MICROBIAL ECOLOGY rn HEALTH AND DISEASE VOL. 8: 243-254 (1995)

Attachment of Streptococcus gordonii HG 222 to Streptococcus oralis Ny 586 and the Influence of Saliva A. J. M. LIGTENBERG*?, E. WALGREEN-WETERINGS?, E. C. I. VEERMAN?, J. 3. DE SOETf and A. V. NIEUW AMERONGEN-f

Departments of ?Oral Biochemistry and $Oral Microbiology, Academic Centre for Dentistry Amsterdam (ACTA), van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

Received 23 March 1995; revised 30 May 1995

The binding of Streptococcus gordonii HG 222 to other streptococci was tested by coaggregation and by binding to immobilised bacteria in an ELISA assay. Out of 17 streptococcal strains, HG 222 coaggregated only with Streptococcus sanguis HG 1470 and Streptococcus oralis Ny 586. Binding of HG 222 to immobilised bacteria also occurred only with HG 1470 and NY 586. Binding of HG 222 to S. oralis Ny 586 was further investigated.

Preincubation of Ny 586 coated microtitreplates with whole saliva, parotid, submandibular and sublingual saliva enhanced binding of HG 222 to Ny 586. Several salivary substances bound to both HG 222 and Ny 586, namely low molecular weight mucins, salivary agglutinin, and IgA.

There were indications that binding of HG 222 to Ny 586 was especially enhanced by IgA. Binding of IgA to both bacteria could be demonstrated with all the types of glandular saliva. In addition, purified colostral IgA enhanced binding of HG 222 to Ny 586. Saliva preincubation of Ny 586 coated microtitreplates in the presence of antiserum against the IgA a-chain inhibited saliva mediated binding of HG 222 to Ny 586. In this case, binding of salivary agglutinin to Ny 586 was also inhibited, suggesting complexation between IgA and agglutinin.

In conclusion, these results indicate that colonisation of dental surfaces by S. gordonii may be accomplished by binding to strains of S. oralis or S. sanguis. This process may be enhanced by IgA in saliva.

KEY WORDS: oral streptococci; saliva; coaggregation; interbacterial binding.

INTRODUCTION The oral cavity is selectively colonised by a variety of bacterial species. An important step in their colonisation process is attachment to the tooth surface that is covered by a biofilm of salivary proteins, termed the acquired enamel p e l l i ~ l e . ' ~ ~ The first layer of attached bacteria consists of approximately 80 per cent of Streptococcus sanguis, Streptococcus mitis and Streptococcus o r a l i ~ . ~ ~ Subsequently, bacterial accumulations of hundreds of microns thickness can be formed, termed dental plaque. A prerequisite for the accu- mulation of bacteria on the tooth surface is inter- bacterial binding. This may be accomplished via direct attachment between bacterial species as has extensively been studied by coaggregation.'2,'8 Interbacterial attachment may also be mediated by salivary components as is underlined by the

*Author to whom correspondence should be addressed.

CCC 089 1-060W95/050243-12 Q 1995 by John Wiley & Sons, Ltd.

presence of salivary components in dental plaque." For example, the attachment of strains of Streptococcus mutans to S. sanguis was en- hanced by saliva.22 This interbacterial attach- ment was especially enhanced by the salivary agglutinin. 23

Streptococcus gordonii, previously included with S. sanguis biotype 1," colonises both dental plaque and soft oral tissues." This s ecies readily adheres to saliva-coated surfaces. However, despite its ability to adhere to saliva-coated sur- faces, this species is not commonly found in early dental plaque, but only in mature dental plaque." Apparently, S. gordonii only colonises the dental surface in vivo when other bacteria are already present. Therefore, in this report the interaction of S. gordonii strain HG 222 with other streptococci has been investigated both by coaggregation studies and by a solid phase assay using bac- teria immobilised on microtitreplates. In addition,

244 A. J. M. LIGTENBERG ET AL.

the effect of saliva was studied by preincubating the bacterial surface with saliva or salivary components.

