consists of a hexamer of 75-kilodalton (kDa) monomers (14a). More than 100 strains of streptococci, including Streptococcus gordonii, S. oralis, Streptococcus SM, and S. sanguis, and nearly 200 strains of actinomyces, including Actinomyces naeslundii and A. viscosus, have been examined. As a result, six coaggregation groups of streptococci (groups 1 to 6) and six coaggregation groups of actinomyces (groups A to F) have been delineated (3, 11-13). Nearly 90% of the streptococci and the strains of A. naeslundii tested and 100% of the strains of A. viscosus tested exhibit coaggregations that are lactose inhibitable. In contrast, the coaggregation of S. gordonii PK488, the reference strain for streptococcal coaggregation group 6, and A. naeslundii PK606, the reference strain for actinomyces coaggregation group D, is not inhibited by lactose (11). Although S. gordonii PK488 does exhibit extensive N-acetylgalactosamine-inhibitable coaggregation with other streptococci (P. E. Kolenbrander, R. N. Andersen, and L. V. H. Moore, unpublished data), it coaggregates only with actinomyces that are members of coaggregation group D. The reference strains for the other five streptococcal coaggregation groups coaggregate with at least three of the reference strains of the six actinomyces coaggregation groups (groups A to F), including coaggregation group D actinomyces (6, 7). Since the coaggregation between group 6 streptococci and group D actinomyces appears to be the *

least complex, we investigated its properties so that the results could be used to unravel some of the more complex coaggregation patterns observed with the other cocci. In this report we identify a 38-kDa protein which we propose is involved in mediating coaggregation between S. gordonii PK488 and A. naeslundii PK606. An immunoreactive protein which may perform a similar function was found in all reference streptococcal strains examined. MATERIALS AND METHODS Bacterial strains and culture conditions. S. gordonii PK488 (previously called S. sanguis PK488 and originally classified as S. sanguis biovar I, VPI strain ElA-lA) and A. naeslundii PK606 are the reference strains for streptococcal coaggregation group 6 and actinomyces coaggregation group D, respectively (6, 7). The other strains used are identified in Table 1. All strains are human oral isolates. The growth medium contained tryptone, yeast extract, Tween 80, and glucose (0.2%) and was buffered to pH 7.5 with K2HPO4 (15). Cells were grown at 37°C under anaerobic conditions with the GasPak system (BBL Microbiology Systems, Cockeysville, Md.), harvested in the late-exponential or earlystationary phase of growth, washed three times, and suspended in coaggregation buffer. Coaggregation buffer consisted of the following (dissolved in 0.001 M Tris adjusted to pH 8.0): CaCl2 (10-4 M), MgCl2 (10-4 M), NaN3 (0.02%), and NaCl (0.15 M). Centrifugation was done at 10,000 x g for 10 min at 4°C; after the cells were washed, they were stored in coaggregation buffer at 4°C until used. Coaggregation assays. (i) Visual assay. The visual assay has been described in detail previously (3, 9). A brief description and some modifications are given here. Suspensions with a final cell density of about 109 cells per ml (260 Klett units, determined with a 660-nm [red] filter in a Klett-Summerson colorimeter; Klett Manufacturing Co., Inc., New York, N.Y.) were prepared for each cell type. Equal volumes (0.1 ml) of each cell type were added to a glass test tube (10 by 75

Corresponding author. 3064

3065

S. GORDON1I ADHESIN

VOL. 58, 1990 TABLE 1. Strains used

Properties or ongin

Strain

A. naeslundii PK606 PK2365a PK1869 PK1878 PK1884

Streptococcus speciesc S. sanguis C104 S. oralis Hi 34 J22 Streptococcus SM PK509 S. gordonii DL1 PK488

PK1701d PK1709e PK1804 PK1817

Reference or source

Coaggregation group

Wild type; reference strain PK606 Rf Smr Gmr Fsr PK2365 (Cog-, selected with heated S. sanguis C104) PK1869 (Cog-, selected with S. gordonii PK488) PK2365 (Cog-, selected with S. gordonii PK488)

NAb NA NA

11 This This This This

Reference strain

3

3

Reference Reference Reference Reference

2 3 4 5

3 3 3 11

1 6 6 6 NA

3 11

strain strain strain strain

Reference strain Wild type; reference strain PK488 RF PK1701 Smr PK1701 (Cog-, selected with A. naeslundii PK606) PK1701 (Cog-, selected with A. naeslundii PK606)

D D

NA

This This This This

study study study study

study study study study

Resistant to rifamycin (25 ,ug/ml), streptomycin (1 mg/ml), gentamicin (1 ,ug/ml), and fusidic acid (10 pg/ml). b NA, Not applicable. c The streptococcal strains Hi, 34, J22, DL1, and PK488 were originally classified as S. sanguis. Streptococcus SM PK509 was formerly classified as Gemella morbillorum and originally classified as Streptococcus morbillorum. d Resistant to rifamycin (25 ,g/ml). e Resistant to streptomycin (25 ,ug/ml). a

