Specificity of Coaggregation Reactions Between Human Oral Streptococci and Strains of Actinomyces viscosus or Actinomyces naeslundii

Transcription

1 INFECTION AND IMMUNITY, June 1979, p /79/ /11$02.00/0 Vol. 24, No. 3 Specificity of Coaggregation Reactions Between Human Oral Streptococci and Strains of Actinomyces viscosus or Actinomyces naeslundii JOHN 0. CISARI* PAUL E. KOLENBRANDER,' AND FLOYD C. McINTIRE2 Laboratory of Microbiology and Immunology, National Institute of Dental Research, Bethesda, Maryland 20205,1 and The Department of Oral Biology, School of Dentistry, University of Colorado Medical Center, Denver, Colorado Received for publication 20 March 1979 Coaggregation reactions between actinomycete and streptococcal cells occurred frequently when human strains of Actinomyces viscosus or A. naeslundii were mixed with human isolates of Streptococcus sanguis or S. mitis, but were infrequent with other oral actinomycetes and streptococci. Two groups of actinomycetes and four groups of streptococci were defined by the patterns of their coaggregation reactions and by the ability of fl-linked galactosides (i.e., lactose) to reverse these reactions. Coaggregations occurred by one of the following three kinds of cell-cell interactions: (i) coaggregation that was blocked by heating the streptococcus but not the actinomycete and was not reversed by lactose; (ii) coaggregation that was blocked by heating the actinomycete but not the streptococcus and was reversed by lactose; and (iii) coaggregation that was blocked only by heating both cell types. The latter reaction was a combination of the first two since lactose reversed coaggregation between heated streptococci and unheated actinomycetes but did not reverse coaggregations between unheated streptococci and heated actinomycetes. Cells that could be heat inactivated also were inactivated by amino group acetylation or protease digestion, whereas cells that were unaffected by heat were not inactivated by these treatments. Coaggregation reactions of each kind were Ca2" dependent and insensitive to dextranase treatment. These findings are consistent with the hypothesis that human strains of A. viscosus and A. naeslundii coaggregate with strains of S. sanguis and S. mitis by a system of specific cell surface interactions between protein or glycoprotein receptors on one cell type and carbohydrates on the other type. Actinomyces viscosus and A. naeslundii occur in human dental plaque and may contribute to various disease states including root surface caries, periodontitis, and actinomycotic lesions (2, 23). Many isolates or oral streptococci belong to the species Streptococcus sanguis and S. mitis (7, 11, 22), and interest in these has come in part from their possible role as early colonizers of the tooth surface (5, 24-26) and from their involvement in bacteremias and bacterial endocarditis (7, 12, 13). Gibbons and colleagues (8, 9) advanced the idea that specific adhesion between different bacterial species contributed to the development of dental plaque. Supporting this concept was the demonstration of coaggregation between paired oral bacteria of different species, for example, strains of A. viscosus and A. naeslundii with S. sanguis (8). Other studies on the nature of these interactions have revealed: (i) a dextranbinding receptor on a hamster strain of A. viscosus apparently mediating coaggregation with sucrose-grown but not glucose-grown isolates of S. sanguis and S. mutans (1); (ii) strain-specific patterns of coaggregation reactions between three human isolates of A. naeslundii and glucose-grown isolates of S. sanguis and S. mitis (6); and (iii) that exposure to heat or proteolytic enzymes reduced the ability of actinomycetes but not streptococci to react with untreated cells of the other type (1, 6). The work of McIntire and colleagues (19) has focused attention on lectin-carbohydrate interactions as the possible mechanism of coaggregation between A. viscosus T14V and glucose-grown S. sanguis 34. Coaggregation required calcium ions, was inhibited by lactose and other fl-linked galacto- 742 sides, and appeared to involve the binding of protein or glycoprotein receptors on the actinomycete with carbohydrate sites on the streptococcus. This communication describes results ob-

2 VOL. 24, 1979 tained from screening several strains of A. viscosus and A. naeslundii, along with certain other species of actinomycetes, for their abilities to coaggregate with a number of human isolates of S. sanguis, S. mitis, S. salivarius, and S. mutans. In addition, the effects of lactose and chelating agents on coaggregation and the susceptibility of each cell type to heat and other treatments were determined in many coaggregation pairs. The findings of this study are consistent with the hypothesis that human strains of A. viscosus and A. naeslundii coaggregate with human strains of S. sanguis and S. mitis by a system of specific cell surface interactions between protein or glycoprotein receptors on one cell type and carbohydrates on the other type. COAGGREGATION BETWEEN ORAL BACTERIA 743 MATERIALS AND METHODS Bacterial strains. The bacterial strain designation and source of each strain used in this study are listed in Tables 1 and 2. Conditions of bacterial growth and coaggregation assays. Coaggregation was studied by two independent procedures which yielded comparable results. The first procedure employed a visual assay for coaggregation. Strains ofactinomyces and Streptococcus were grown in a complex medium (hereafter referred to as the complex medium) containing tryptone, yeast extract, Tween 80, and glucose (0.2%) buffered to ph 7.5 with K2HPO4 (16). Cultures were incubated at 37 C without shaking in screw-capped test tubes or bottles and harvested during the mid-exponential phase of growth (about 100 to 130 Klett units) as determined with a Klett-Summerson colorimeter fitted with a red filter (660 nm). The ph of cultures at the time of harvest varied from 6 to 7. Harvested cells were prepared for coaggregation assays by three cycles of centrifugation (10,000 x g for 10 min at 4 C) and suspension of the pellet in coaggregation buffer which consisted of the following (dissolved in M tris(hydroxymethyl)aminomethane adjusted to ph 8.0): CaCl2 (1 x 10-4 M), MgCl2 (1 x 10-4 M), NaN3 (0.02%), and NaCl (0.15 M). Cells either were used immediately