Adhesive Properties of Viridans Streptoccocal Species

MICROBIAL ECOLOGY IN HEALTH AND DISEASE VOL. 7: 125-137 (1994)

Adhesive Properties of Viridans Streptoccocal Species S. D. HSUT, J. 0. CISAR*t, A. L. SANDBERGt and M . KILIANS

TLaboratory of Microbial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892 and $Institute of Medical Microbiology, University of Aarhus, DK-8000 Aarhus C, Denmark

Received 16 April 1993; revised 10 October 1993; accepted 2 December 1993

Seventy-one strains of viridans streptococci, classified as Streptococcus sanguis, S. gordonii, S. oralis, S. mitis or S. anginosus by a revised taxonomic scheme, were characterized and compared by their specific adhesive properties. The frequency of bacterial adhesion to saliva-coated hydroxyapatite (SHA) was greater among strains of S. sanguis, S. gordonii and S. oralis than among those of S. mitis and S. anginosus. Similarly, the expression of sialic acid reactive adhesins, detected by neuraminidase sensitive bacterial haemagglutination, was noted more frequently with strains of S. sanguis (19 of 21), S. gordonii (14 of 16) and S. oralis ( 8 of 11) than those of S. mitis (2 o f 12) and S. anginosus (0 of 11). Most strains of S. gordonii (14 of 16) and S. oralis (7 of 11) also aggregated acidic proline rich protein-coated latex beads, but this activity was observed rarely with strains of S. sanguis (2 of 21), S. mitis (1 o f 12) and S. anginosus (0 of 11). Strains of S. anginosus (6 of 11) participated in lactose resistant coaggregations with actinomyces in coaggregation groups A (e.g. Actinomyces viscosus T14V-J1) and B (e.g. A. naeslundii WVU45). Lactose resistant coaggregations were also observed between strains of S. gordonii (9 of 16) and actinomyces in coaggregation group A. Lactose sensitive coaggregations occurred between actinomyces and each of 11 S. oralis strains but less frequently with strains of S. sanguis (6 o f 21), S. gordonii ( 3 of 16), S. mitis ( 3 of 12) and S. anginosus (1 o f 11). Certain streptococcal strains with receptors for the lactose sensitive lectins of actinomyces, including 9 of 11 S. oralis, also coaggregated frequently with strains of either S. sanguis (10 o f 21) or S. gordonii (9 of 16). Further studies with representatives of these latter three streptococcal species suggested that the streptococci with receptors for the GalNAc sensitive lectins of S. sanguis and S. gordonii were those with GalNAcP1+3Gal- rather than GalP 1 +3GalNAc-containing cell wall polysaccharides.

KEY woms-Streptococci; Adhesion; Coaggregation; Adhesins; Lectins; Receptors.

INTRODUCTION Recent studies of the taxonomy of viridans streptococci have provided improved criteria for distin- guishing the species of these bacteria in accordance with established genetic relationship^.^^ This has involved the use of various enzymes including IgA protease, neuraminidase and other glycosidases as characteristic traits for the identification of species.5326 As a result, certain organisms previously identified as S. sanguis have been placed in a new species, S. gordonii, and the descriptions of 5’. sun uis, S. mitis and S. oralis have been emended3 The heterogeneous ‘S. milleri group’ has also been separated into three more homoge- neous species, one of which is S. a n g i n o ~ u s . ~ ~ Ecological significance is attached to these findings

*Author to whom correspondence should be addressed: Build- ing 30, Rm 302, NIDR, NIH, Bethesda, MD 20892, USA.

CCC 0891-060X/94/030125-13 0 1994 by John Wiley & Sons, Ltd.

since different taxonomic groups presumably have evolved to fill characteristic niches within the oval environment.

Of the species considered above, all are found in mature dental plaque but only some participate in initial colonisation of the tooth surface.” Thus, S. sanguis, S. oralis and arginine-negative S. mitis (biovar 1). were recovered in greatest numbers from test squares of enamel following incubation in vivo for periods of time ranging from four to 24 h o ~ r s . ~ ~ , ~ When examined ultrastructurally after four hours, enamel squares were sparsely popu- lated by individual microorganisms attached to the acquired salivary pellicle and after eight or 12 hours, spreading microcolonies were observed.41342 Individual colonies were composed of a peripheral monolayer of dividing cocci and rods and a central region of multilayered bacteria. Most of the rod- shaped bacteria were Actitaomyces viscosus and

126

A. n a e ~ l u n d i i , ~ ~ species previously identified along with the different streptococci as primary colonisers of teeth.45 These and earlier observations of dental plaque morph~genesis~~ have equated colonisation with bacterial adhesion and growth rather than aggregation and accretion.

A number of adhesive properties have been associated with various streptoccocci that colonise teeth.,, Many of these bacteria are known to attach avidly to saliva-treated hydroxyapatite (SHA), an in vitro model for bacterial interactions with salivary components of the acquired pellicle. Such interactions involve sialic acid reactive streptococcal adhesins12~*3~’9~30~33~36.40 and other adhesins for proline rich proteins (PRP).2”32 Col- onisation may also be promoted by specific interbacterial adhesion between different streptococcal speciesz8 or between these bacteria and other early colonisers such as actin~myces.~.’,~~

While many studies of viridans streptococci have revealed molecular mechanisms of in vitro adhesion, efforts to relate specific interactions to in vivo colonisation have been limited by uncertain- ties in the classification of species. The current investigation was initiated to examine possible correlations between a number of well- documented adhesive activities and taxonomy of 7 1 streptococcal strains representing the species S. sanguis, S. gordonii, S. oralis, S. mitis and S. anginosus.