mm) and vortex mixed for 5 s. Coaggregation scoring was done by viewing the tube above an indirect light source (illuminated magnifier no. 39-101; Stocker and Yale, Inc., Beverly, Mass.) while placing the tube on a glass surface attached to the light source and rocking the tube gently to maximize the interaction between the two cell types. The change from an evenly turbid suspension before mixing to the formation of visible aggregates consisting of both cell types (coaggregates) either occurred immediately during vortex mixing or developed within a few seconds during the gentle rocking procedure. A coaggregation score ranging from 0 (no change in turbidity and no visible coaggregates) to +4 (maximum coaggregation, with large coaggregates settling immediately, leaving a water-clear supematant) was given for each pair. A score of +3 indicated the formation of large settling coaggregates but a slightly turbid supematant, and a +2 score was given when definite coaggregates were visible but did not settle immediately. The weakest score (+1) represented finely dispersed coaggregates in a turbid

background. The reversal of coaggregation was determined by adding lactose to a final concentration of 0.06 M and rescoring the coaggregating pair. The effect of temperature was determined by heating a cell suspension at 85°C for 30 min before mixing it with heated or unheated cells of the partner. Protease treatment of cells was done by incubating cell suspensions (109 cells per ml) in coaggregation buffer with 0.5 mg of pronase (Calbiochem, La Jolla, Calif.) per ml at 50°C for 60 min. The cells were washed three times with coaggregation buffer and suspended to the original volume in coaggregation buffer. (ii) Radioactivity assay. Either [methyl-3H]thymidine (2.96 TBq/mmol [80 Ci/mmol]; New England Nuclear Corp., Boston, Mass.) or [2-14C]uracil (2.2 GBq/mmol [59.9 mCi/ mmol]; New England Nuclear) at 10 or 2 ,Ci, respectively, per ml of growth medium was used to label cells. After four

to six cell doublings, cells were harvested and washed (as described above) by centrifugation, suspended in coaggregation buffer, and stored at 4°C (specific radioactivity, about 103 bacteria per cpm). Details of the radioactivity assay for coaggregation have been described previously (9, 10). Cog- mutant selection. An antibiotic-resistant strain of either S. gordonii PK488 or A. naeslundii PK606 was used to select coaggregation-defective (Cog-) mutants by a method described earlier (5). Cell suspensions were made in coaggregation buffer without azide. Antibiotic-resistant S. gordonii PK488 cells (S. gordonii PK1709, about 109 cells) were mixed with an excess (about 1010 cells) of antibiotic-sensitive A. naeslundii PK606 cells. The same procedure was used for selecting Cog- mutants of A. naeslundii PK2365 (antibioticresistant A. naeslundii PK606), except that the streptococcus was the antibiotic-sensitive selection partner. Anti-PK488 serum production. Cell walls of S. gordonii PK488 were prepared by sonication (4 min at 70% maximum output with a 350-W Sonifier cell disrupter (Branson Sonic Power Co., Danbury, Conn.). After 20 cycles of washing with distilled water and centrifugation (10,000 x g for 10 min), and lyophilization, the cell walls were adjusted to a density of 380 Klett units in distilled water (15.5 mg/ml). A coaggregation score of +3 was observed when the cell wall suspension was mixed with whole cells of A. naeslundii PK606. A volume of 0.5 ml of cell wall suspension was injected into the marginal ear vein of a female New Zealand White rabbit twice weekly for 4 weeks; the rabbit was bled from the central ear artery 7 days after the final injection. Subsequent bleedings were done 7 days after a booster injection of the cell wall suspension. Serum was stored at -40°C. Rabbit immunoglobulin G (IgG) was purified by a two-step procedure involving ammonium sulfate precipitation and DEAE ion-exchange chromatography (21). Cog- mutant absorption of anti-PK488 serum. Cells of Cog- mutant S. gordonii PK1804 were suspended to a

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TABLE 2. Coaggregation of A. naeslundii PK606 with reference strains that represent the streptococcal coaggregation groups 1 through 6 Treatment of A. naeslundii PK606streptococcus paira

Both strains Streptococcus Actinomyces Neitherstrain

S.

gordonii (1)

DL1

0 0 3, 3 3,3

S. oralis Hi (2)

0 0 4, 4

4,4

S. oralis 34 (3)

0

3, 0 4, 4 4,4

Coaggregation score" with: S. oralis S. sanguis J22 (4) C104 (3)

0 3, 0 3, 3 4,3

0 2, 0 4, 4 4,4

Streptococcus

SM PK509 (5)

S. gordonii PK488 (6)

0 3, 0 3, 3 3,3

0 0 2, 2 2,2

Treatment consisted of heating at 85°C for 30 min before strains were mixed. b The method for assigning coaggregation scores is described in Materials and Methods. Coaggregation scores are given in two parts: the first score is that given after mixing the two strains together, and the second score is that given after adding lactose to a final concentration of 60 mM. Coaggregations that were reversed by lactose are indicated with coaggregation scores in boldface type. Numbers in parentheses are streptococcal coaggregation groups. a