or were stored in the same buffer at 4 C; after 2 months at 4 C, stored cells coaggregated in the same fashion as freshly harvested cells. Bacterial cell suspensions were adjusted to a turbidity of 260 Klett units (equivalent to an optical density of 2.0 at 660 nm using a Gilford model spectrophotometer and contained about 10'0 cells per ml). Equal volumes (0.2 ml) of each cell suspension were mixed for at least 10 s on a Vortex mixer, allowed to stand at room temperature for 1 to 2 h, mixed again, and scored for coaggregation. The tubes were allowed to stand at room temperature overnight, mixed the next day for at least TABLE 1. Strains ofactinomyces species Strain Other identification Culture source Host origin A. viscosus Keyes Human MG1 A. L. Delisle Human W1557 CDC Human R28 Rat X602 Hamster T WVU 745 Hamster A828 ATCC CDC Hamster W1628 CDC Human T14AV B. Hammond Human T14V B. Hammond Human M100 B. Hammond Human 5-5S Human AlB1 S. Socransky Human A. naeslundii WVU820 M. A. Gerencser Human WVU45 ATCC M. A. Gerencser Human W826 ATCC CDC Human W1527 CDC Human WVU398 A M. A. Gerencser Human W1544 CDC Human W752 CDC Human W1096 ATCC CDC Human I S. Socransky Human A. israelii W1011 ATCC M. A. Gerencser Human WVU307 ATCC M. A. Gerencser Human A. parabifidus Bifidobacterium bifidum Human A. odontolyticus ATCC B. Williams Human a Abbreviations:, National Institute of Dental Research, Bethesda, Md.; CDC, Center for Disease Control, Atlanta, Ga.

3 744 CISAR, KOLENBRANDER, AND McINTIRE TABLE 2. Strains of Streptococcus species Strain a Biotype Serotvpeb Culture sources S. sanguis M5 DL1 (Challis) D105 G102 C Kl K4 Hl J22 B2 C (ATOC) S. mitis 9811 (ATCC) S. mutans I I I I B. Rosan R. Cole B. Rosan R. Gibbons B. Rosan AHT a E49 a BHT b FAl b (NCTC) c LM7 e g S. salivarius A5 B101 H120 Ii a Based upon the classification outlined by Facklam (7). b Based upon the classification reported by Bratthall (4) and Perch et al. (21). Abbreviation:, National Institute of Dental Research, Bethesda, Md. 10 s, and scored again. Tubes containing each cell suspension alone (0.2 ml) plus 0.2 ml of buffer were included as controls. Scores for the degree of coaggregation ranged from "zero" to "four plus" and were assigned by the following criteria: zero-no visible aggregates in the cell suspension; plus one-small uniform coaggregates in suspension; plus two-definite coaggregates easily seen but suspension remained turbid without immediate settling of coaggregates; plus three-large coaggregates which settled rapidly leaving some turbidity in the supernatant fluid; plus fourclear supernatant fluid and large coaggregates which settled immediately. The second procedure employed a spectrophotometric assay to determine the percentage of coaggregation between representative bacterial pairs (19). Actinomycetes were grown in a medium containing (per liter of distilled water): NaCl, 5 g; K2HPO4, 2.5 g; the dialyzable portion of 17 g of Trypticase (BBL, Cockeysville, Md.) and 4 g of yeast extract (BBL, Cockeysville, Md.). Streptococci were grown in the same medium with the addition of glucose at a final concentration of 0.5%. Cultures were grown under an atmos- INFECT. IMMUN. phere of nitrogen and harvested when the optical density at 650 nm reached 1.0 to 1.5. The buffer used to suspend cells after collection by centrifugation and for coaggregation studies was M potassium phosphate (ph 8.0) containing M NaCl. Washed cells were used either immediately for coaggregation or stored at -20'C in 50% glycerol. The determination of percentage of coaggregation has been outlined in detail (19). Bacterial cells of a coaggregation pair were mixed, allowed to stand at room temperature for 10 min, mixed again, allowed to stand for 2 h, and centrifuged at about 7 x g for 2 min. The supernatants were analyzed spectrophotometrically at 650 nm, and the percent of coaggregation was calculated from these values along with readings of absorbance at 650 nm of controls containing each individual cell suspension alone. Assays for the effects of sugars and chelating agents on coaggregation. In the visual assay procedure, the ability of sugars and ethylenediaminetetraacetic acid to reverse coaggregation was determined after overnight incubation and scoring. To the suspension (0.4 ml) of coaggregated bacteria was added either 1 M lactose, 1 M methyl a-d-galactoside, 1 M methyl /?-D galactoside, or 0.01 M ethylenediaminetetraacetic acid (adjusted to ph 7.6 with sodium hydroxide) to give final concentrations of 0.06 M for the sugars and 0.6 mm ethylenediaminetetraacetic acid. Reversal of coaggregation was judged by the complete disappearance of bacterial aggregates. The spectrophotometric procedure described previously (19) was used to determine the percentage of inhibition of coaggregation by lactose at various concentrations and by 0.1 mm magnesium ethyleneglycolbis (8-aminoethyl ether) NN'-tetraacetic acid. These substances were added to assay tubes prior to the addition of either bacterial suspension. Heat treatment of cells. Actinomycetes and streptococci at cell densities of about 1010 cells/ml in coaggregation buffer were heated separately at 850C for 30 min and then cooled in a water bath at room temperature. Coaggregation assays were performed by the visual method using heated and unheated cells of each type. Similar experiments also were performed by heating cells at 950C for 6 min and measuring coaggregation by the spectrophotometric method (19). Amino group acetylation of cells. Bacterial cells were reacted with N-acetyl succinimide as described previously (3, 19). This treatment was performed twice on each cell suspension before visual assays for coaggregation. Enzymatic treatment of cells. Selected actinomycetes and streptococci were grown in the complex medium plus 0.2% glucose, harvested at mid-exponential growth, washed by repeated centrifugation and suspension in 0.1 M potassium phosphate buffer (ph 6.0), containing 1.0 mm CaCl2, and adjusted to a density of about 1010 cells/ml. Dextranase (D-105, Sigma Chemical Co., St. Louis, Mo.) was added at a final concentration of 0.5 mg/ml, and the suspension was incubated at 370C for 60 min with intermittent agitation. The dextranase-treated cells were washed by repeated centrifugation in coaggregation buffer, and tested in the visual assay for their ability to coaggregate.