S. D. HSU ET AL.

MATERIALS AND METHODS

Bacterial strains and growth conditions The streptococcal isolates indicated by the prefix

SK have been previously described;26 S. anginosus SK215, SK216, SK218 and SK221 were isolated from subgingival dental plaque. Other strains previously are listed in Table 1. Strepto- coccal strains 10556, 10557, 10558, 6249, 9811, 15914 and 903 were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The current species designation of each streptococcal strain was made in accordance with results from recent taxonomic s t ~ d i e s . ‘ ~ , ~ ~

The actinomyces included in this study were A. viscosus T14V-J1; its mutant lacking type 1 and type 2 fimbriae, strain 147;’’ A. viscosus WVU627; A. viscosus 19246 obtained from ATCC; A. naeslundii strains W1544, I, WVU45 (ATCC 12104) and the fimbriae-deficient mutant, strain WVU45M.8 All these actinomyces are members of

Table 1. Source of the streptococci

Strain Previous Source designation (reference)

S. gordonii Biovar 1

38 Biovar 2 D105 DL1 (Challis) GI02 K1 K4 M5

S. oraiis 34 981 1 B2 C104

S. mitis Biovar 1

15914 HI 522

Biovar 2 H127 118 K103

S. sanguis I

S. sanguis I S. sanguis I S. sanguis I S. sanguis I1 S. sanguis I1 S. sanguis I

S. sanguis I1 S. miris S. sanguis I1 S. sanguis I1

S. sanguis I1 S. sanguis I1 S. sanguis I1

S. mitis S. mitis S. sanguis I1

9

9, 24 9 9, 24 9, 24 9, 24 9

9 9 9, 24 9, 24

9 9, 24 9, 24

24 24 24

A. naeslundii genospecies 2 except strain w W 4 5 which is in genospecies l.25

The bacteria used in the adhesion assays were grown overnight in complex medium containing tryptone (0.5 per cent), yeast extract (0.5 per cent), Tween 80 (0.5 per cent) glucose (0-2 per cent) and K,HPO, (0.5 per cent),’ washed and adjusted to a turbidity of 260 Klett units (approximately 2 x lo’ celldml) in Tris-buffered saline (TBS; 0.15 M NaCl, 0.02 M Tris HCl, 10 - M CaCl,, M MgCl,, 0.02 per cent NaN,, pH 7.8).

Bacteria-mediated haemagglutination Human type 0, goat, dog or guinea-pig eryth-

rocytes (RBC) from heparinised or citrated blood were washed three times with 0.02 M phosphate buffered saline, pH 7.2, containing 2 mg/ml bovine serum albumin (PBS-BSA). Where indicated, the RBC (3 per cent vol./vol.) were treated with 0.04 U/ml Type V neuraminidase (NANase) from Clostridium perfringens (Sigma Chemical Co., St Louis, MO, USA) for 2 h at 37°C with gentle

ADHESION OF VIRIDANS STREPTOCOCCI 127

mixing and washed to remove the enzyme. Two- fold serial dilutions of each bacterial cell suspension were prepared in PBS-BSA and mixed with equal volumes (25 pl) of 0.5 per cent untreated or NANase-treated RBC in round bottom wells of polyvinyl microtitration plates. The plates were incubated 30 min at room temperature and overnight at 4°C. Endpoint titres were determined microscopically from the presence of agglutinated RBC following gentle resuspension at 4°C.

Aggregation of PRP-coated latex beads Polystyrene latex beads (15.8 pm diameter, Poly-

sciences, Inc., Warrington, PA, USA) were coated by incubation in 20 pg/ml of purified acidic PRP- 1 (provided by D. Hay, Forsyth Dental Center, Boston, MA, USA) or BSA in 0.05 M carbonate buffer, pH 9.5 as previously described.,' The beads were washed and suspended to 0.5 per cent (vol./ vol.) in 1 mM phosphate, 0.05 M KC1, 1 mM CaCl,, 1 mM MgC1, (buffered KCl), pH 6.0, containing 2 mg/ml BSA. Aggregation assays were performed in flat bottom microtitration plates by mixing 100 pl of protein-coated bead suspension with 5 p1 of bacterial cell suspension (260 Klett units). Aggregation of PRP coated beads, as described previously,20 was scored from '+' (weak) to '+ + +' (strong). Bacterial aggregation of BSA- coated beads was rarely noted.

Coaggrega t ion Coaggregation was determined visually.' A

quantitative assay similar to that described previ- 0us1y~~ was also used to determine percent coaggregation between selected streptococcal strains and percent inhibition by saccharides. Saccharides assayed as inhibitors of coaggregation included GalNAcPl -3GalaOMe (Sockerbolaget, Arlov, Sweden) and Galpl -3GalNAcaOC,H,NO, (p) (Sigma). Quantitative assays were performed in TBS containing 0.05 per cent Tween 20 (Sigma).

The coaggregation group of each streptococcal strain was determined by its interactions with actinomyces from coaggregation group A ( A . viscosus strains T14V-J1, WVU627, 19246 and A. naeslundii W1544) and coaggregation group B (A . naeslundii strains WVU45 and I).' Lactose sensitive and lactose resistant coaggregations were distinguished as previously described' and con- firmed by results with fimbriae-deficient mutant strains that participate in lactose resistant but not lactose sensitive coaggregations with strepto-

Table 2. Properties of streptococcal coaggregation groups

StrePtococcal Coaggregation with actinomyces coaggregation group Group A Group B

LR LR LR LS LS LR LS

LS

-

~

LR, lactose-resistant coaggregation; LS. lactose-sensitive coaggregation; -. no coaggregation.

cocci." The characteristic Drouerties of each streD- . I

tococcal coaggregation are given in Table 2.