concentration of 0.5 g (wet weight) per ml of coaggregation buffer. A volume of 1 ml was pelleted by centrifugation (10,000 x g for 10 min at 4°C), and the pellet was mixed with 5 ml of either rabbit whole anti-PK488 serum (42 mg of protein per ml) or purified IgG (4.5 mg of protein per ml) with constant rotation for 12 h at 4°C. The procedure was repeated eight times by pelleting the cells from the serum and then adding the serum to another 0.5-g cell pellet. Both absorbed anti-PK488 serum and absorbed IgG failed to agglutinate S. gordonii PK488 or any of the other streptococci used in this study. ELISA. For the enzyme-linked immunosorbent assay (ELISA), whole cells of S. gordonii PK488 or its Cogmutant PK1804 were suspended to 10 Klett units (2 x 107 cells per ml) in 0.2 M sodium carbonate buffer (pH 9.6), and 0.1 ml was added to each well of a 96-well microtiter plate as a solid-phase antigen. After overnight incubation at 4°C, the attached cells were washed with three 0.1-ml volumes of sodium carbonate buffer. Purified IgG or Cog- mutantabsorbed IgG was added in the range of 1.5 pLg to 1.5 ng of protein per well. Whole antiserum or Cog- mutant-absorbed whole antiserum was used at 14 p.g to 14 ng of protein per well. These concentrations of protein fell in the range of twofold dilutions of antiserum and IgG between 1:300 and 1:300,000. After 1 h, biotinylated sheep anti-rabbit IgG was added to each well, followed by streptavidin-biotinylated horseradish peroxidase complex, in accordance with the instructions of the manufacturer (Amersham Corp., Arlington Heights, Ill.). Chromophore generation was measured with the Titertek Multiskan MC (Flow Laboratories, Inc., McLean, Va.). Streptococcal surface preparations. For each of the three following methods, cells (600 mg [wet weight]) of S. gordonii PK488 (wild type), antibiotic-resistant parent (PK1701), and Cog- mutant (PK1804) were washed three times with distilled water by centrifugation (10,000 x g for 10 min at 4°C). (i) Mild sonication. A 3-ml volume of cell suspension was sonicated in an ice bath for 1 min at maximum power (50 W) with a microultrasonic cell disrupter (Kontes, Vineland, N.J.). The sonicated suspension was centrifuged at 20,000 x g for 10 min, and the supernatant was stored frozen until used. (ii) Sodium barbital. After the final wash in distilled water, the cell pellet was suspended in 2 mM sodium barbital (pH 8.6) by the method of Lamont et al. (14), except that the concentration of cells used here was 60 mg/ml instead of 25

mg/ml. (iii) CHAPS. The distilled water-washed cell pellet was suspended in 10 ml of 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) dissolved in coaggregation buffer. Extraction was done at room temperature for 15 h on a rotary shaker. The clear supernatant after

centrifugation of the extract at 15,000 x g for 10 min was saved. To reduce the amount of CHAPS in the extract, we applied a portion of the extract to a 1ODG column packed with Bio-Gel P6 (Bio-Rad Laboratories, Inc., Richmond, Calif.) and eluted it with coaggregation buffer. Samples before and after desalting with Bio-Gel yielded identical results on immunoblots of polyacrylamide gels described below. Immunoblots. Streptococcal extracts made by mild sonication, by extraction with 2 mM sodium barbital (pH 8.6), or by extraction with 0.5% CHAPS were subjected to sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (4). The separated proteins were transferred to nitrocellulose filters (2, 17). Filters were treated with a 1:1,000 dilution of rabbit anti-PK488 serum or purified IgG at a final concentration of 40 or 7 ,ug of protein, respectively, per ml of buffer. In most experiments the antiserum or the IgG was absorbed with Cog- mutant PK1804 before use. Immune complexes were visualized with alkaline phosphatase-conjugated antirabbit IgG and a dye indicator system supplied by Promega Biotech, Madison, Wis. Chemicals. All sugars were purchased from Sigma Chemical Co., St. Louis, Mo. Solutions of amino sugars and N-acetylated amino sugars were adjusted to a pH of 7.4 with 1 N NaOH. Protein determinations. Protein concentrations were determined with a protein assay kit from Bio-Rad. RESULTS General properties of coaggregation of A. naeslundii PK606 with reference strains of the six streptococcal coaggregation groups. Coaggregation group D actinomyces, represented by A. naeslundii PK606, coaggregated with reference strains of all six streptococcal coaggregation groups (Table 2). Heat or protease treatment of the actinomyces did not change the lactose-nonreversible nature of the interaction. Identical treatment of the streptococci abolished coaggregation of group 1, 2, and 6 streptococci with A. naeslundii PK606 and changed coaggregation of the other three groups to lactose reversible. Protease treatment of the streptococci and actinomyces produced the same results as heat treatment. These results indicate the presence on the streptococci of at least one protein that mediates coaggregation, since treatment of the streptococci either prevented coaggregation with A. naeslundii PK606 (as seen with S. oralis Hi and S. gordonji DL1 and PK488) or made detectable a lactose-sensitive lectin on the actinomyces (as seen with S. oralis 34 and J22, S. sanguis C104, and Streptococcus SM PK509). The lactose-insensitive coaggregations were also insensitive to the following sugars tested at a 60 mM final concentration: N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, D-ga-

VOL. 58, 1990

S. GORDONII ADHESIN

3067

TABLE 3. Coaggregation of A. naeslundii PK2365 and its Cog- mutants with reference strains of Streptococcus species that represent the streptococcal coaggregation groups 1 through 6 Coaggregation score' with:

Treatment of actinomycesstreptococcus pair'

S. gordonii

DL1 (1)

Hi (2)

S. oralis 34 (3)

S. sanguis C104 (3)

S. oralis J22 (4)

Streptococcus SM PK509 (5)

S. gordonii PK488 (6)

PK2365 (parent)

Both strains Streptococcus Actinomyces Neither strain

0 0 3, 3 3, 3

0 0 3, 3 3, 3

0 4, 1 4, 4 4, 4

0 4, 0 3, 2 4, 3

0 3, 0 3, 3 4, 4

0 3, 0 3, 3 3, 3

0 0 2, 2 2, 2

PK1869 (Cog-)