MATERIALS AND METHODS

Bacteria and culture conditions The organisms used in this study are listed in

Table 1. All strains were stored at - 70°C in Protect bacterial preservers (Technical Service con- sultants, Bury Lancs, UK). For each experiment, the bacteria were subcultured on blood agar plates for 48 h at 37°C aerobically with 5 per cent C02. Subsequently, an isolated colony was used to inoculate 100ml of Todd Hewitt Broth (Difco Laboratories, Detroit, Michigan, USA). Cultures were grown overnight at 37°C in completely filled 100 ml flasks under air without shaking. Cells were harvested by centrifugation (3000 g at 4°C for 10 min), washed in PBS (150 mM sodium chloride, 10 mM potassium phosphate buffer, pH 7.4) and resuspended in PBS with 0-5 mM calcium chloride (PBS-Ca) to an optical density at 700 nm (OD,,,) of 1.0.

Coaggregation assay Bacterial suspensions of 150 p1 each were mixed

in microtitreplates (Greiner, Recklinghausen, Germany) and incubated for 2 h at 37°C. As a control, 300 pl of the separate bacterial suspen- sions of a coaggregation pair was incubated. Coaggregates sedimented to the bottom of the microtitreplate wells, resulting in a lower optical density of the upper suspension. Non- coaggregating bacteria and control bacteria re- mained in suspension. Subsequently, 200 pl of the upper suspensions was transferred to another microtitreplate and the optical density at 570 nm (OD,,,) was measured with a microtitreplate reader (Dynatech MR 7000, Billinghurst, UK). Coaggregation was expressed as the percentage decrease in absorbance compared to the control, calculated as follows:

Percentage coaggregation = [ 1 - ODs7, sample/ OD,,, control] x 100 per cent

The control was formed by the average OD,,, of the separate bacterial suspensions of a coaggrega- tion pair. This method was analogous to the method described for saliva-induced aggregation.'