Bacterial udhesion to SHA This assay was performed as previously de-

scribed:" spheroidal HA beads (10 mg, Gallard- Schlesinger Chemical Corp., Carle Place, NY, USA) were equilibrated with buffered KCl (pH 7.0) for 1 h prior to overnight incubation with 150 pl of freshly collected clarified whole saliva from a single donor. Prior to use, saliva was heated for 30 rnin at 60°C centrifuged at 23,OOOg for 10 min at 4°C and sodium azide added to a final concentration of 0.04 per cent. Washed SHA and untreated HA were blocked for 1 h with 2 mg/ml BSA in buffered KCI (KCl-BSA)" and subsequent steps were performed in KC1-BSA. SHA was treated with 0.05 U/ml of Type X NANase from CI. perfringens (Sigma) for 90 min at 37°C and washed twice. Streptococcal cells were tritium- labelled by growth in the presence of 20 pCi/ml of ['H-methyflthymidine (60-90 Ci/mmol; ICN Bio- medicals, Inc., Costa Mesa, CA, USA). Specific activities of labelled bacteria, determined by liquid scintillation counting, ranged from approximately 2000 to 100,000 c.p.m./107 cells. SHA, NANase treated SHA, BSA-blocked HA and BSA-blocked tubes without HA were incubated with 1 x lo7 'H-labelled cells in a final volume of 0.1 ml. After incubation for 60 min at room temperature with constant mixing, the beads were allowed to settle and 5Opl of each supernatant, containing unbound bacteria, was removed from each tube for scintillation counting. Percentage bacterial adhesion was calculated from the total radioactivity added minus the amount unbound. Radioactivity

128 S. D. HSU ET AL.

Table 3. Adhesive properties of viridans streptococci

Bacterial haemagglutination (log, dilution ~ ') Bacterial adhesion (%)

NANase BSA Aggregation treated blocked of PRP Coaggregation

Strain Human Goat Dog Guinea-pig SHA SHA HA latex beads group

S. sanguis Biovar 1

10556 SK1 (10556) SK36 SK37 SK72 SK75 SK76 SK77 SK85 SK108

Biovar 2 SK4 SK156 SK157 SKI58 SK164

Biovar 3 SK150 SKI 59 SK160 SK162 SK163

Biovar 4 SK45 SK112

S. gordonii Biovar 1

38 SK6 SK120 SK121

Biovar 2 10558 D105 DL 1 G102 K1 K4 M5 SK184

Biovar 3 SK9 SK12 SK33 SK186

5

7 6 7 8 6 6 7 6

-

- 3

4 -

-

- 8 6 6 4

4 3*

6 5* 5* 5*

- 7 7* 6 6 7

7

6 8 7 3'

-

5

6 4 4 4

3 6 6

-

-

-

5 4 7 5

- 5 5 -

-

6 6

5 6 6 6

~

6 6 6 6 6

6

5 6 6 6

-

86 54 76 90 93 88 70 60 83 86

27 73 39 74 30

50 92 91 55 31

15 87

95 74 88 87

9 97 95 93 90 92 21 95

99 98 94 81

7 63 18 61 63 56 28 13 10 41

8 15 15 17 11

57 24 6

13 9

35 84

85 17 69 58

8 62 76 59 60 63 25 82

87 76 77 75

10 10 32 63 41 50 20 9

12 36

2 12 9 9 5

12 8 3

10 8

21 19

25 9

25 29

9 74 17 75 58 72 17 85

68 43 46 10

ADHESION OF VIRIDANS STREPTOCOCCI I29

Table 3. Continued

Bacterial haemagglutination (log, dilution ~ ') Bacterial adhesion (%)

NANase BSA Aggregation treated blocked of PRP Coaggregation

Strain Human Goat Dog Guinea-pig SHA SHA HA latex beads group

S. oralis 10557 82 M9811 SK2 (10557) SK23 SK92 SKI 11 6249 C104 34 SK143 SK 144

Biovar 1 15914 HI J22 SK137 SK141 SKI42

Biovar 2 903 H127 I18 K103 SK96 SK148

SK14 SK47 SK52 SK63 SK64 SK66 SK8 1 SK215 SK216 SK218 SK221

S. mitis

S. anginosus

- 89 96 94 90 94 96 89 71 50 81 84 66

~

-

~

-

-

~

-

-

-

-

-

- 96 49 89 28 53 33

~

-

-

-

-

~ 61 32 45

- 14 12

3 55

-

~

-

76 94 95 90 90 79 62 70 47 22 38 31

96 23 89 20 48 36

58 31 28 12 1 1 34

7 13 4

28 5

16 16 9 9

12 2

42 43 49 62 89 62 51 47 38 30 38 22

84 29 59 17 53 37

56 33 18 9

82 30

6 11 10 20

5 10 12 5 8 8 8

3 4 4 3 3 3 3 5 3 3 3 3

3 2 4 - - -

- 3 - - - -

- - 5

2 2

2 2 2 2

-

-

*Similar endpoints were obtained with NANase treated and untreated RBCs.

bound in the absence of HA was neglible and was performed in duplicate with a different culture of ignored. Each assay included S. oralis 34 as an labelled bacteria) of this strain to SHA, NANase internal control; the adhesion (mean % f standard treated SHA and BSA-blocked HA were 80 f 8, deviation, n=30 independent determinations, each 22 f 6 and 30 f 8, respectively. Corresponding


values of percentage adhesion for the other strains were means of up to five independent determinations each performed in duplicate. Variations in percentage adhesion between different cultures of the same strain generally fell within a range of f 15% and duplicates of each determination within * 5%.

Inhibition of adhesion was determined by the sequential addition of saccharides and 3H-labelled bacteria to BSA-blocked SHA in a final volume of 0.1 ml KCl-BSA. Stock solutions of N- acetylneuramin-lactose from bovine colostrum (Sigma), N-acetylneuraminic acid from Escherichia coli (Sigma) and D-glucuronic acid (Sigma) were neutralised with NaOH.