Both strains Streptococcus Actinomyces Neither strain

0

0

0

0

0

0

0

0 3, 2 3, 2

0 4, 4 4, 4

0 3, 3 3, 3

0 1, 1 1, 1

0 2, 2 3, 2

0 1, 1 2, 2

0 2, 1 2, 2

PK1878 (Cog-)

Both strains Streptococcus Actinomyces Neither strain

0 0 4, 0 4, 0

0 0 4, 4 4, 4

0 0 2, 0 2, 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

PK1884 (Cog-)

Both strains Streptococcus Actinomyces Neither strain

0 0 2, 0 2, 0

0 0 3, 3 3, 3

0 4, 0 3, 0 4, 0

0 4, 0 0 4, 0

0 4, 0 0 4, 0

0 3, 0 0 3, 0

0 0 0 0

A. naeslundii strain

S. oralis

aSee Table 2, footnote a. "Table 2, footnote b.

lactose, fructose, L-rhamnose, D-fucose, L-fucose, D-mannose, 2-deoxy-D-glucose, D-galacturonic acid, 1-o-methyl-aD-galactopyranoside, methyl-p-D-galactopyranoside, or 6-o-methyl-D-galactose. Use of Cog- mutants to study coaggregation properties. To study the A. naeslundii PK606-S. gordonii PK488 interaction more closely, we isolated mutants of both strains. The properties of these derivatives will be discussed in detail later (see Fig. 7). Heat-treated S. sanguis C104 (streptococcal coaggregation group 3) was mixed with PK2365, a multiple-antibiotic-resistant derivative of PK606, to select for a Cog- mutant. A. naeslundii PK1869 was isolated by this procedure (Table 3). It did not coaggregate with any of the heat-treated cocci. To isolate a double Cog- mutant, we mixed strain PK1869 with S. gordonii PK488. A. naeslundii PK1878 was one of the mutants obtained. Coaggregation with streptococci of coaggregation groups 3, 4, 5, and 6 was absent. S. oralis 34, one of the two reference strains for coaggregation group 3, exhibited lactose-reversible coaggregation with the double mutant, as did S. gordonii DL1 (coaggregation group 1), indicating lactose-sensitive lectin activity on these two streptococci. Coaggregation with S. oralis Hi (coaggregation group 2) remained unchanged. A third Cog- mutant, strain PK1884, was selected by mixing strain PK2365 with S. gordonii PK488. The coaggregation properties of PK1884 indicated that a receptor that functioned in recognizing an adhesin on group 1, 3, 4, 5, and 6 streptococci was altered or missing. The absence of this receptor on the actinomyces led to lactose-inhibitable coaggregation with strains DL1, 34, C104, J22, and PK509 but not strain Hi (coaggregation group 2). The latter strain appeared to participate in additional lactose-noninhibitable interactions with the actinomyces. The results obtained with the actinomyces Cog- mutants suggested that each streptococcus possessed an adhesin that recognized the same actinomyces receptor. To provide evidence for this idea, we chose to study the S. gordonji PK488-A. naeslundii PK606 interaction. Cog- mutants of S. gordonii PK1709, an antibiotic-resistant derivative of

PK488, were selected by mixing PK1709 with A. naeslundii PK606. All Cog- mutants, for example, S. gordonii PK1804 (Fig. 1), exhibited the same Cog- phenotype (absence of coaggregation with actinomyces) but maintained coaggregation with other strains of streptococci (Kolenbrander et al., unpublished data). Whereas the parent streptococcus formed extensive coaggregates with the actinomyces (Fig. 1A), the mutant streptococcus did not (Fig. 1B). Quantitative assay of coaggregation and competition. Coaggregation of S. gordonii PK488 and A. naeslundii PK606 appeared relatively weak in the visual assay in comparison with that of other coccus-actinomyces pairs (Table 2). To quantify the coaggregation and more rigorously examine the Cog- mutants, we mixed increasing numbers of unlabeled actinomyces with a constant number of labeled streptococci and measured the distribution of radioactivity in coaggregates that sedimented upon low-speed centrifugation. A ratio of actinomyces to wild-type S. gordonii PK488 of about 5:1 yielded the maximum number of streptococci in coaggregates (data not shown). The streptococcal Cog- mutant PK1804 did not coaggregate even in the presence of a 19-fold excess of actinomyces, indicating the absence of functional adhesins on the surface of PK1804. To determine the potential usefulness of the Cog- mutant PK1804 in preparing an absorbed anti-wild-type serum, we investigated its ability to compete with the wild-type strain PK488 for binding to the actinomyces (Fig. 2). The addition of increasing numbers of unlabeled PK488 to a constant number of radioactive PK488 resulted in the expected competition for coaggregation with the partner, A. naeslundii PK606. Maximal competition was observed at a ratio of unlabeled to labeled streptococci of 14:1, the highest ratio tested. No competition was evident when the Cog- mutant was used in place of the parent strain. Coaggregation-blocking properties of Cog- mutant-absorbed anti-S. gordonii PK488 serum. The titer of both whole antiserum and IgG in the ELISA was 1:300,000 (50% of maximum color development) for both PK488 and PK1804 (data not shown). Absorbed whole antiserum and absorbed

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INFECT. IMMUN.

KOLENBRANDER AND ANDERSEN

9.