4 VOL. 24, 1979 RESULTS Coaggregations between human strains of A. viscosus or A. naeslundii and S. sanguis or S. miti. Many isolates of A. viscosus and A. naeslundii coaggregated with a number of S. sanguis and S. mitis strains, and about half of these interactions were reversed completely by lactose (Table 3). Two groups of actinomycetes (groups A and B) and four groups of streptococci (groups 1, 2, 3, and 4) were recognized by the patterns of their coaggregation reactions and by the ability or inability of lactose to reverse these interactions. The group 1 streptococci contained four strains of S. sanguis I and two strains of S. sanguis. These six streptococcal strains coaggregated with nine strains of A. viscosus and four strains ofa. naeslundii (group A) but failed to react with five strains ofa. naeslundii (group COAGGREGATION BETWEEN ORAL BACTERIA 745 Streptococci and actinomycetes selected for protease digestion were grown as described above for between the streptococci in group 1 and the B). Lactose did not reverse any coaggregations dextranase treatment. Protease treatments were performed on cells in coaggregation buffer at a density of tions developed gradually over a period of 2 h actinomycetes in group A. Many of these reac- 10'0 cells/ml. Protease from Streptomyces griseus (P- 5130, Sigma Chemical Co., St. Louis, Mo.) at 20 mg of and were promoted by repeated mixing of the enzyme per ml in coaggregation buffer was self digested at 370C for 2 h followed by heating at 80'C for The group 2 streptococci were represented by bacterial pairs. 5 min. This solution was added to the cell suspensions a single strain (S. sanguis H1) which was unique to give a final enzyme concentration of 2 mg/ml, and in that it coaggregated with each of the actinomycetes in both groups A and B, and none of digestion of the cells was conducted at 50'C. The, effect of the elevated temperature (500C) on coaggregation of the cells was tested using the undigested The group 3 streptococci contained two these reactions was reversed by lactose. control cells. None of the strains exhibited altered strains of S. sanguis I and three strains of S. coaggregation after 60 min at 50'C, and only S. sanguis M5 showed reduced coaggregation after 2 h. sanguis. These coaggregated to varying extents with the actinomycetes in group A and Therefore, strain M5 was treated with protease for 60 min while the remainder of the strains were incubated strongly with those in group B. All coaggregations with group 3 streptococci were reversed for 120 min. The treated cells were washed by centrifugation to remove protease and tested by the visual completely by lactose and methyl,f-d-galactoside but not by methyl a-d-galactoside. This assay for coaggregation. Bacterial agglutination assays with dextran ability of lactose to reverse all coaggregations and levan. Agglutination assays were employed to distinguished the group 3 streptococci from the detect receptors for dextran or levan on actinomycete other groups. A. viscosus T14AV, an avirulent and streptococcal cells. Bacteria grown in the complex medium plus 0.2% glucose were adjusted to a cell mutant of the T14V strain, was shown previously (19) to lack coaggregation activity with S. density of about 1010 cells/ml in coaggregation buffer. Threefold serial dilutions of Dextran 2000 (Sigma sanguis 34, a member of the present group 3 Chemical Co., St. Louis, Mo.) and a high-molecularweight, branched levan were prepared in coaggrega- coaggregate with the other streptococci in group streptococci. The T14AV strain also failed to tion buffer and covered a range of concentrations from 3, but reacted with all streptococci in the other 0.8 pg/ml to 500,ug/ml. The levan was kindly provided groups. by Michael Pabst, National Jewish Hospital, Denver, The group 4 streptococci contained two Colo., and was prepared by incubating sucrose with a strains of S. sanguis and one strain of S. mitis. small amount of purified levansucrase from A. viscosus T14V (20). Agglutination assays were performed in Although these streptococci coaggregated round-bottom microtiter plates, and individual wells strongly with all the actinomycetes in groups A contained one drop (25 Ad) of the polysaccharide solution or buffer and one drop (25,l) of the bacterial and B, the ability of lactose to reverse these suspension. The plates were shaken on a rotary microshaker for 30 min at room temperature and then examined under a dissecting microscope for the presence of agglutinated bacterial cells. reactions differentiated group A from group B. Coaggregations with group A actinomycetes were not lactose reversible, but those with group B strains were reversed by lactose and methyl fl-d-galactoside but not by methyl a-d-galactoside. Comparison of the visual and spectrophotometric methods of determining coaggregation. Along with the experiments summarized in Table 3, coaggregation between representatives of the various groups and the inhibition of these reactions by lactose were measured spectrophotometrically (19). A comparison of the two sets of data revealed the following: (i) coaggregation reactions which were scored as 2 plus or greater yielded values of approximately 40% coaggregation or greater when measured spectrophotometrically whereas bacterial pairs which gave no visible coaggregation (i.e., group 1 streptococci with group B actinomycetes) gave zero or a negligible percent of coaggregation; and (ii) coaggregation reactions which were reversed completely by 0.06 M lactose were inhibited 90% or greater by 0.10 M lactose, whereas reactions which appeared to be nonreversible by lactose