RESULTS

S. sunguis Bacteria-mediated haemagglutination of RBC

from at least one species was observed with 19 of 21 S. sunguis strains and was not observed with NANase treated RBC except in one instance (SK112 with goat RBC) (Table 3). The adhesion of all S. sunguis strains to SHA was greater than their adhesion to HA preincubated with only BSA (Table 3). With most of these strains (18 of 21), specific adhesion was reduced or abolished by NANase pretreatment of SHA. Similarly, adhesion of strain 10556 was inhibited approximately 65 per cent by 10 mM N-acetylneuramin-lactose, less effectively by N-acetylneuraminic acid and not at all by lactose or D-glucuronic acid (Figure 1, top panel). Bacterial aggregation of PRP-coated latex beads was noted only with the two biovar 4 strains of S. sanguis (Table 3).

Only six of 23 S. sunguis strains coaggregated with actinomyces (Table 3). These interactions were all lactose sensitive (streptococcal coaggregation group 3) and did not occur when streptococci were incubated with fimbriae-deficient mutants of actinomyces. The six positive S. sunguis strains were among these previously distinguished from the other strains of S. sunguis by their lack of reactivity with group H (Blackburn) antiserum.26

Sixteen of 21 S. sunguis strains coaggregated with at least one other streptococcus and this interaction was most frequently observed with strains of S. oralis (Figure 2). The coaggregation of strain 10556 with S. oralis 34, an interaction previously shown to be GalNAc sensitive,28 was further examined. GalNAcPl+3GaluOMe was

N-Acetylneuramin-bctose 0 N-Acetylneuraminc Acid A Lactose

80 A D-Glucurone Acid

70 0 a

I 0

-10 O V -

S. oralis 34

20

S. oralis 34

20

P

-10 O P 0.5 1 2 5 1 0 2 0 50

Inhibitor (mM)

Figure 1. Inhibition of bacterial adhesion to SHA by saccharides. In the absence of inhibitors, adhesion of 1 x lo7 radiola- belled S. sunguis 10556, S. gordonii DL1 or S. oralis 34 to S H A (10 mg) was 84 per cent, 95 per cent or 83 per cent, respectively

a more effective inhibitor of coaggregation than GalPl +3GalNAcaOC,H,NO, which was slightly more active than GalNAc on a molar basis. Lactose and galactose were less inhibitory (Figure 3, top panel). Comparable inhibition data were obtained for the coaggregation of S. sanguis SK4 or SK36 with S. oralis 34 (results not shown). Strain 10556 and the other S. sanguis strains that coaggregated with S. oralis 34 each failed to coaggregate with S. oralis 34M,6 a spontaneous mutant


M5 K4 K l G1 O2 DLl D105 10558 SK6

S. g o h n i i

SK45 - * a - - - - - - - - sK163 - .. - - .. - - - - 0 - . S K I M ) - I - - - - - - - I - - - SK159 - - - - - - - S K l s - - - - - - - S K l W - - - - - - -

s,

SK4 - .. - - - - Figure 2. Coaggregations observed among viridans streptococci when each strain listed in Table 3 was incubated with all other strains. Symbols identify streptococcal pairs that coaggregated ( 0 ) and pairs that did not coaggregate (-). Streptococcal strains not included in the figure did not coaggregate with other streptococci

lacking the strain 34 receptor po lysa~cha r ide .~~~~ Certain S. sanguis strains, most notably SK163, SK45 and SK112, failed to interact with S. oralis, but instead coaggregated with other strains of S. sanguis or S. gordonii expressing GalNAc sensitive adhesins (Figure 2).

The in vitro loss of specific adhesive properties from S. sanguis 10556 was evident. Although this strain from ATCC agglutinated RBC, the same organism maintained in a separate culture collection, SK1, was inactive. Likewise, certain cultures of strain 10556 failed to exhibit the GalNAc sensitive lectin detected by coaggregation with other streptococci. Observations similar to these have been made by other investigator^.^^

S. gordonii

Bacteria-mediated haemagglutination was a property of most S. gordonii strains (14 of 16) and,

except for certain reactions with goat RBC, all were NANase sensitive (Table 3). Each strain with haemagglutinating activity adhered well to SHA, while the two strains that failed to haemagglutinate (10558 and M5) were non-adherent. As noted above for S. sanguis 10556, the failure of S. gordonii M5 to adhere may be attributed to an in vitro loss of this activity since strain M5 has previously been shown to express a sialic acid reactive l e ~ t i n . ' ~ , ~ ~ Adhesion of most strains of S. gordonii to SHA was reduced by only 10-30 per cent by NANase treatment of SHA. This reduction in adhesion of DL1 was comparable to the percentage inhibition of adhesion to SHA observed in the presence of N-acetylneuramin-lactose (Figure 1, middle panel). Several S. gordonii biovar 1 and biovar 3 strains, as well as strain DL1, were considerably more adherent to NANase treated SHA than to the control HA beads blocked with BSA (Table 3). These bacteria exhibited strong


W GalNAcP 1-3 Gala -0-Me 0 Gal pl-3 GalNAcn -O-C&Nq

GalNAc 0 Gal

v

c m 20

8

0.1 0.3 0.5 1 3 5 10 30 50 100 300

Inhibitor (mM)

Figure 3. Inhibition of the coaggregation of S. sanguis 10556 (top panel) or S. gordonii DLl (bottom panel) with S. oralis 34 by saccharides. In the absence of inhibitor, coaggregation between strains 10556 or DLI and 34 was 54 per cent and 56 per cent, respectively

aggregating activities for PRP-coated latex beads. Positive, but weaker, interactions with PRP-coated beads were detected with most other S. gordonii strains (Table 3).

Three of four S. gordonii biovar 1 strains were members of coaggregation group 3, i.e. lactose sensitive coaggregation with actinomyces (Table 3). In contrast, seven of eight S. gordonii biovar 2 strains and two of four biovar 3 strains were included in coaggregation group 1 as determined by their lactose resistant coaggregation with actinomyces.

All eight S. gordonii biovar 2 strains and one biovar 1 strain (SK6) coaggregated with strains of S. oralis (Figure 2) and, in addition, certain S, gordonii biovar 2 strains also interacted with S. sanguis SK163, SK45 and SK112 as well as S. gordonii 38. The GalNAc sensitive coaggregation of S. gordonii DL1 with S. oralis 3428 was inhibited more effectively by GalNAcPl+3GalaOMe than by GalNAc which was more active than GalP 1 + 3GalNAcaOC,H4N0, lactose or galactose on a molar basis (Figure 3, bottom panel). Similar results (not shown) were obtained for the coaggregation of S. gordonii 10558 with S. oralis 34. Each strain of S. gordonii that coaggregated with S. oralis 34 failed to coaggregate with a

receptor deficient mutant, S. oralis 34M (data not shown).

S. oralis

These bacteria did not react with human, dog or guinea-pig RBC, although eight of 12 strains agglutinated untreated but not NANase treated goat RBC (Table 3). These included S. oralis 10557 from ATCC but not strain SK2, the same organism maintained in a separate culture collection.26 The S. oralis strains, with the exception of SK23, were more adherent to SHA than BSA-HA. Of the other 11 strains, adhesion of six was reduced following treatment of SHA with NANase, a hd ing that correlated with inhibition of the interaction of S. oralis 34 with SHA by N-acetylneuramin-lactose (Figure 1, bottom panel). Seven of the S. oralis strains aggregated PRP-coated latex beads. Three of the strongest aggregating strains (B2, M9811 and SK2) were among those that exhibited NANase insensitive binding to SHA. In contrast, the binding to SHA of three strains (34, SK143 and SK144) that failed to aggregate PRP-coated beads was considerably reduced by NANase treatment of SHA.

ADHESION OF VIRlDANS STREPTOCOCCI 133

Each of the 12 S. oralis strains was a member of coaggregation group 3, 4 or 5 and, thus, participated in lactose sensitive coaggregation with actinomyces (Table 3). Nine S. oralis strains also coaggregated with at least four S. sanguis and S. gordonii strains (Figure 2) and, of five examined, all were GalNAc sensitive.

S. mitis Most strains of this species lacked haemaggluti-

nating activity, failed to adhere to SHA signifi- cantly more than to BSA-HA and did not aggregate PRP-coated latex beads (Table 3). Four S. mitis strains coaggregated with actinomyces with patterns characteristic of groups 2, 3 and 4 (Table 3). S. mitis H1 was the only member of coaggregation group 2 among the S. mitis, S. sanguzs, S. gordonii and S. oralis strains. Only one strain of S. mitis (SK141) coaggregated with more than one other streptococcal strain (Figure 2).

S. anginosus With few exceptions, strains of this species did

not interact with RBC, SHA, PRP-coated latex beads (Table 3) or other streptococci (Figure 2). However, lactose resistant coaggregations with actinomyces, like those noted for S. mitis HI (i.e. coaggregation group 2), were observed with six of the 11 S. anginosus isolates including each of the four (i.e. SK215, SK216, SK218 and SK221) from subgingival dental plaque.

DISCUSSION

These studies extend the recent reclassification of viridans streptococci to include the activities of adhesins and receptors. Other cell surface properties previously examined for their association with different species include bacterial binding of salivary a-amylase'4327 and the expression of IgA protease, neuraminidase and certain other glycosi- d a s e ~ . ~ ~ ~ ~ Considered in conjunction with results from studies of in vivo c o l ~ n i s a t i o n , ' ~ ~ ~ ~ ~ the present findings provide an improved basis for defining the ecological properties of the different species and the involvement of specific cellular and molecular interactions in microbial colonisation of the tooth surface.

Sialic acid reactive adhesins were detected on most strains of S. sanguis, S. gordonii and S. oralis but not on those of S. mitis or S. anginosus. Most of the strains positive for NANase sensitive

haemagglutination also exhibited NANase sensitive adhesion to SHA. In addition, four strains that failed to haemagglutinate (i.e. S. sanguis SK4, S. mitis H1, S. mitis I18 and S. anginosus SK81) appeared to possess sialic acid reactive adhesins based on their greater adhesion to SHA than NANase treated SHA. Haemagglutinating activities of various strains were observed more frequently in assays using goat or dog than guinea- pig or human RBC. The patterns of reactivity observed with these RBC were of interest since they distinguished the lectin activities of different strains and to some extent species. For example, S. gordonii strains generally reacted with each of the RBC tested while S. oralis strains reacted only with goat RBC. The distinct patterns of these reactions may reflect differences in bacterial lectin specificity like those observed in studies of NANase sensitive viral haemaggl~tination.~' Alternatively, they may depend on differences in the density and distribution of similar lectins and their complimentary receptors on bacterial and RBC surfaces, respectively. Either possibility could influence the selectivity of streptococci for distinct ecological niches. The production of NANase by S. oraiis and some S, mitis strains26 represents yet another factor that may modulate the selectivity of lectin-mediated adhesion.

Other streptococcal adhesins for pellicle receptors include those for acidic proline rich proteins.21732 In the present investigation, aggregation of PRP-coated latex beads was observed with most S. gordonii, about half of the S. oralis and both biovar 4 strains of S. sanguis, but not with other S. sanguis biovars, most S. mitis and all S. anginosus strains. The expression of PRP binding activity by the various strains was associated in most cases with NANase resistant bacterial adhesion to SHA. Thus, the 21 strains that were most adherent to NANase treated SHA compared to BSA blocked HA included each of the 15 with strong or moderate aggregating activities for PRP-1 coated latex beads and only five that were negative for this activity. Adhesins for other pellicle receptors may be present on the latter strains and perhaps on at least some of those positive for PRP-1 binding.

Comparisons of the adhesion and colonisation properties of different streptococcal species sug- gest that the sialic acid reactive adhesins present on most S. sanguis and S. oralis strains and the PRP-1 binding activities expressed by some of these may be involved in the initial appearance of

134

these species on the tooth s u r f a ~ e . ~ ~ . ~ Likewise, the apparent absence of either specific activity on strains of S. anginosus is consistent with the absence of this s ecies during the primary phase of colonisation.’ Direct correlations of adhesive and colonisation properties were not obvious with the strains of other species. Thus, the initial colonisation of teeth by S. mitis biovar 1 strains, including SK137 and SK141,26,43 was not associated with any adhesive property considered. These and other observations made with strains designated S. mi ti^^^ encourage the further study of the ecological properties of these bacteria. The frequent interactions of S. gordonii strains with pellicle receptors is also of interest since this species was identified more frequently in mature supragingival plaque than during initial colonisation of the tooth surface.17 These findings impli- cate cell surface activities in addition to those of adhesins as determinants of colonisation and in vivo distribution. Indeed, IgA protease is pro- duced by the streptococcal species that initiate colonisation but not by S. gordonii.26 Thus, the sialic acid and PRP-1 binding adhesins of S. gordonii have ecological roles that extend beyond the recognition of pellicle receptors during initial colonisation. Examples of interactions that could contribute to later phases of colonisation include binding of soluble salivary macromolecules or insoluble components associated with mature plaque.

Coaggregation properties may also be correlated with patterns of colonisation. Virtually all streptococci placed in coaggregation groups 1 or 2 by their lactose resistant coaggregations were species that appear to colonise preexisting supragingival or subgingival plaque rather than the clean tooth surface. l7 Coaggregation group 1 was composed of S. gordonii strains in biovars 2 or 3, many of which were previously classified as S. sanguis I,9 and coaggregation group 2 was represented primarily by strains of S. unginoszds. Earlier studies have associated coaggregation group 2 with streptococci identified as S, MG-intermediu?’ and S. milleri, l ’ ~ ~ , ~ ~ bacteria similar to those presently designated S. a n g i n o s ~ s . ~ ~ ~ ~ ~ In contrast to the properties of the above species, all strains of S. oralis, an initial coloniser of the tooth s u r f a ~ e , 4 ~ ’ ~ participated in lactose sensitive coaggregations with actinomyces as did certain strains of S. mitis, S. sanguis and S. gordonii (i.e. those in coaggregation groups 3 or 4). Many of these streptoccocal strains, including S. oralis 34, also participated in

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S. D. HSU ET AL.

GalNAc sensitive coaggregations with various S. sanguis (10556, SK36 or SK4) or S. gordonii (DLl or 10558) strains, interactions like those previously described.28 However, other streptococci, such as S. mitis 522, were recognised by actinomyces lectins but not by GalNAc sensitive lectins of S. sanguis or S. gordonii strains.

The A. viscosus, A. naeslundii, S. sanguis and S, gordonii strains that coaggregated with S. oralis 34 failed to coaggregate with strain 34M, a spontaneous mutant lacking the strain 34 receptor polysaccharide.6 Recognition of this molecule by actinomyces lectins was previously attributed to GalNAcPl+3Gal within the oligosaccharide repeating unit of the p o l y s a ~ c h a r i d e . ~ ~ ~ ~ ~ ~ ~ Simi- larly, this receptor structure may also be recognised by streptococci since GalNAc sensitive coaggregations between these bacteria and S. oralis 34 were inhibited most effectively by GalNAcPl+3GalaOMe. Sigdicantly, two other streptococcal strains, S. oralis C1043 and S. gordonii 38 (manuscript submitted), expressing different receptor polysaccharides, each containing GalNAcpl-+3Gal, coaggregated frequently with other streptococci and actinomyces. However, two strains, S. mitis J22l and S. oralis 10557? with GalP 1 +3GalNAc-containing receptor polysaccharides, coaggregated with actinomyces but not with other streptococci. Thus, the coaggregation properties of these bacteria appear to be closely correlated with recognition of receptor polysaccharides containing either GalNAcp 1 + 3Gal or GalP1 +3GalNAc by actinomyces lectins but only GalNAcPl+3Gal-containing polysaccharides by the streptococcal lectins. In spite of their common GalNAc sensitivity, the lectin activities of S. sanguis and S. gordonii were not identical as indicated by differences in the relative inhibitory effects of GalNAc and Galj31+ 3GalNAcaOC6H4N02. The hgh degree of structural complementarity that characterises these coaggregations supports the view that specific polysaccharides present on some streptococci are natural receptors for lectins expressed by other streptococci and actinomyces.

Microbial colonisation of tooth surfaces is initiated by attachment of individual bacteria to the acquired pellicle and subsequent growth of these or anisms as a mixed colony of cocci and rod^.^',^' The characteristic tendency of these bacteria to grow in close association may depend on the expression of specific bacterial lectins and their receptors. The extent to which lectin-


mediated cell-cell recognition favours other interbacterial interactions such as the formation of food chains and the mutual utilisation of different cell surface enzymatic activities remains to be determined.

ACKNOWLEDGEMENTS

their criticial reviews of the manuscript. We wish to thank Stefan Ruhl and Si Young Lee for 12.

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2.

3.

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5.

6.

7.

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9.

10.

REFERENCES 13. Abeygunawardana C, Bush CA, Cisar JO. (1990). Complete structure of the polysaccharide from Streptococcus sanguis 522. Biochemistry 29, 234- 248. Abeygunawardana C, Bush CA, Cisar JO. (1991). Complete structure of the cell surface polysaccharide of Streptococcus oralis ATCC 10557: a receptor for lectin-mediated interbacterial adherence. Bio- chemistry 30, 6528-6540. Abeygunawardana C, Bush CA, Cisar JO. (1991). Complete structure of the cell surface polysaccharide of Streptococcus oralis C104: a 600-MHz NMR study. Biochemistry 30, 8568-8577. Abeygunawardana C, Bush CA, Tjoa SS, Fennessey PV, McNeil MR. (1989). The complete structure of the capsular polysaccharide from Streptococcus sanguis 34. Carbohydrate Research

Beighton D, Hardie JM, Whiley RA. (1991). A scheme for the identification of viridans streptococci. Journal of Medical Microbiology 35, 367- 372. Cisar JO, Brennan MJ, Sandberg AL. (1985). Lectin-specific interaction of Actinomyces jimbriae with oral streptococci. In: Mergenhagen SE, Rosan B (eds) Molecular Basis of Oral Microbial Adhesion. American Society for Microbiology, Washington, DC, pp. 159-163. Cisar JO, Brennan MJ, Sandberg AL. (1989). Bac- terial and host cell receptors for the Actinomyces spp. fimbrial lectin. In: Switalski L, Hook M, Beachey E (eds) Molecular Mechanisms of Micro- bial Adhesion. Springer-Verlag, New York, pp. 164-170. Cisar JO, David VA, Curl SH, Vatter AE. (1984). Exclusive presence of lactose-sensitive fimbriae on a typical strain (WVU45) of Actinomyces naeslundii. Infection and Immunity 46, 453458. Cisar JO, Kolenbrader PE, McIntire FC. (1979). Specificity of coaggregation reactions between human oral streptococci and strains of Actinom,vcr.r viscosus or Actinomyces naeslundii. Infecfion and Immunit-v 24, 742-752. Cisar JO, Vatter AE, Clark WB, Curl SH, Hurst- Calderone S, Sandberg AL. (1988). Mutants of

191, 279-293.

14.

15.

16.

17.

18.

19.

20.

21.

22.

Actinomyces viscosus T14V lacking type 1, type 2, or both types of fimbriae. Infection and Immunity 56, 2984-2989. Clark WB, Wheeler TT, Cisar JO. (1984). Specific inhibition of adsorption of Actinomyces viscosus T14V to saliva-treated hydroxyapatite by antibody against type 1 fimbriae. Infection and Immunity 43, 497-501. Cowan MM, Taylor KG, Doyle RJ. (1987). Role of sialic acid in the kinetics of Streptococcus sanguis adhesion of artificial pellicle. Infection and Immu- nit-v 55, 1552-1557. Demuth DR, Golub EE, Malamud D. (1990). Streptococcal-host interactions. Structural and functional analysis of a Streptococcus sanguis receptor for a human salivary glycoprotein. Journal of Biological Chemistry 265, 7120-7126. Douglas CWI, Pease AA, Whiley RA. (1990). Amylase-binding as a discriminator among oral streptococci. FEMS Microbiology Letters 66, 193- 198. Eifuku H. Kitada K, Yakushiji T, Inoue M. (1991). Lactose-sensitive and -insensitive cell surface interactions of oral Streptococcus milleri strains and actinomyces. Injection and Immunity 59, 460- 463. Eifuku H, Yakushiji T, Mizuno J, Kudo N, Inoue M. (1990). Cellular coaggregation of oral Strepto- coccus milleri with actinomyces. Infection and Immunity 58, 163-168. Frandsen EVG, Pedrazzoli V, Kilian M. (1991). Ecology of viridans streptococci in the oral cavity and pharynx. Oral Microbiology and Immunology 6, 129-133. Gibbons RJ, Etherden I. (1985). Albumin as a blocking agent in studies of streptococcal adsorption to experimental salivary pellicles. Infection and Immunity 50, 592-594. Gibbons RJ, Etherden I, Moreno EC. (1983). Association of neuraminidase-sensitive receptors and putative hydrophobic interactions with high- affinity binding sites for Streptococcus sanguis C5 in salivary pellicles. Inj2ction and Immunitv 42, 1 0 0 ~ 1 0 12. Gibbons RJ, Hay DI, Cisar JO, Clark WB. (1988). Adsorbed salivary proline-rich protein 1 and statherin: receptors for type 1 fimbriae of Actino- myces viscosus T14V-JI on apatitic surfaces. Znfec- tion and Immunity 56, 2990-2993. Gibbons RJ, Hay DI, Schlesinger DH. (1991). Delineation of a segment of adsorbed salivary acidic proline-rich proteins which promotes adhesion of Streptococcus gordonii to apatitic surfaces. Infection and Immunity 59, 2948-2954. Gibbons RJ, van Houte J. (1980). Bacterial adherence and the formation of dental plaques. In: Beachey EH, (ed) Bacterial Adherence. Chapman and Hall, London.

136 S . D. HSU ET AL..

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

Gilmour MN, Whittam TS, Kilian M, Selander RK. (1987). Genetic relationships among the oral streptococci. Journal of Bacteriology 169, 5247- 5257. Hawley RJ, Lee LN, LeBlanc DJ. (1980). Effects of tetracycline on the streptococcal flora of periodontal pockets. Antimicrobial Agents and Chemo- therapy 17, 372-378. Johnson JL, Moore LV, Kaneko B, Moore WE. (1990). Actinomyces georgiae sp. nov., Actinomyces gerencseriae sp. nov., designation of two genospecies of Actinomyces naeslundii, and inclusion of A. naeslundii serotypes I1 and 111 and Actinomyces viscosus serotype I1 in A. naeslundii genospecies 2. International Journal of Systematic Bacteriology 40, 273-286. Kilian M, Mikkelsen L, Henrichsen J. (1989). Taxonomic study of viridans streptococci: descrip- tion of Streptococcus gordonii sp. nov. and emended descriptions of Streptococcus sanguis (White and Niven, 1946), Streptococcus oralis (Bridge and Sneath, 1982), and Streptococcus mitis (Andrewes and Horder, 1906). International Jour- nal of Systematic Bacteriology 39, 471484. Kilian M, Nyvad B. (1990). Ability to bind salivary alpha-amylase discriminates certain viridans group streptococcal species. Journal of Clinical Microbiol- ogy 28,25762577. Kolenbrader PE, Andersen RN, Moore LV. (1990). Intrageneric coaggregation among strains of human oral bacteria: potential role in primary colonization of the tooth surface. Applied and Environmental Microbiology 56, 3890-3894. Kolenbrander PE, Williams BL. (1983). Prevalence of viridans streptococci exhibiting lactose- inhibitable coaggregation with oral actinomycetes. Infection and Immunity 41, 449452. Levine MJ, Herzberg MC, Levine MS, Ellison SA, Stinson MW, Li HC, van Dyke T. (1978). Specific- ity of salivary-bacterial interactions: role of termi- nal sialic acid residues in the interaction of salivary glycoproteins with Streptococcus sanguis and Streptococcus mutans. Infection and Immunity 19, 107-1 15. Lie T. (1977). Early dental plaque morphogenesis. A scanning electron microscope study using the hydroxyapatite splint model and a low-sucrose diet. Journal of Periodontal Research 12, 73-89. Ligtenberg AJM, Walgreen-Weterings E, Veerman ECI, de Soet JJ, de Graaff J , Nieuw Amerongen AV. (1992). Influence of saliva on aggregation and adherence of Streptococcus gordonii HG 222. Znfec- tion and Immunity 60, 3878-3884. Liljemark WF, Bloomquist CG, Fenner LJ, Antonelli PJ, Coulter MC. (1989). Effect of neuraminidase on the adherence to salivary pellicle of Streptococcus sanguis and Streptococcus mitis.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

Malamud D, Appelbaum B, Kline R, Golub EE. (1981). Bacterial aggregating activity in human saliva: comparisons of bacterial species and strains. Infection and Immunity 31, 1003-1006. Markwell MAK. (1986). Viruses as hemagglutinins and lectins. In: Mirelman D. (ed) Microbial Lectins and Agglutinins. John Wiley, New York, pp. 21-53. McBride BC, Gisslow MT. (1977). Role of sialic acid in saliva-induced aggregation of Streptococcus sanguis. Infection and Immunity 18, 3540. McIntire FC, Bush CA, Wu SS, Li SC, Li YT, McNeil M, Tjoa SS, Fennessey PV. (1987). Struc- ture of a new hexasaccharide from the coaggregation polysaccharide of Streptococcus sanguis 34. Carbohydrate Research 166, 133-143. McIntire FC, Crosby LK, Vatter AE. (1982). In- hibitors of coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34: beta-galactosides, related sugars, and anionic am- phipathic compounds. Infection and Immunity 36, 371-378. McIntire FC, Crosby LK, Vatter AE, Cisar JO, McNeil MR, Bush CA, Tjoa SS, Fennessey PV. (1988). A polysaccharide from Streptococcus sanguis 34 that inhibits coaggregation of s. sanguis 34 with Actinomyces viscosus T14V. Journal of Bacteriology 170, 2229-2235. Murray PA, Levine MJ, Tabak LA, Reddy MS. (1982). Specificity of salivary-bacterial interactions: 11. Evidence for a lectin on Streptococcus sanguis with specificity for a NeuAc alpha 2, 3Gal beta 1, 3GalNAc sequence. Biochemical and Biophysical Research Communications 106, 390- 396. Nyvad B, Fejerskov 0. (1987). Transmission electron microscopy of early microbial colonization of human enamel and root surfaces in vivo. Scandina- vian Journal of Dental Research 95, 291-307. Nyvad B, Fejerskov 0. (1987). Scanning electron microscopy of early microbial colonization of human enamel and root surfaces in vivo. Scandinavian Journal of Dental Research 95, 287-296. Nyvad B, Kilian M. (1987). Microbiology of the early colonization of human enamel and root surfaces in vivo. Scandinavian Journal of Dental Research 95, 369-380. Nyvad B, Kilian M. (1990). Comparison of the initial streptococcal microflora on dental enamel in caries-active and in caries-inactive individuals. Caries Research 24, 267-272. Socransky SS, Manganiello AD, Propas D, Oram V, van Houte J. (1977). Bacteriological studies of developing supragingival dental plaque. Journal of Periodontal Research 12, 90-106. Whiley RA, Fraser H, Hardie JM, Beighton D. (1990). Phenotypic differentiation of Streptococcus

Caries Research 23, 141-145. intermedius, Streptococcus constellatus, and Strep-


tococcus anginosus strains within the ‘Streptococcus of the Streptococcus sanguis group. Microbial milleri group’. Journal of Clinical Microbiology 28, Ecology in Health and Disease 4, 61-12. 1497-1501. 48. Yakushiji T, Katsuki M, Yoshimitsu A, Mizuno J, Willcox MDP, Knox KW. (1991). A comparison of Inoue M. (1988). Isolation and physiological char- the adhesion profiles and cell surface characteristics acterization of Streptococcus milleri strains from of Streptococcus mitis with those of other members human dental plaque. Microhios 55, 161-171.

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Adhesive Properties of Viridans Streptoccocal Species

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