FIG. 1. Phase-contrast photomicrographs of coaggregation between S. gordonii PK488 and A. naeslundii PK606 (A), absence of coaggregation between Cog- mutant S. gordonii PK1804 and A. naeslundii PK606 (B), and cell suspensions of A. naeslundii PK606 (C), S. gordonii PK488 (D), and Cog- mutant S. gordonii PK1804 (E). The adherence of streptococci (arrowheads in panel A) to the filamentous actinomyces is shown. Bar, 10 p.m.

IgG exhibited titers of 1:75,000 with PK488 cells (wild type) and 1:600 with PK1804 cells (Cog- mutant), while the titer of preimmune serum was 1:300 with both cell types. Absorbed antiserum blocked the coaggregation of S. gordonii PK488 and A. naeslundii PK2365 (Table 4). The coaggregations with the reference strains for streptococcal groups 1 and 5 were lactose reversible in the presence of antiserum. Coaggregation of A. naeslundii Cog- mutant PK1869 and S. gordonii PK488 was also completely blocked by the absorbed antiserum. In addition, coaggregation of this mutant, PK1869, with Streptococcus SM PK509 was completely blocked, while coaggregation with S. gordonii DL1 was altered in the same way as with the wild-type strain. As compared with the coaggregation pattern shown in Table 3, no change in the coaggregation of either A. naeslundii Cogmutant PK1878 or PK1884 with their coccal partners was observed in the presence of absorbed antiserum. Coaggregation between S. oralis Hi and the actinomyces did not change after preincubation of the streptococci with absorbed antiserum, indicating no obvious steric effects of the preincubation of cells with antiserum on the ability of cells to coaggregate. Furthermore, preincubation of strains DL1, Hi, 34, C104, J22, PK509, or PK488 with preimmune serum had no effect on their ability to coaggregate with A. naeslundii PK2365. Absorbed antiserum inhibited coaggregation between S. gordonii PK488 and A. naeslundii PK606 by 50% at 1.5 ,ug of protein per ml (108 streptococcal cells per ml) (Fig. 3).

\0COG -

0 cJ 0 40

c

0)

a.01) 0

0

2

4

6

Ratio of cells (

10

8

labeled

)

FIG. 2. Competition between radioactively labeled S. gordonii PK488 and either unlabeled PK488 (0) or its Cog- mutant PK1804 (0) for coaggregation with A. naeslundii PK606. Competitor cell types were mixed together before addition of the actinomyces. The input radioactivity was 2,755 cpm, and the percentage of input counts per minute in coaggregates was 67. Coaggregates were separated from free cells by low-speed centrifugation as described in the text.

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TABLE 4. Antibody and lactose inhibition of coaggregation between A. naeslundii PK2365 or its Cog- mutants PK1869, PK1878, and PK1884 and reference strains that represent streptococcal coaggregation groups 1 through 6 A. naeslundii strain

Coaggregation scorea with: S. gordonii DL1 (1)

PK2365 (parent) PK1869 (Cog-) PK1878 (Cog-) PK1884 (Cog-)

3, 3, 3, 3,

PK2365 (no antiserum)

3, 3

S. oralis Hi (2)

4, 4, 4, 4,

0 0 0 0

S. oralis J22 (4)

2 2 0 0

4, 1 1, 1 0 4, 0

3, 1 2, 1 0

0 0

4, 0

4, 0

0 0

4, 4

4, 3

4, 4

3, 3

2, 2

3, 3, 2, 4,

4 4 4 4

3, 3

S. gordonji PK488 (6)

S. sanguis C104 (3)

S. oralis 34 (3)

Streptococcus SM PK509 (5)

2,0

0

See Table 2, footnote b. Streptococci were preincubated for 30 min at room temperature with absorbed antiserum (456 ,ug of protein per 108 cells). Antiserum (5 ml) was absorbed with eight changes (the tube was continuously inverted for 12 h at 4°C) of 0.25 g (wet weight) of S. gordonii PK1804, a Cog- mutant of S. gordonii PK488, and used at a 1:10 dilution. a

Preimmune serum had no effect on this coaggregation. In addition, no inhibition of coaggregation between S. gordonji PK488 and other streptococci (Kolenbrander et al., unpublished data) was observed at any concentration of absorbed antiserum tested. Thus, it appeared that the absorbed antiserum specifically reacted with a surface component on S. gordonii PK488 that was mediating coaggregation with A. naeslundii PK606. Identification of an S. gordonii PK488 immunoreactive surface protein with Cog- mutant-absorbed antiserum. Three methods to release bacterial surface components were evaluated. By using immunoblotting techniques, the supernatants obtained after sonication of the wild type (PK488), antibiotic-resistant parent (PK1701), and Cog- mutant (PK1804) were compared with the supernatants obtained after treatment of the cells with sodium barbital (2 mM, pH 8.6, 30 min, 4°C) or 0.5% CHAPS (15 h at room temperature) (Fig. 4). Anti-wild-type IgG revealed a protein band at 38 kDa in the lanes containing the wild type (Fig. 4A, lanes 2, 5, and 8) and antibiotic-resistant parent (Fig. 4A, lanes 3, 6, and

9) that was faintly visible in the lanes containing the Cogmutant (Fig. 4A, lanes 4, 7, and 10). The 38-kDa band was clearly seen in the wild-type and parent preparations (Fig. 4B, lanes 2 and 3, 5 and 6, and 8 and 9, respectively) when IgG prepared from Cog- mutant-absorbed antiserum was used. Little or no evidence of the 38-kDa band was observed with PK1804 (Fig. 4B, lanes 4, 7, and 10). The dilution of the absorbed antiserum used in these immunoblots was sufficient to block coaggregation completely (Fig. 3). 1

kDa 110 -

2

3 4 5 6 7 8 9 10

A

84-

_

47

=

24-

*.