5 746 CISAR, KOLENBRANDER, AND McINTIRE INFECT. IMMUN. TABLE 3. Actinomycetes Group A A. viscosus MG1 Coaggregation reactions involving human strains of A. viscosus or A. naeslundii with human strains of S. sanguis or S. mitis and the reversal of these reactions by lactose W1557 W1628 T14V T14AV M100 Keyes 5-5S AlB1 A. naeslundii W1527 Group B W1544 WVU820 W752 A. naeslundii WVU45 W82 WVU398 W1096 S. sanguis Group 1 Streptococci Group 2 Group 3 S. sanguis 11 S. sanguis 11 S. sanguis S. sanguis 11 M5 DL1 D105 G102 Kl K4 Hi C C J22 B b O 2 O O 3 O O 3 O 2 Coaggregation Scorea O o o a0 for no coaggregation to 4 for maximum coaggregation. b Coaggregation not reversed by 0.06 M lactose and blocked by heating (85 C for 30 min) the streptococcus but not the actinomycete. Coaggregation reversed completely by 0.06 M lactose and blocked by heating (85 C for 30 min) the actinomycete but not the streptococcus. d Coaggregation not reversed by 0.06 M lactose. All coaggregations except those with A. viscosus T14AV (see text) were blocked only by heating (850C for 30 min) both the actinomycete and the streptococcus. were either not inhibited or were inhibited only heated actinomycetes (Table 4). partially (20 to 60%) by lactose at concentrations In every coaggregation where lactose caused up to 0.1 M. Of the coaggregations in Table 3 complete reversal, the reaction was blocked by which were scored as being lactose reversible, heating the actinomycete but not the streptomany were inhibited more than 90% by 0.01 M coccus (Table 3). Thus, heated cells of A. vislactose (a relatively low concentration). cosus W1628 (group A) and unheated cells of S. Effect of heat on the ability of cells to sanguis (group 3) did not interact, but coaggregate. All coaggregations with group 1 lactose-reversible coaggregation occurred beand group 2 streptococci in Table 3 were pre- tween unheated actinomycete cells and heated vented by heating (850C for 30 min) the strep- streptococcal cells (Table 4). Likewise, this pattococcal cells but not the actinomycete cells. For tern was observed for the interaction of A. naesexample, coaggregation did not occur when lundii W826 (group B) with S. sanguis heated cells of S. sanguis DL1 (group 1) were (group 3) or S. mitis 9811 (group 4) (Table 4). mixed with unheated cells of A. viscosus W1628 Coaggregations between group 4 streptococci (group A), but occurred between unheated strep- and group A actinomycetes were abolished when tococci and heated actinomycetes (Table 4). both cell types were heated but not when only Similarly, heated S. sanguis H1 cells (group 2) one cell type was heated. This is shown by the did not coaggregate with A. viscosus W1628 coaggregation reactions with heated and un- (group A) or A. naeslundii W826 (group B), but heated cells of A. viscosus W1628 and S. sanguis the unheated streptococci reacted with the 9811 (Table 4). Significantly, lactose reversed Group 4 S. sanguis 11 S. mitis d

6 VOL. 24, 1979 coaggregation between unheated actinomycete cells and heated streptococcal cells but failed to reverse the converse reaction of unheated streptococci with heated actinomycetes. A. viscosus T14AV differed from the other group A actinomycetes in its interactions with the group 4 streptococci (data not shown). Unheated T14AV cells did not react with heated group 4 streptococci although heated T14AV cells coaggregated with unheated streptococci. Thus, A. viscosus T14AV, unlike the other group A strains, failed to give lactose-reversible coaggregation with heated streptococcal cells from group 4. This difference was consistent with the failure of T14AV cells to react with group 3 streptococci (Table 3). Heat inactivation experiments like those presented in Table 4 were performed with each coaggregation pair listed in Table 3, and, with the exception ofa. viscosus T14AV, all members within each group reacted alike. In addition, concordant results were obtained from spectrophotometric assays with representative members of each group. Thus, three kinds of cell-cell interactions were identified: (i) coaggregation that was blocked by heating the streptococcus but not the actinomycete and was not reversed by lactose; (ii) coaggregation that was blocked by heating the actinomycete but not the streptococcus and was reversed by lactose; and (iii) coaggregation that was a combination of the first two kinds and was blocked only by heating both cell types. Effect of amino group acetylation and protease digestion on the ability of cells to coaggregate. In coaggregations between representative group 1 streptococci and group A actinomycetes, the streptococcal cells were inactivated by amino group acetylation (Table 5) COAGGREGATION BETWEEN ORAL BACTERIA 747 and by protease digestion (Table 6), whereas treatment of the actinomycete cells did not affect their reactions with untreated streptococci. Likewise, protease digestion abolished the reactivity of S. sanguis H1 (group 2) with untreated actinomycetes from group A and group B but did not affect the actinomycetes in their coaggregations with untreated S. sanguis H1 (Table 6). Aminoacetylation of S. sanguis H1 caused a significant decrease in its coaggregation with A. naeslundii W752 but not with the other actinomycetes studied (Table 5). These results from aminoacetylation of S. sanguis H1 suggested that its coaggregations with different actinomycetes may be mediated by various mechanisms. In coaggregations which were reversed completely by lactose (i.e., group A or B actinomycetes with group 3 streptococci and group B actinomycetes with the group 4 streptococcus), aminoacetylation (Table 5) and protease digestion (Table 6) of actinomycetes eliminated or reduced coaggregations with untreated streptococci. Similar treatment of the streptococci did not alter their reactions with untreated actinomycetes. Coaggregations between S. sanguis J22 (group 4) and the group A actinomycetes were abolished or greatly reduced only when both cell types were aminoacetylated (Table 5) or digested with protease (Table 6). Coaggregations between untreated actinomycetes and treated streptococci were reversed completely or almost completely by lactose, but lactose reversal was not observed when untreated streptococci coaggregated with treated actinomycetes. Thus, cells that could be heated inactivated also were inactivated by protease digestion and in most cases by amino group acetylation, whereas cells which were unaffected by heat TABLE 4. Coaggregation reactions with heat-treated and untreated cells of representative actinomycetes and streptococci Coaggregation score' with streptococci: Actinomycetes Coaggregation mixture Group I Group 2 Group 3 Group 4 (DL1)' (HI) (15914) (9811) Group A: A. viscosus W1628 A(t) + S(c) A(c) + S(t) 0 0 4* 4* A(t) + S(t) A(c) + S(c) 3 4 4* 4 Group B: A. naeslundii W826 A(t) + S(c) A(c) + S(t) 0 0 4* 4* A(t) + S(t) A(c) + S(c) 0 2 4* 4* Abbreviations: A, actinomycete; S, streptococcus; (t), treated cells (85"C for 30 min); (c), control cells. 'Score: 0 for no coaggregation to 4 for maximum coaggregation; asterisk indicates that coaggregation was reversed completely by 0.06 M lactose. 'Strain number in parentheses.

7 748 CISAR, KOLENBRANDER, AND McINTIRE TABLE 5. Coaggregation reactions with N-acetyl succinimide-treated and untreated cells of representative actinomycetes and streptococci Coaggregation scored for streptococci: Actinomycetes Coaggregation mixture' Group 1 Group 2 Group 3 Group 3 Group 4 (M5)c (Hi) (34) (C104) (J22) Group A A. naeslundii W752 A(t) + S(c) A(c) + S(t) 0 1 4* 1* 4* A(t) + S(t) A(c) + S(c) 2 3 4* 1* 4 A. viscosus T14V A(t) + S(c) A(c) + S(t) 0 3 4* 1* 4d A(t) + S(t) A(c) + S(c) 3 4 4* 1* 4 Group B A. naeslundii WVU45 A(t) + S(c) 0 0 1* 0 0 A(c) + S(t) 0 1 4* 4* 4* A(t) + S(t) A(c) + S(c) 0 2 4* 4* 4* A. naeslundii W826 A(t) + S(c) 0 0 2* 0 1* A(c) + S(t) 0 1 4* 4* 4* A(t) + S(t) 0 0 1* 0 1* A(c) + S(c) 0 2 4* 4* 4* a Abbreviations: A, actinomycete; S, streptococcus; (t), treated cells (amino group acetylation with N-acetyl succinimide); (c), control cells. b Score: 0 for no coaggregation to 4 for maximum coaggregation; asterisk indicates that coaggregation was reversed completely by 0.06 M lactose. 'Strain numbers are in parentheses. d Coaggregation reversed partially by lactose. were not inactivated by these treatments. These results on the mechanism of various coaggregations are consistent with cell surface interactions which require proteins or glycoproteins on one cell type and carbohydrate on the other. Effect of Ca2" on coaggregation. All coaggregations given in Table 3 were reversed by ethylenediaminetetraacetic acid, and all of those studied by the spectrophotometric method were inhibited 100% by 0.1 mm magnesium ethyleneglycol-bis (,B-aminoethyl ether) N,N'-tetraacetic acid. These findings indicate an essential role for Ca2". An especially interesting example of the function of Ca2" is its enhancement of coaggregation at ph 4.5 as shown by the data in Table 7. The percent of coaggregation at ph 4.5 was less than that at ph 8.0 in every instance and was greatly decreased in 8 of the 10 interactions shown. The addition of Ca2" (1 mm) always increased the coaggregation, and in most cases the coaggregation at ph 4.5 with 1 mm Ca2" was equal or nearly equal to the coaggregation at ph 8.0. Thus, in the presence of the amount of Ca2" found in saliva and in dental plaque, coaggregation of actinomycetes with streptococci proceeds INFECT. IMMUN. readily even at the low ph values often recorded in plaque. Effect of cell age on the ability of cells to coaggregate. In an earlier report, A. viscosus T14V cells cultured under different conditions varied in their ability to coaggregate with S. sanguis 34 cells (19). These results prompted an investigation of the effect of physiological cell age on the coaggregation reactions between actinomycetes and streptococci from each of the coaggregation groups. Strains of Streptococcus (strains M5, H1, J22, 34, and C104), A. viscosus (strains MG1 and T14V), and A. naeslundii (strains W1544, WVU820, W752, W1096, and I) were inoculated into the complex medium containing 0.2% glucose and harvested at various points in the growth cycle. The first sample was taken at the early exponential phase of growth, and the final sample was harvested 48 h after each culture reached stationary phase. Actinomycete cells grown to the mid-exponential phase were used to test the coaggregation ability of streptococcal cells harvested at various cell ages, and streptococcal cells from the mid-exponential phase were