Table 1. Streptococcal strains used in this study

Species Strain

Streptococcus gordonii

Streptococcus mitis Streptococcus mutans serotype c Streptococcus oralis

Streptococcus rattus Streptococcus sanguis

Streptococcus salivarius Streptococcus sobrinus

~

HG 222" HG 384' HG 1280" Ny 346b HG 179" Ny 582' Ny 583b Ny 586' HG 1277" HG 59 (BHT)" Ny 584' HG 1470" HG 1471" HG 1473" HB' HG 456(B- 13)" OMZ 176"

~~~

aFrom J. de Graaff, Amsterdam. bFrom J. S. van der Hoeven, Nijmegen. 'From H. J. Busscher, Groningen.

Collection of saliva All saliva samples were collected in ice-cooled

vessels after brushing the teeth and at least 1 h after the previous meal. Human whole saliva was collected by expectoration and after centrifugation designated clarified human whole (CHW) saliva. Parotid (PAR) saliva was collected with a Lashley cup under stimulation with citric acid or paraffin chewing. Saliva from the submandibular (SM) and sublingual (SL) glands of an individual with blood group B reactivity was segregated with a device adapted individually for the mouth with separate chambers over the ductal orifices of the sub- mandibular and sublingual gland^.^' Both types of glandular saliva were obtained without stimu- lation. After collection the saliva samples were homogenised by shaking on a Vortex-genie mixer (Scientific Industries, Springfield, USA) to reduce viscosity, and subsequently clarified by centrifu- gation (4000g at 4°C for 10 min) to remove cellular debris.

Isolation of salivary proteins a-Amylase, acidic proline-rich proteins (PRPs)

and proline-rich glycoprotein (PRG) were purified from PAR saliva by anion exchange chromatogra- phy with a preparative Mono Q HR loll0 column

INTRAGENERIC BINDING OF S. GORDON11

on a FPLC apparatus (Pharmacia LKB, Uppsala, Sweden). To 30 ml of PAR saliva, 3-3 ml 0.2 M Tris-HC1 pH 8.4 was added, and this was applied to a Mono Q HR 10/10 anion exchange column. After passage of unbound proteins, anions were eluted with a sodium chloride gradient (0-0.2 M NaCl in 0.02 M Tris-HC1 pH 8.4) at a flow rate of 4mUmin. Peak fractions were pooled and after dialysis rechromatographed on the same column. Again peak fractions were pooled and purity was checked by electrophoresis. Another sample of PRG was a generous gift from M. J. Levine (State University of New York at Buffalo, Buffalo, NY).

High molecular weight salivary mucins (MG- 1) were isolated as described previously4 by ultra- centrifugation (100 000 g, 48 h) in dissociative buffer (8 M urea, 0.5 M NaC1, 10mM EDTA) followed by gel filtration over Sephacryl S-500 HR (Pharmacia LKB) in 4 M guanidine-HC1

Low molecular weight mucins (MG-2) were isolated by the following procedure. Fifty ml CHW saliva was mixed with 50 ml 8 M guanidine- HCl (Gu-HC1) (pH 7.4) and incubated overnight at 4°C. The suspension was then centrifuged at 4°C for 24 h at 100 000 g to remove MG-1. CsCl was added to the supernatant under gentle stirring to a density of 1-45 gfml. The resulting solution was centrifuged at 100 000 g for 72 h. Subsequently, 1 ml fractions with a density varying between 1.65 and 1.28 were screened in an ELISA with mono- clonal antibodies E9 and F2. Both antibodies were produced by immunisation of mice with MG-1 and partly characterised in our lab0ratory.4~ F2 recog- nises epitopes only present on MG-1, whereas E9 recognises a class of sialic acid containing epitopes on both MG-1 and MG-2.45 E9-positive/F2- negative fractions were pooled and again centri- fuged at 4°C for 72 h at 100 000 g. EPpositive fractions were pooled, dialysed and freeze-dried, and resuspended in 4 M Gu-HC1. The sample was fractionated on a Sephacryl S-500 HR column and E9-positive fractions were again pooled, freeze- dried and resuspended in 4 M Gu-HC1. Subse- quently, the sample was fractionated onto a Sephacryl S-200 HR column (Pharmacia LKB) and E9-positive fractions were pooled and dialysed.

Salivary agglutinin was enriched as described previously by affinity adsorption of the agglutinin to S. gordonii HG 222 followed by desorption in the presence of EDTA.'03'6 Bacteria were grown in Todd Hewitt (300 ml), harvested by centrifugation,

(Gu-HC1).

245