-

.

.

100k 0

/ 0, mf

° 80

2

1

kDa 110 8447-

CD m/ 8 60 0,

0X

3

4

5

6

7

8

9 10

B

20-

3324-

Co

0

01

2

3

4

5

(*g of protein/ml) COG- mutant-absorbed anti-PK488 serum FIG. 3. Inhibition of S. gordonii PK488-A. naeslundii PK606 coaggregation by Cog- mutant-absorbed anti-PK488 serum. A 1:5 ratio of labeled streptococci to unlabeled actinomyces was used. Labeled streptococci, coaggregation buffer, and antiserum were mixed by vortexing and incubated for 30 min at room temperature before unlabeled actinomyces cells were added. Other procedures are as described in Materials and Methods. The input radioactivity was 5,367 cpm.

FIG. 4. Immunoblot analysis of proteins released from S. gordonii PK488 (wild type), S. gordonii PK1701 (antibiotic-resistant parent), and S. gordonii PK1804 (Cog- mutant) by sonication (lanes 2 to 4), sodium barbital extraction (lanes 5 to 7), or CHAPS treatment (lanes 8 to 10). All lanes were loaded with 1.5 ,ug of protein. (A) The immunoblot was developed with IgG (4.5 mg of protein per ml, diluted 1:1,000) prepared from unabsorbed antiPK488 serum. (B) The immunoblot was developed with Cogmutant-absorbed IgG (4.2 mg of protein per ml, diluted 1:1,000) prepared from anti-PK488 serum. Lane 1 contains prestained lowmolecular-mass standards (Bio-Rad).

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INFECT. IMMUN.

KOLENBRANDER AND ANDERSEN 2

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FIG. 5. Comparison by protein staining (lanes 1 to 4, 6.0 ,ug of protein loaded on lanes 1 to 3) and immunoblotting (lanes 5 to 8, 1.5 ,ug of protein loaded on lanes S to 7) of sonicated cell supernatants from S. gordonii PK488 (wild type, lanes 1 and 7), S. gordonii PK1701 (parent, lanes 2 and 6), and S. gordonii PK1804 (Cogmutant, lanes 3 and 5). The immunoblot (lanes 5 to 8) was developed with Cog- mutant-absorbed IgG (4.2 mg of protein per ml, diluted 1:1,000) prepared from anti-PK488 serum. Unstained low-molecular-mass protein standards (Bio-Rad) with molecular masses of 97.4 (phosphorylase b, rabbit muscle), 66.2 (bovine serum albumin), 45.0 (ovalbumin, hen egg white), 31.0 (carbonic anhydrase, bovine), and 21.5 (soybean trypsin inhibitor) kDa were applied to lane 4. Proteins were visualized by the Pro-Blue staining system (Integrated Separation Systems, Hyde Park, Mass.). Lane 8 contains the corresponding prestained low-molecular-mass standards (Bio-Rad) with apparent masses of 110.0 (phosphorylase b, rabbit muscle), 84.0 (bovine serum albumin), 47.0 (ovalbumin, hen egg white), 33.0 (carbonic anhydrase, bovine), and 24.0 (soybean trypsin inhibitor) kDa.

Protein profile of surface components. A comparison of the total protein profile with the profiles of the immunoreactive proteins (Fig. 5) in sonicated cell supernatants of the wild type, the parent, and the Cog- mutant revealed no significant difference in the protein profiles of the wild type (lane 1), the parent (lane 2), and the mutant (lane 3). However, the mutant (lane 5) clearly possessed less (or none) of the 38-kDa immunoreactive protein than did the parent (lane 6) or the wild type (lane 7). Examination of an independently isolated Cog- mutant, S. gordonii PK1817, yielded the same results as those obtained with Cog- mutant PK1804 (data not shown). Presence of an immunoreactive protein in other streptococcal partners of A. naeslundii PK606. The observation that preincubation of the reference streptococci with absorbed anti-PK488 serum had profound effects on their coaggregation with A. naeslundii PK2365 (Table 4) as well as with A. naeslundii PK606 (data not shown) suggested that a similar surface protein on these streptococci may be involved in coaggregation with the actinomyces. Supernatants of sonicated cells of the six reference streptococci were compared by immunoblot analysis with absorbed antiserum (Fig. 6). An immunoreactive protein of about the same size as that found with PK488 was observed. A slightly larger protein was present in the extracts of both coaggregation group 3 streptococci (lanes 6 and 7). Thus, it appears that all streptococci that coaggregate with A. naeslundii PK606 express a functionally equivalent protein of about 38 kDa, which we propose is an adhesin mediating the coaggregation. DISCUSSION

S. gordonii PK488 (reference strain for streptococcal coaggregation group 6) was chosen to study an adhesin that

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FIG. 6. Immunoblot analysis of proteins released by sonication from S. gordonii PK488, PK1701, and DL1; S. oralis H1; S. oralis 34; S. sanguis C104; S. oralis J22; and Streptococcus SM PK509 (lanes 2 to 9, respectively). The strains represent coccal coaggregation groups 6, 6, 1, 2, 3, 3, 4, and 5, respectively. All lanes were loaded with 1.5 ,ug of protein. The immunoblot was developed with Cog- mutant-absorbed IgG (4.2 mg of protein per ml, diluted 1:1,000) prepared from anti-PK488 serum. Lane 1 contained prestained low-molecular-mass standards (Bio-Rad).