8 VOL. 24, 1979 TABLE 6. COAGGREGATION BETWEEN ORAL BACTERIA 749 Coaggregation reactions with protease-treated and untreated cells of representative actinomycetes and streptococci Coaggregation scoreb with streptococci: Actinomycetes Coaggregation mixture Group 1 Group 1 Group 2 Group 3 Group 3 Group 4 (M5)c (DL1) (H1) (34) (C104) (J22) Group A A. viscosusw1628 A(t) + S(c) A(c) + S(t) * 1* 2* A(t) + S(t) A(c) + S(c) * 1* 4 A. viscosus T14V A(t) + S(c) A(c) + S(t) * 1* 2* A(t) + S(t) A(c) + S(c) * 1* 4 Group B A. naeslundii WVU45 A(t) + S(c) A(c) + S(t) * 4* 4* A(t) + S(t) A(c) + S(c) * 4* 4* A. naeslundii W826 A(t) + S(c) A(c) + S(t) * 4* 4* A(t) + S(t) A(c) + S(c) * 4* 4* a Abbreviations: A, actinomycete; S. streptococcus; (t), treated cells (protease digested at 500C); (c), control cells incubated at 500C. b Score: 0 for no coaggregation to 4 for maximum coaggregation; asterisk indicates that coaggregation was reversed completely by 0.06 M lactose. 'Strain numbers are in parentheses. TABLE 7. Enhancement of coaggregation at low ph by Ca2+ Coaggregation (%)' Actinomycete Streptococcus H 8 ph ph PI 4.5 Ca2+ b A. viscosus S. sanguis T14V M T14V G T14V H T14V J T14V B T14V T14V MG A. naeslundii WVU I '0.025 M potassium phosphate M sodium chloride buffer. b 1.0 mm Ca2+. used to test the reactivity of actinomycete cells taken at various phases of growth. No significant change in the coaggregations of either actinomycetes or streptococci was observed using cells of various ages, with the exception ofa. viscosus T14V cells taken 48 h into stationary phase. These exhibited a reduced ability to coaggregate with S. sanguis 34 cells but not with the other streptococci tested. Thus, with one exception, it appeared that cell age was not a critical factor in coaggregation reactions. Similar results were obtained by Gibbons and Nygaard (8) in their study of coaggregation between A. naeslundii I and S. sanguis 34. Effects of dextran, levan, and dextranase on coaggregation reactions. Small amounts of sucrose are known to be present in various complex media (10), and this could result in limited synthesis of dextran or levan by streptococci or actinomycetes. Moreover, receptors for dextran have been detected on certain streptococci (18) and various strains of A. viscosus (1, 17). Consequently, attempts were made to determine whether dextran or levan mediated any of the coaggregations described in this study. The results of various experiments showed that: (i) glucose-grown cells ofthe actinomycetes and streptococci utilized in the experiments shown in Table 3 were not agglutinated by highmolecular-weight dextran or levan at concentrations of 0.4 to 250,ug/ml, whereas certain S. mutans strains and rodent strains ofa. viscosus

9 750 CISAR, KOLENBRANDER, AND McINTIRE were agglutinated by dextran; (ii) coaggregations of six representative actinomycetes with eight representative streptococci from the various coaggregation groups were not inhibited or enhanced by the presence of Dextran 2000 at 10 mg/ml; and (iii) treatment of the eight representative streptococci with dextranase at 0.5 mg/ ml did not alter their coaggregation reactions with the six representative actinomycetes which also were dextranase treated. Thus, no evidence was obtained for the participation of dextran or levan in these coaggregation reactions. Coaggregation studies with other actinomycetes and streptococci. The streptococcal and actinomycete strains in Table 3 were studied for their ability to coaggregate with other oral actinomycetes and streptococci. When four rat or hamster strains of A. viscosus, two human strains of A. israelii, and human strains of A. odontolyticus and A. parabifidus (Table 1) were tested for coaggregation with each member of the four streptococcal groups, only A. odontolyticus and A. viscosus X602, a hamster strain, reacted with more than five streptococcal strains. The patterns of coaggregation reactions for these two actinomycetes differed from the ones displayed by group A and group B actinomycetes, and furthermore, none of the reactions were reversed by lactose. Likewise, when five strains of S. mutans (serotypes a, b, c, e, and g) and four isolates of S. salivarius (Table 2) were tested for coaggregation with 11 group A and 4 group B actinomycetes, no reactions were observed. Thus, the scheme of coaggregation reactions shown in Table 3 involved group-specific interactions between human strains of A. viscosus or A. naeslundii with S. sanguis I, S. sanguis, or S. mitis, whereas other actinomycetes and streptococci reacted differently or not at all. DISCUSSION The present findings suggest that human strains of A. viscosus and A. naeslundii coaggregate with isolates of S. sanguis and S. mitis by a system of specific but varied cell surface interactions. The interactions which account for coaggregations between the two groups of actinomycetes and the four groups of streptococci are illustrated diagramatically in Fig. 1. Three kinds of cell-cell interactions were recognized. The first involved a reaction of heat-sensitive receptors on streptococcal cells with heat-stable sites on the actinomycetes and included all coaggregations involving the group 1 and group 2 streptococcal strains. With these coaggregations, the addition of lactose did not cause complete reversal. The second type of interaction was like that described in a previous investigation (19) E 0 2 -/ {Gr Het Seniv Stieptococci 1 Hoe Sabs INFECT. IMMUN. 