washed in PBS and resuspended in 50 ml PBS-Ca. Then, 50 ml paraffin-stimulated CHW saliva was added. After 1 h of absorption at 37°C bacteria were harvested by centrifugation, washed in PBS-Ca and resuspended in 5 ml PBS with 5 mM EDTA. After 1 h of incubation the bacteria were pelleted and the supernatant was dialysed against demineralised water and subsequently lyophilised. The lyophilisate was resuspended in 2 ml PBS and analysed by SDS-PAGE (4-15 per cent) and staining with Coomassie Brilliant Blue. Besides agglutinin (>300 kD) the preparation contained slgA as was verified by ELISA. Several other bands were probably membrane components of S. gordonii as could be demonstrated from a control suspension that was not incubated with saliva. These components showed no reactivity with OMVU 31, the antibody used for recognising S. gordonii.

Binding assay for S. gordonii to bacterial surfaces Binding of S. gordonii strain HG 222 to other

oral bacteria was tested in an ELISA system.6 Bacteria were detected with a monoclonal anti- body OMVU 31.8 OMVU 31 was produced by immunisation of mice with S. mutans and recog- nised antigen B on the surface of S. mutuns. It also recognises epitopes on the surface of S. gordonii HG 222.8 All incubations were performed at 37°C unless stated otherwise and between all incu- bations microtitreplates were washed with PBS with 0.1 per cent Tween 20 (PBS-T). Of the bacteria listed in Table 1, bacterial surfaces were developed by incubating flat bottom Immulon (Dynatech, Chantilly USA) or Greiner high affin- ity microtitreplates (Recklinghausen, Germany) overnight at 4°C with 100 p1 bacterial suspension in PBS-Ca. The presence of a layer of bacteria after the washing procedures was checked by staining with crystal violet and by microscopic observation. Uncoated microtitreplate wells were used as controls. Subsequently, 100 pl bacterial suspension was added to each well and incubated for 1 h followed by 100 pl OMVU 31 in PBS-T with 1 per cent bovine serum albumin (BSA). Rabbit-anti-mouse conjugated horse radish per- oxidase (HRP) in PBS-T with 1 per cent BSA was then added and incubated for 1 h. Substrate con- sisted of 0.75 mg 3,3',5,5' tetramethyl benzidine dihydrochloride (Pierce, Rockford, USA), dis- solved in 250 pl dimethyl sulfoxide, which was mixed with 10 ml sodium acetate buffer (pH 5.5) together with 0 . 2 ~ 1 30 per cent H,O,. Ninety pl

246 A. J . M. LIGTENBERG ET AL.

substrate was added to the microtitreplate wells and after 30min of incubation at room temperature the reaction was stopped with 50 p1 0.05 M H,SO,. The substrate coloration was measured with a Dynatech microtitreplate reader at 450 nm.

Binding of HG 222 to saliva-coated bacteria Binding of HG 222 to bacteria-coated micro-

titreplates was also tested after preincubation with saliva, salivary components or colostral IgA (Sigma). For this purpose, 100 p1 saliva, salivary components, or colostral IgA was serially diluted in PBS-T with 0.5 mM calcium chloride and added to Ny 586 coated microtitreplates. After incu- bation for 1 h at 37°C the microtitreplates were washed with PBS-T to remove unbound salivary components. Hereafter, binding of HG 222 was assayed as described above. Salivary preincubation of microtitreplates that were not coated with Ny 586 revealed no binding of HG 222.

Binding of salivary components by bacteria Saliva was depleted of bacterium binding sub-

stances by taking 10 ml bacterial suspension which was pelleted and resuspended in 1 ml PAR or SM saliva. The samples were incubated at 37°C for 1 h and subsequently centrifuged. The supernatants were analysed by SDS-PAGE with 4-15 per cent gradient gels with the Pharmacia Phast system (Pharmacia LKB). Gels were stained with Coomassie Brilliant Blue R 250 (Pierce) or silver-stained.3

Extraction of bacteria binding substances was carried out as follows. Five ml bacterial suspen- sions were incubated at 37°C for 1 h with 5 ml PAR or SM saliva or 2ml SL saliva. After cen- trifugation and washing in PBS-Ca bacteria were resuspended in 100 p1 sample buffer containing 2 per cent SDS and gently mixed for 1 h. Hereafter, bacteria were pelleted and supernatant containing salivary proteins was analysed by SDS-PAGE and immunoblotting. After SDS-PAGE, proteins were transferred to nitrocellulose (Schleicher & Schuell, Dassel, Germany) or Immobilon (Millipore, Bedford, USA) filters by diffusion blotting for 2 h. Filters were blocked in PBS-T with 2 per cent BSA and subsequently incubated with antibody and conjugate. Blots were developed in 10 ml PBS with 2 ml methanol containing 6 mg 4-chloro- 1 - naphthol (Pierce) and 20p1 35 per cent H,O,. Development was stopped by transfer to distilled

water. Antibodies and conjugate did not show any reactivity with extracts of bacteria not incubated with saliva.

ELISA Binding of salivary components to bacteria was

also demonstrated by ELISA. Saliva was serially diluted in PBS-T with 0.5 mM CaCI, in bacteria- coated microtitreplates and incubated at 37°C for 1 h. Unbound salivary components were removed by washing three times with PBS-T. Bacteria- bound salivary components were demonstrated with the antibodies F2 recognising MG-1, E9 recognising sialic acid acid epitopes on MG-1 and MG-2, and anti-Secretory Component of IgA (Dakopatts, Glostrup, Denmark). HRP-labelling and substrate coloration were carried out as described in the binding assay for HG 222. Controls were formed by microtitreplate wells that were not incubated with saliva, but further were treated identically.

Bacterial binding of a-amylase Bacterial binding of a-amylase was assayed

as described previously.26 A pellet from 10 ml bacterial suspension was suspended in 100 p1 PAR saliva and incubated at 37°C for 30min. After centrifugation the supernatant was tested for a-amylase activity with amylase 10 reagent (Sigma) according to the manufacturer’s instructions. PAR saliva that was not incubated with bacteria was used as a control. Bacteria resuspended in 10011 PBS instead of saliva showed no a-amylase activity with amylase 10 reagent.

Inhibition of IgA binding Fifteen pl antiserum against the IgA a-chain,

the IgA Secretory Component or iysozyme (Dakopatts, Hagersten, Sweden) was added to 600 pl PAR saliva and incubated for 15 min at room temperature. Subsequently, these samples were tested in a binding assay for HG 222 to Ny 586 and for binding of salivary agglutinin to S. oralis NY 586.

RESULTS

Coaggregation of S. gordonii HG 222 with various streptococci and attachment to bacteria-coated surfaces

All strains listed in Table 1 were tested for coaggregation with S. gordonii HG 222. HG 222

INTRAGENERIC BINDING OF S. GORDON11

1 4

13-

1 2 -

11-

1 0 -

0 9-

0 8 -

0 7 -

247

. CHW saliva x PAR saliva + SM saliva * SL saliva E

C

0 In d

a, 0 C m I 0 u1

Q

v

n

n

1 I I I I I I I , 0 6’ I IF- 1 2 3 4 5 6 7 8 9 10 Control

210g[dilut~on]

Figure 1. Binding of S. gordonii HG 222 to S. oralis Ny 586 after preincubation of Ny 586 coated microtitreplates with CHW and glandular saliva samples. The control value was obtained after preincubation with PBS-T with 0.5 mM CaC1,. HG 222 was detected in an ELISA assay. The substrate coloration was taken as a measure for HG 222 binding

only coaggregated with S. sanguis HG 1470 and S. oralis Ny 586 resulting in 10 per cent (HG 1470) and 12 per cent (Ny 586) decrease in optical density compared to the separate bacterial suspensions.

Bacterial strains listed in Table 1 were immo- bilised on microtitreplates and tested for subse- quent binding of S. gordonii HG 222. In agreement with the results of the coaggregation experiments, HG 222 only bound to S. sanguis HG 1470 and S. oralis Ny 586. The binding of HG 222 to Ny 586 was studied more extensively.

Influence of saliva The influence of glandular saliva on the binding

of HG 222 to Ny 586 was investigated by pre- incubating the Ny 586 coated microtitreplates with CHW, PAR, SM and SL saliva. After washing away unbound salivary substances, binding of HG 222 to Ny 586 was tested (Figure 1). All types of saliva were able to enhance the attachment of HG 222 to Ny 586 coated microtitreplates. Salivary preincubation of microtitreplates not coated with Ny 586 revealed no binding of HG 222 indicating no binding of salivary components to the microtitreplates that promote adhesion of HG 222.

Binding of salivary components to S . gordonii and S . oralis

To identify salivary components mediating attachment of S. gordonii to S. oralis binding of several salivary constituents to these bacteria was investigated. For this purpose, PAR and SM saliva were incubated with HG 222 and Ny 586. The depleted saliva samples were analysed by SDS- PAGE followed by Coomassie Brilliant Blue stain- ing and silver staining (Figure 2A and B). Several proteins disappeared from PAR and SM saliva after incubation with both HG 222 and Ny 586. MG-2 disappeared from SM saliva after incu- bation with HG 222 and Ny 586 (Figure 2A, lanes 6 and 7). MG-1 did not disappear from SM saliva after incubation with either H G 222 or Ny 586 suggesting no binding of MG-1. This was con- firmed by ELISA with CHW saliva on HG 222 and Ny 586-coated microtitreplates. A 350- 400 kD protein from PAR saliva, probably the salivary agglutinin, also disappeared after incu- bation with HG 222 and Ny 586 (Figure 2B, lanes 3 and 4). Saliva was depleted of a-amylase after incubation with HG 222, but not after incubation with Ny 586 (Figure 2A, lanes 3 and 4). This was confirmed by assaying a-amylase activity in saliva. After incubation with HG 222 saliva showed no

248 A. J. M. LIGTENBERG ET AL.

Figure 2. SDS-PAGE profiles (4-15 per cent acrylamide) of PAR and SM saliva before and after depletion of bacterium- binding substances. Gels were Coomassie Brilliant Blue stained (A) or silver stained (B). Lane 1, Marker, molecular weights are expressed in kilodaltons; lane 2, PAR saliva; lane 3, PAR saliva after adsorption with H G 222; lane 4, PAR saliva after adsorption with Ny 586; lane 5, SM saliva; lane 6, SM saliva after adsorption with HG 222; lane 7, SM saliva after adsorption with Ny 586

further a-amylase activity, whereas after incuba- tion with Ny 586 the salivary a-amylase activity was unaffected.

Adsorbed salivary proteins were extracted from the bacterial surface and these extracts were ana- lysed by SDS-PAGE and immunoblotting. After incubation of PAR saliva with Ny 586 and HG 222 a protein was extracted with a Mr of 350400 kD, probably the parotid agglutinin, which could be demonstrated by immunoblotting with the mono- clonal antibody E9 (Figure 3, lanes 5 and 6), recognising sialic acid epitopes. In the original PAR saliva sample the concentration of salivary agglutinin was too low to demonstrate this pro- tein by immunoblotting. After SDS-PAGE and immunoblotting of extracts from bacteria incu- bated with SM saliva, MG-2 binding was demon- strated for HG 222 (Figure 3, lane 3) but not for Ny 586 (lane 2). In contrast, MG-2 was demon- strated in extracts of Ny 586 incubated with SL saliva (lane 8).

Binding of IgA from saliva to HG 222 and Ny 586 was demonstrated by immunoblotting (Figure 4) and ELISA (Figure 5). SM saliva was depleted of IgA after incubation with Ny 586, but not after incubation with HG 222. IgA could be released from both bacteria by SDS extraction. Colostral IgA was not completely absorbed by either HG 222 or Ny 586, but could be released from the surface of both bacteria.

Involvement of salivary components Several saliva derived components were tested

for their ability to enhance the binding of S. gordonii HG 222 to S. oralis Ny 586. Microtitre- plates coated with Ny 586 were incubated with salivary components and subsequently tested for binding HG 222. Especially sIgA, MG-2 and agglutinin enhanced the attachment of HG 222 to Ny 586 (Table 2). The preparations of MG-2 and agglutinin were shown to contain IgA which might be responsible for the enhancing effect on HG 222 binding. PRPs and PRG also slightly enhanced attachment of HG 222 to Ny 586, whereas MG-I and a-amylase had no effect.

Inhibition of HG 222 binding and agglutinin binding to Ny 586 with IgA spec$c antibodies

The role of IgA in binding of HG 222 to Ny 586 was tested with several anti-IgA antibodies. Anti- a-chain serum, anti-Secretory Component serum or anti-lysozyme serum as a control were added to PAR saliva. These samples were tested for their influence on the binding of HG 222 to Ny 586. The saliva-mediated binding of HG 222 to Ny 586 was inhibited by anti-a-chain serum suggesting that IgA is involved in binding of HG 222 to Ny 586 (Figure 6B). Agglutinin binding to Ny 586 was also inhibited by anti a-chain serum (Figure 6A), whereas anti-secretory component and

INTRAGENERIC BINDING OF S. GORDON11 249

Figure 3. SDS-PAGE (4-15 per cent) and immunoblotting with the monoclonal antibody E9 directed against sialic acid epitopes on MG-1 and MG-2. E9 also recognises a 350-400 kD protein eluted from bacteria after incubation in PAR saliva. Lane 1, SM saliva; lane 2, Ny 586 eluate SM saliva, lane 3, HG 222 eluate SM saliva; lane 4, PAR saliva; lane 5 , Ny 586 eluate PAR saliva; lane 6, HG 222 eluate PAR saliva; lane 7, SL saliva; lane 8, Ny 586 eluate SL saliva. The molecular weight of the Marker proteins is expressed in kilodaltons

anti-lysozyme did not. Verification that the anti a-chain serum showed no crossreactivity with sali- vary agglutinin was obtained by immunoblotting. This result suggests that IgA and agglutinin are complexed in saliva.

Influence of EDTA on the binding of salivary components to Ny 586 and binding of HG 222

Since complexation of salivary agglutinin and IgA was previously demonstrated to be calcium- dependent,35 experiments were carried out to investigate whether EDTA abolished agglutinin binding to Ny 586 and the binding of HG 222 to Ny 586. For this purpose, Ny 586 coated microti- treplates were incubated with PAR saliva in PBS-T with 2 mM EDTA or PBS-T with 0.5 mM CaCl,. Hereafter, binding of agglutinin, IgA and HG 222 to Ny 586 was measured. EDTA inhibited binding of salivary agglutinin from PAR saliva to Ny 586 (Figure 7A). On the other hand, IgA binding to Ny 586 (Figure 7A) and saliva-mediated binding of HG 222 to Ny 586 were not inhibited by preincu- bation with EDTA (Figure 7B). These results

suggest that binding of HG 222 to Ny 586 is primarily mediated through IgA, and not through an IgA-agglutinin complex.

DISCUSSION

Intergeneric and interspecies coaggregation is a widely studied subject in oral research.'* Well known combinations of species that coaggregate are S. gordonii and Actinomyces naeslundii," Streptococcus salivarius and Veillonella alcales- ens,^^ and Porphyromonas gingivalis and A. viscosus.25 Intrageneric coaggregation between species of streptococci also O C C U ~ S . ~ ~ , ~ ~ There are several indications that coaggregation between species plays a role in the formation of dental plaque. Coaggregation between oral bacteria only occurs between species that are found together in vivo in the same stage of formation of dental plaque. In addition, many species recognise strep- tococci which are primary colonisers of the dental surface. In contrast, Skopek et d4* found little difference in bacterial accumulations on surfaces of

250

1 4 -

1 2 - - E

0 m = 1 0 -

5 08- RJ u 5 06- n

0 4 -

0 2 -

00

i

D <

A. J . M. LIGTENBERG ET AL.

. CHW s a l i v a x PAR s a l i v a + SM sal iva * SL s a l i v a

k-3 ' ' ' ' ' ' ' ' ' ' I t--

1 2 3 4 5 6 7 8 9 1 0

Figure 4. SDS-PAGE and immunoblotting with antibodies against the Secretory Component of IgA. Lane 1, SM saliva; lane 2, SM saliva after HG 222 absorption; lane 3, SM saliva after Ny 586 absorption; lane 4, SDS extract of HG 222 after incubation in SM saliva; lane 5, SDS extract of the Ny 586 surface after incubation in SM saliva: lane 6, colostral IgA; lane 7, colostral IgA after incubation with HG 222; lane 8, colostral IgA after incubation with Ny 586; lane 9, SDS extract of HG 222 after incubation with colostral IgA; lane 10, SDS extract of Ny 586 after incubation with colostral IgA

S. oralis and S. gordonii which showed different coaggregation characteristics in vitro. Both strains, however, showed saliva-induced aggregation which might influence the bacterial accumulation in vivo.

In this paper intrageneric coaggregation is described between S. gordonii HG 222 and S. oralis Ny 586. In solution, the coaggregation between HG 222 and Ny 586 is difficult to observe. There- fore, a more sensitive assay was used by measuring binding of HG 222 to Ny 586 that was immobi- lised on microtitreplates. This assay better mimics the in vivo situation of plaque formation in which bacteria also attach to bacteria on a surface. Solid-phase assays to study intergeneric bacterial attachment have been described previously, using bacteria immobilised on tissue culture plates or bovine enamel slabs,30 nitrocellulose membranes,22

1 4

p 0 4 n <

0 2

A . CHW sal iva x PAR sal iva t SM s a l i v a % SL sal iva

agarose beads43 or saliva-coated hydr~xyapatite.~' The assay described in this paper was carried out in microtitreplates, which enabled rapid screening of many parameters. Because monoclonal anti- bodies specific to S. gordonii were available,' this assay could be conducted using standard ELISA equipment without special requirements for radio- labelling. The microtitreplates assay supported the initial findings of the coaggregation experiments since only S. oralis strain Ny 586 and S. sanguis strain HG 1470 were able to bind HG 222.

The solid-phase assay used in this study also makes it possible to study the influence of saliva on the interbacterial attachment without interference of saliva-induced aggregation. Since saliva strongly aggregates both Ny 58628 and HG 222,'' the effect of saliva on interbacterial binding could not be studied in a coaggregation assay. Up to

INTRAGENERIC BINDING OF S. GORDON11 25 1

Table 2. Binding of S. gordonii HG 222 to S. oralis Ny 586 preincubated with salivary components. The percentage substrate coloration towards the control as measured in an ELISA assay was taken as measure for HG 222 binding. Binding of HG 222 to Ny 586 preincubated in PBS-T with 0.5 mM CaCl, was set at 100 per cent

Substance HG 222 binding

PBS-T with 0-5 mM CaC1, CHW saliva MG-1 (1 mglml) MG-2 (25 pglml) a-Amylase PRPs (1 00 pglml) Agglutinin (6 pglml)

PRG (100 pglml) slgA (3 Pdml)

100 163 96

166 116 132 160 160 120

now, the effect of saliva on interbacterial binding has been studied primarily in aggregation as- s a y ~ . ~ ' , ~ ~ , ~ ~ In these cases, saliva mediated binding between bacteria of the same species was tested. Salivary components mediating binding between bacteria of the same species possibly also mediate binding between bacteria of different species. A relationship has been shown between saliva- mediated interbacterial binding and saliva- mediated aggregation of the species of a coaggregation pair.40 Preincubation of a bacteria- coated surface with saliva strongly enhanced binding of a second bacterium, when one or both species of a coaggregation pair were aggregated by saliva. Saliva did not enhance interbacterial bind- ing, when the species of a coaggregation pair were not aggregated by saliva. Likewise, the major salivary component mediating binding of S. mutans to S. sanguis was salivary agg~ut in in ,~~ previously described as an aggregating factor in saliva for these two b a ~ t e r i a . ~ . ~ ~ In this study, several salivary components were tested for their influence on the binding of S. gordonii HG 222 to S. oralis Ny 586. IgA, in particular, enhanced binding of HG 222 to Ny 586 and this immuno- globulin has been described previously as a component mediating bacterial agg rega t i~n .~~ Preparations of MG-2 and salivary agglutinin were also demonstrated to enhance interbacterial binding, but both preparations contained IgA (not shown). From inhibition studies with anti- serum against the a-chain of IgA, indications were

0 4

0 2

A

4

0 0L a IF-- 0 1 2 3 4 5 6 7 Control

6

rol

Figure 6. Influence of antibodies on binding of agglutinin and HG 222 to Ny 586. PAR saliva was incubated with anti-a- chain, anti-secretory component and anti-lysozyme and subse- quently serially diluted in Ny 586 coated microtitreplates. Hereafter, agglutinin binding (A) and binding of HG 222 (B) were tested as described in the Materials and methods section. Symbols: x , PAR saliva; 0, PAR saliva+anti-a-chain; 4, PAR saliva+anti-Secretory Component; A, PAR saliva+anti- lysozyme

obtained that agglutinin binding to Ny 586 was mediated through IgA. IgA-mediated binding to bacteria was previously demonstrated for aggluti- nin to Pseudomonas aeruginosa,2 and for MG-2 to P. aeruginosa and Staphylococcus a u ~ e u s . ~ Saliva- mediated binding was also inhibited by antibodies against the a-chain of IgA, indicating that IgA or an agglutinin-IgA complex were involved in inter- bacterial binding. The complexation between IgA and agglutinin was previously shown to be calcium

Therefore, Ny 586 coated micro- titreplates were incubated with PAR saliva in the presence of EDTA. In this case, agglutinin binding to Ny 586 was inhibited, but IgA binding and HG 222 binding were not (Figure 7), suggesting that

252 A. J. M. LIGTENBERG ET AL.

demonstrated to bind to S. g ~ r d o n i i , ' ~ ~ ~ ~ and S. orali~.'~ MG-1 did not bind to Ny 586 or HG 222 and therefore could not mediate interbacterial binding.

In conclusion, this paper reports strain-specific binding of S. gordonii to S. oralis. This process is enhanced by preincubation with saliva or salivary components, especially IgA.

A

1 4 , I

" - 0 1 2 3 4 5 6 7 C o n t r o l

210g[d~lution]

B

07 081

06l " " ' ' ' ' ' ' 1 I 2 3 4 5 6 7 a 9 10

'log[ dilution]

Figure 7. Influence of EDTA on binding of salivary agglutinin and IgA to Ny 586 coated microtitreplates. Ny 586 coated microtitreplates were incubated with PAR saliva that was serially diluted in PBS-Ca or PBS with 2 m M EDTA. Here- after, binding of agglutinin and IgA (A), and binding of HG 222 (B) were assayed in an ELISA as described in Materials and methods. The substrate coloration was taken as a measure for binding. Symbols. A: IgA binding from PAR saliva in PBS-Ca (.) or PBS with EDTA ( x ); Agglutinin binding from PAR saliva in PBS-Ca (+) or PBS with EDTA (*). B: H G 222 binding after incubation of PAR saliva in PBS-Ca (m) or PBS with EDTA (0)

agglutinin was not involved with interbacterial binding.

Binding of HG 222 to Ny 586 was only slightly or not at all enhanced by PRG, PRPs, a-amylase or MG-1. Of these substances, a-amylase has been demonstrated to bind to S. gordonii, but not to S. o r a l i ~ . ~ ? ~ ~ a-Amylase could therefore not mediate binding between S. gordonii and S. oralis. PRG has been demonstrated to bind to S. sanguis ATCC 10557 (renamed S. oralis) and S. sanguis G9B (renamed S. gordonii) without inducing aggre- gation of these b a ~ t e r i a . ~ ~ , * ~ * ~ ~ PRPs have been

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