mediates coaggregation with A. naeslundii PK606 (reference strain for actinomyces coaggregation group D) because this is the simplest kind of interaction that occurs between the actinomyces and six known streptococcal coaggregation groups (6, 7). PK488 only coaggregated with the group D actinomyces, the interaction was lactose noninhibitable, and heat treatment of the streptococcus was sufficient to prevent coaggregation. However, under certain conditions the other streptococci exhibited coaggregation properties similar to those of S. gordonii PK488. These similar properties were proposed to be manifested by the same adhesin-receptor complementary pairs to keep the number of streptococcal adhesins to a minimum (6, 7). The coaggregation properties were based on four parameters for each streptococcusactinomyces pair tested: (i) coaggregation occurring between a bacterial pair, (ii) inhibition of coaggregation by the addition of lactose, (iii) inhibition of coaggregation by heat or protease treatment of one or both partners, and (iv) altered coaggregation properties for Cog- mutants of either or both partners. By using Cog- mutants of both S. gordonii PK488 and A. naeslundii PK606 and coaggregation-blocking antiserum, we developed a model concordant with the results of the streptococcus-actinomyces coaggregations (Fig. 7). To keep the discussion of this model lucid, we will use symbols to describe cell-cell interactions. It should be noted that, except for the set of obelisk symbols (e.g., PK488-PK606), symbols that have the same shape on different cells are functionally related and are likely to be structurally related as well, but direct evidence to support this proposal is sketchy. For example, the carbohydrate receptors shown as rectangles (receptors are symbols without stems, whereas complementary adhesins are symbols with stems [i.e., heat and protease inactivated]) on streptococcal strains 34, J22, and PK509 all mediated lactose-inhibitable interactions with A. naeslundii PK606 which were detectable when the cocci were heat or protease treated (Table 2). A polysaccharide was isolated from strain 34, and a structurally related one was isolated from strain J22. They consisted of repeating hexasaccharide or heptasaccharide units joined together by phosphodiester bridges (1, 16). Both were composed of the same sugars, N-acetylgalactosamine, galactose, rhamnose,

S. GORDONII ADHESIN

VOL. 58, 1990

PK1878

FIG. 7. Diagrammatic representation of the coaggregations between A. naeslundii PK606 or its Cog- mutants, PK1869, PK1878,

and PK1884, and the reference strains of streptococcal tion groups 1 to 6.

coaggrega-

and glucose, with only a slight rearrangement of the sugar sequence. They were probably related to the receptor depicted on strain PK509, since strains J22 and PK509 were simultaneously unable to exhibit lactose-inhibitable coaggregation with the Cog- mutants PK1869 and PK1878 (Table 3). Furthermore, both of these actinomyces mutants were selected with a different coccal partner strain, C104, which exhibited coaggregation properties identical to those of strains J22 and PK509 (Table 3). Thus, the four streptococci, 34, C104, J22, and PK509, possess functionally related receptors involved in lactose-sensitive coaggregations, but direct evidence of structural relatedness is incomplete. The second kind of lactose-sensitive interaction depicted here involves a receptor (rectangle) on the actinomyces and its complementary lectin on the streptococcal strains DL1 and 34. Prior to this study, this kind of lactose-inhibitable coaggregation was only shown with coaggregation group C actinomyces (6, 7) because, with coaggregation group D actinomyces, it was masked by the lactose-noninhibitable interactions mediated by the components represented by obelisks. When Cog- mutants PK1878 and PK1884 were selected, the second kind of lactose-inhibitable interaction became detectable by a standard coaggregation assay and exhibited coaggregation properties identical to those found with group C actinomyces (6, 7). The loss of the receptor represented by obelisks on the actinomyces was obtained either directly by mutation from the parent, as with PK1884, or indirectly through Cogmutant PK1869, as with PK1878, by selecting an actinomyces that was unable to coaggregate with PK488. Both PK1884 and PK1878 were simultaneously altered in their ability to coaggregate with the other streptococci, except for strain Hi, which possesses additional lactose-insensitive mechanisms (semicircle and M complementary symbols) of coaggregation with the actinomyces (8). On the basis of the results of the standard visual coaggregation assay, the same lactose-insensitive mechanisms on S. oralis Hi appear to mask detection of the adhesin, represented by an obelisk, that mediates the recognition of the actinomyces receptor represented by an obelisk. We did not expect to find the 38-kDa protein in preparations of S. oralis Hi (Fig. 6, lane 5), since this strain does not exhibit altered coaggregation with A. naeslundii PK606 in the presence of antiserum, nor is it agglutinated by the anti-PK488 serum. We have not added the obelisk symbol to the Hi surface in this model,

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because we have not been able to detect the coaggregation function independently of the other coaggregation mediators. A study of Cog- mutants of S. oralis Hi will be necessary to delineate the components mediating this interaction. The presence of a related surface component on the other reference streptococci is dramatically demonstrated by the ability of Cog- mutant-absorbed anti-PK488 serum to block coaggregation between the other cocci and their actinomyces coaggregation partners (Table 4). This same antiserum does not block coaggregation between PK488 and its streptococcal coaggregation partners (Kolenbrander et al., unpublished data). Thus, the ability of an antiserum against one streptococcal strain, PK488, to block specifically coaggregation between other cocci and their common actinomyces partner indicates that the proposed streptococcal adhesins represented by obelisks are functionally similar. The size of the adhesin determined by denaturating gel electrophoresis appears to be about 38 kDa in all the streptococci (Fig. 6). All of the cocci express this immunoreactive protein, and all except strain Hi, discussed above, have altered properties of coaggregation with actinomyces after preincubation with Cog- mutant PK1804-absorbed anti-PK488 serum. We propose that the 38-kDa protein is a streptococcal adhesin that mediates coaggregation with group D actinomyces and that a functionally related adhesin is present on streptococci of all coaggregation groups. ACKNOWLEDGMENTS We thank C. Roseberry for isolating Cog- mutant A. naeslundii PK1884. We thank J. Donkersloot, N. Ganeshkumar, C. Hughes, J. London, and S. Robrish for helpful comments on the manuscript. LITERATURE CITED 1. Abeygunawardana, C., C. A. Bush, and J. 0. Cisar. 1990. Complete structure of the polysaccharide from Streptococcus sanguis J22. Biochemistry 29:234-248. 2. Batteiger, B., W. J. Newhall, and R. B. Jones. 1982. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J. Immunol. Methods 55:297-307. 3. Cisar, J. O., P. E. Kolenbrander, and F. C. McIntire. 1979. The specificity of coaggregation reactions between human oral streptococci and strains of Actinomyces viscosus or Actinomyces naeslundii. Infect. Immun. 24:742-752. 4. King, J., and U. K. Laemmli. 1971. Polypeptides of the tail fibers of bacteriophage T4. J. Mol. Biol. 62:465-477. 5. Kolenbrander, P. E. 1982. Isolation and characterization of coaggregation-defective mutants of Actinomyces viscosus, Actinomyces naeslundii, and Streptococcus sanguis. Infect. Immun.

37:1200-1208.

6. Kolenbrander, P. E. 1988. Intergeneric coaggregation among human oral bacteria and ecology of dental plaque. Annu. Rev.

Microbiol. 42:627-656.

recognition among oral bacteria: multigeneric coaggregations and their mediators. Crit. Rev. Microbiol. 17:137-159. 8. Kolenbrander, P. E., and R. N. Andersen. 1985. Use of coaggregation-defective mutants to study the relationship of cell-tocell interactions and oral microbial ecology, p. 164-171. In S. E. Mergenhagen and B. Rosan (ed.), Molecular basis of oral microbial adhesion. American Society for Microbiology, Washington, D.C. 9. Kolenbrander, P. E., and R. N. Andersen. 1986. Multigeneric aggregations among oral bacteria: a network of cell-to-cell interactions. J. Bacteriol. 168:851-859. 10. Kolenbrander, P. E., and R. N. Andersen. 1989. Inhibition of coaggregation between Fusobacterium nucleatum and Porphyromonas (Bacteroides) gingivalis by lactose and related sugars. 7. Kolenbrander, P. E. 1989. Surface

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Infect. Immun. 57:3204-3209. 11. Kolenbrander, P. E., Y. Inouye, and L. V. Holdeman. 1983. New Actinomyces and Streptococcus coaggregation groups among human oral isolates from the same site. Infect. Immun. 41:501506. 12. Kolenbrander, P. E., and B. L. Williams. 1981. Lactose-reversible coaggregation between oral actinomycetes and Streptococcus sanguis. Infect. Immun. 33:95-102. 13. Kolenbrander, P. E., and B. L. Williams. 1983. Prevalence of viridans streptococci exhibiting lactose-inhibitable coaggregation with oral actinomycetes. Infect. Immun. 41:449-452. 14. Lamont, R. J., B. Rosan, G. M. Murphy, and C. T. Baker. 1988. Streptococcus sanguis surface antigens and their interactions with saliva. Infect. Immun. 56:64-70. 14a.London, J., and J. Allen. 1990. Purification and characterization of a Bacteroides loeschei adhesin that interacts with procaryotic and eucaryotic cells. J. Bacteriol. 172:2527-2534. 15. Maryanski, J. H., and C. L. Wittenberger. 1975. Mannitol transport in Streptococcus mutans. J. Bacteriol. 124:1475-1481. 16. McIntire, F. C., L. K. Crosby, A. E. Vatter, J. 0. Cisar, M. R. McNeil, C. A. Bush, S. S. Tjoa, and P. V. Fennessey. 1988. A

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polysaccharide from Streptococcus sanguis 34 that inhibits coaggregation of S. sanguis 34 with Actinomyces viscosus T14V. J Bacteriol. 170:2229-2235. 17. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 18. Weiss, E. I., P. E. Kolenbrander, J. London, A. R. Hand, and R. N. Andersen. 1987. Fimbria-associated proteins of Bacteroides loescheii PK1295 mediate intergeneric coaggregations. J. Bacteriol. 169:4215-4222. 19. Weiss, E. I., J. London, P. E. Kolenbrander, R. N. Andersen, C. Fischler, and R. P. Siraganian. 1988. Characterization of monoclonal antibodies to fimbria-associated adhesins of Bacteroides loescheii PK1295. Infect. Immun. 56:219-224. 20. Weiss, E. I., J. London, P. E. Kolenbrander, A. R. Hand, and R. Siraganian. 1988. Localization and enumeration of fimbriaassociated adhesins of Bacteroides loescheii. J. Bacteriol. 170: 1123-1128. 21. Williams, C. A., and M. W. Chase (ed.). 1967. Purification of antibody. Methods Immunol. Immunochem. 1:321-326.