00 C) -< *1 fe + FIG. 1. Diagrammatic representation of the specific interactions which mediate coaggregations between the groups of actinomycetes and streptococci. The figure is not intended to imply identity of either the heat-sensitive receptors or heat-stable sites among the different groups of bacteria. and occurred between heat-stable components on streptococcal cells and heat-sensitive receptors on the actinomycetes. This was exemplified by coaggregations of group 3 streptococci with group A or B actinomycetes and by coaggregation of group 4 streptococci with group B actinomycetes; all of these reactions were reversed completely by lactose. The third kind of cell-cell interaction occurred when group 4 streptococci reacted with group A actinomycetes. With these, heating of both the streptococcal and actinomycete cells was required to abolish coaggregation. Lactose reversed the interaction of heated group 4 streptococci with unheated group A actinomycetes but did not reverse coaggregations between unheated streptococci and heated or unheated actinomycetes. Thus, the third type of interaction appeared to be a combination of the first two. The cell surface interactions depicted in Fig. 1 may be mediated by lectin-like receptors which are heat sensitive and carbohydrates which are heat stable. This hypothesis is consistent with the following observations: (i) cells which could CD c0 0

10 VOL. 24, 1979 be heat inactivated also were inactivated by protease digestion and in most cases by amino group acetylation, whereas cells which were unaffected by heat were not affected by the other treatments; (ii) coaggregation reactions, like certain lectin-carbohydrate interactions (14), were Ca2" dependent; and (iii) all coaggregations which were prevented by heating the actinomycete but not the streptococcus were reversed completely by lactose and methyl,b-d-galactoside but not by methyl a-d-galactoside. The latter finding strongly favors the presence of lectin-like receptors on the actinomycetes. However, saccharides have not yet been found which give complete reversal of those coaggregations in which heat-sensitive receptors on the streptococci react with heat stable-sites on the actinomycetes. It is unlikely that dextran mediates any of the interactions summarized in Fig. 1, because sucrose was not added to culture media; receptors for dextran were not detected on any of the actinomycetes or streptococci examined; and dextranase digestion of representative isolates did not affect their coaggregation reactions. Thus, the present results with many coaggregation pairs are consistent with and extend the findings of McIntire and co-workers (19) who proposed that coaggregation between A. viscosus T14V and S. sanguis 34, which was inhibited completely by lactose, involved the interaction of a protein or glycoprotein on the actinomycete with a carbohydrate other than dextran on the streptococcus. Further studies are needed to characterize the receptors on the actinomycetes and on the streptococci and to chemically define the components to which they bind on the other cell type. The specific coaggregation reactions investigated in the current study appeared to be limited to reactions between human isolates of A. viscosus or A. naeslundii and S. sanguis or S. mitis. Thus, of eight actinomycetes, which were not human strains of A. viscosus or A. naeslundii, only two coaggregated with a significant number of the S. sanguis and S. mitis isolates given in Table 3, and these two actinomycetes differed from the group A and group B human strains in the patterns of their coaggregation reactions. In addition, human strains of A. viscosus and A. naeslundii (groups A and B) failed to react with four isolates of S. salivarius and strains of S. mutans representing five of the serotypes. This was consistent with results from other investigations (6, 8) in which several isolates of S. salivarius and S. mutans also failed to coaggregate. The group A actinomycetes contained strains of A. viscosus and A. naeslundii, and strains COAGGREGATION BETWEEN ORAL BACTERIA 751 classified as S. sanguis I, S. sanguis, and S. mitis were distributed throughout the streptococcal coaggregation groups. The distribution of more than one bacterial species within a single coaggregation group is not surprising since species designations are generally based on physiological properties whereas coaggregation groups presumably reflect cell surface properties. A similar type of disparity between schemes of classification based on cell surface and physiological properties was evident when the serological reactions of viridans streptococci were compared to species designations assigned by the physiological classification of Facklam (7). It is interesting that strains of A. naeslundii serotype 1 (15) were confined to the group B actinomycetes, and this suggests a correlation between serological reactions and coaggregation reactions within this group of actinomycetes. Correlations between coaggregation reactions and cell surface antigens remain to be established for the actinomycetes and streptococci in other coaggregation groups. It is significant that many of the actinomycetes and streptococci studied could be included in the present scheme. Of twenty-three human strains of A. viscosus or A. naeslundii from various culture collections, 19 were placed in group A or group B; the other strains either gave variable results or appeared to be slightly different from those grouped in Table 3. Of 25 strains classified physiologically as S. sanguis or S. mitis, 8 gave no coaggregations, but 15 of the remaining 17 were placed into one of the four groups described. Although the present scheme (Fig. 1) includes the majority of coaggregations studied to date, it is likely that additional groups exist and that at least some of the present groups will be subdivided as additional information becomes available on the specificity of various coaggregation reactions and the structural components which mediate these interactions. The coaggregation reactions observed between the strains of actinomycetes and streptococci used in this study exhibit two potentially important attributes: coaggregation was not affected by the composition of media in which pairs of bacteria were cultivated, and it was independent of the physiological age of the respective microorganisms. Freedom from such physiological constraints increases the likelihood that interactions between pellicle-bound cells of S. sanguis and unbound cells of A. viscosus and A. naeslundii occur in the oral cavity at significant frequency, thereby aiding the colonization of the latter two microorganisms. This speculation is strengthened by the observation that human strains of S. sanguis

11 752 CISAR, KOLENBRANDER, AND McINTIRE and S. mitis coaggregated more frequently with human than with nonhuman strains of actinomycetes. In addition, the observation of efficient coaggregation at ph values as low as 4.5 in the presence of Ca2" ions indicates that these interactions could proceed in an acidic environment like that present in plaque. ACKNOWLEDGMENTS We thank Shelley Berg and Jean Baughman for their help during the course of this study, and Carol Oesch for her help in preparing the manuscript. This work was supported in part by Public Health Service grant 1 RO DE from the National Institute of Dental Research to F.C.M. INFECT. IMMUN. LITERATURE CITED 1. Bourgeau, G., and B. C. McBride Dextran-mediated interbacterial aggregation between dextran-synthesizing streptococci and Actinomyces viscosus. Infect. Immun. 13: Bowden, G. H., and J. M. Hardie Commensal and pathogenic Actinomyces species in man, p In G. Sykes and F. A. Skinner (ed.), Actinomycetales: characteristics and practical importance. Society for Applied Bacteriology Symposium Series No. 2. Academic Press Inc. London. 3. Boyd, H., S. J. Leach, and B. Milligan N-acylsuccinimides as acylating agents for proteins: the selective acylation of lysine residues. Int. J. Peptide Protein Res. 4: Bratthall, D Immunofluorescent identification of Streptococcus mutans. Odont. Revy 23: Carlsson, J., H. Grahnen, and G. Jonsson Lactobacilli and streptococci in the mouth of children. Carries Res. 9: Ellen, R. P., and I. B. Balcerzak-Raczkowski Interbacterial aggregation of Actinomyces naeslundii and dental plaque streptococci. J. Periodont. Res. 12: Facklam, R. R Physiological differentiation of viridans streptococci. J. Clin. Microbiol. 5: Gibbons, R. J., and M. Nygaard Interbacterial aggregation of plaque bacteria. Arch. Oral Biol. 15: Gibbons, R. J., and J. van Houte On the formation of dental plaques. J. Periodontol. 44: Hamada, S., and M. Torii Effect of sucrose in culture media on the location of glucosyltransferase on Streptococcus mutans and cell adherence to glass surfaces. Infect. Immun. 20: Hardie, J. M., and G. H. Bowden Physiological classification of oral viridans streptococci. J. Dent. Res. 55:A166-A Horstmeier, C., and J. A. Washington, Microbiological study of streptococcal bacteremia. Appl. Microbiol. 26: Kast, A Comparative statistical investigations regarding incidence, etiology and topography of subacute bacterial endocarditis. Jpn. Circ. J. 35: Us, H., and N. Sharon The biochemistry of plant lectins. Annu. Rev. Biochem. 42: Marucha, P. T., P. H. Keyes, C. L. Wittenberger, and J. London Rapid method for identification and enumeration of oral Actinomyces. Infect. Immun. 21: Maryanski, J. H., and C. L. Wittenberger Mannitol transport in Streptococcus mutans. J. Bacteriol. 124: McBride, B. C., and G. Bourgeau Dextran-induced aggregation of Actinomyces viscosus. Arch. Oral Biol. 20: McCabe, M. M., and E. E. Smith Carbohydrate receptors of oral streptococci, p In W. H. Bowen, R. J. Genco, and J. C. O'Brien (ed.), Immunologic aspects of dental caries. Special Supplement of Immunological Abstracts. Information Retrieval, Inc., Washington, D. C. 19. McIntire, F. C., A. E. Vatter, J. Baros, and J. Arnold Studies on the mechanism of coaggregation between Actinomyces viscosus T14 and Streptococcus sanguis 34. Infect. Immun. 21: Pabst, M. J Levan and levansucrase ofactinomyces viscosus. Infect. Immun. 15: Perch, B., E. Kjems, and T. Ravn Biochemical and serological properties of Streptococcus mutans from various human and animal sources. Acta Pathol. Microbiol. Scand. Sect. B 82: Rosan, B Absence of teichoic acids in certain oral streptococci. Science 201: Slack, J. M., and M. A. Gerencser (ed.) Actinomyces, filamentous bacteria: biology and pathogenicity. Burgess Publishing Co., Minneapolis. 24. Socransky, S. S., A. D. Manganiello, D. Propas, V. Oram, and J. van Houte Bacteriological studies of developing supragingival dental plaque. J. Periodont. Res. 12: Theilade, E., 0. Fejerskov, M. Prachyabraed, and M. Kilian Microbiologic study on developing plaque in human fissures. Scand. J. Dent. Res. 82: Tinanoff, N., A. Gross, and J. M. Brady Development of plaque on enamel. Parallel investigations. J. Periodont. Res. 11: