INFECTION AND IMMUNITY, Feb. 1980, p. 335-3430019-9567/80/02-0335/09$02.00/0

Vol. 27, No. 2

Sialidase-Enhanced Lectin-Like Mechanism for Actinomycesviscosus and Actinomyces naeslundii Hemagglutination

R. P. ELLEN,* E. D. FILLERY, K. H. CHAN, AND D. A. GROVE

Faculty of Dentistry, University of Toronto, Toronto, Canada M5G 1G6

Laboratory strains representing six numerical taxonomy clusters and freshisolates ofhumanActinomyces viscosus and Actinomyces naeslundii were studiedby standard flocculation slide tests for the ability to hemagglutinate erythrocytes(RBC) from various animal species. Human AB and horse RBC were agglutinatedmore frequently and rapidly than others; guinea pig RBC were agglutinated byonly a few strains. Human AB RBC were selected for studies of hemagglutinationmechanisms. Treatment ofRBC with clostridial neuraminidase (NTRBC) greatlyenhanced hemagglutination for almost all strains. In hapten inhibition experi-ments in which various concentrations of sugars were used, ,8-galactosides werethe most effective inhibitors of hemagglutination for both RBC and NTRBC;inhibition of NTRBC agglutination required higher concentrations. Soybeanlectin agglutinated both RBC and NTRBC but not Actinomyces cells. NTRBCagglutinated at a 125-fold-lower concentration. Hemagglutination was sensitiveto ethylenediaminetetraacetate for one strain tested. Hemagglutination reactionswere reversible by addition of f8-galactosides. The ability of Actinomyces strainsto "prime" RBC for hemagglutination by removing sialic acid to expose morepenultimate fl-galactoside sites was studied by recycling Actinomyces-agglutin-ated RBC which were dispersed with a lactose solution and washed free ofbacteria (primed RBC). Priming in this manner augmented subsequent hemag-glutination by indicator Actinomyces strains and made the RBC more sensitiveto agglutination by soybean lectin. The priming ability of Actinomyces strainsgenerally correlated with the amount of sialic acid removed from primed RBC.Strains representing the numerical taxonomy clusters differed in both theirhemagglutinating and priming activities. Cluster 5 strains (typical A. naeslundii)were good agglutinators of RBC, NTRBC, and primed RBC but were poorprimers. Cluster 3 strains (atypical A. naeslundii) were the weakest hemagglutin-ators but could prime RBC adequately for subsequent agglutination by otherstrains. Together, these data indicate that Actinomyces hemagglutination pro-ceeds via a two-step mechanism: (i) neuraminidase removal of terminal sialic acidand (ii) lectin-like binding to exposed ,B-galactoside-associated sites on the RBC.Strains differ in the extent to which they can perform the two functions, and thisspecificity may relate to their taxonomic classification.

Actinomyces viscosus and Actinomyces naes-lundii are closely related species indigenous tothe mouths of most humans and several animalspecies (2, 6). Human isolates have been segre-gated by numerical taxonomy into six clustersby considering overall phenotypic similarity(10). Actinomyces species have been implicatedin the pathogenesis of inflammatory periodontaldiseases by virtue of their numerical associationwith clinical lesions in humans and their con-sistent ability to induce periodontal lesions inexperimental animals. In addition to elicitingmeasurable immune responses which may me-diate tissue injury, Actinomyces cells or subcel-lular fractions thereof have been reported tointeract directly with mammalian cell systems

thought to be important in oral colonization andthe pathogenesis of periodontal lesions. Theseinclude their attachment to oral epithelial cellsand fibroblasts (8, 9), their phagocytosis by poly-morphonuclear leukocytes with subsequentrelease of hydrolytic enzymes (24), their directmitogenic stimulation of B lymphocytes (3), andtheir enhancement of bone resorption in organculture (14). Since it is likely that cell surfacereceptor interactions may act to influence tissuetropisms or to trigger biological responses inthese mammalian cell systems, investigationsinto the mechanisms by whichActinomyces cellsseek receptor-like molecules on mammalian cellswere initiated. This report deals with what isprobably the simplest of such systems, the inter-

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336 ELLEN ET AL.

action with erythrocytes (RBC) as measured byhemagglutination.

(The results of this investigation were pre-sented in part at the 57th General Session of theInternational Association for Dental Research,New Orleans, La., 29 March to 1 April 1979).

MATERLALS AND METHODSCultures and cultural conditions. A total of 25

laboratory strains representing six clusters in the nu-merical taxonomy scheme of Fillery et al. (10) wereselected to screen for hemagglutinating activity. Thesestrains (see Table 1) were derived from the lyophilizedstock culture collection of the Faculty of Dentistry,University of Toronto. A total of 17 representativelaboratory strains (see Table 2) were used for subse-quent experiments on the mechanism of hemaggluti-nation. Working stock cultures were maintained bymonthly transfers on slants of brain heart infusionagar. An additional 115 strains were freshly isolated;11 were from human root surface dental plaques, 60were from smooth surface coronal plaques, and 44were from tongue dorsa. These were identified as A.viscosus and A. naeslundii by agglutination of wholecells with species-specific antisera, analysis of acid endproducts by gas chromatography, catalase activity,and detection of the specific cell wall sugar 6-deoxy-L-talose by paper chromatography (E. D. Fillery, D. A.Grove, K. H. Chan, and R. P. Ellen, Int. Assoc. Dent.Res., abstr. no. 1112, 1978). Bacterial cells for allhemagglutination experiments were grown for 2 daysin a chemically defined medium for oral gram-positiverods (S. S. Socransky, C. Smith, and A. D. Mangan-iello, Int. Assoc. Dent. Res., abstr. no. 120, 1973) inanaerobic jars containing 80% N2, 10% H2, and 10%CO2 with a palladium catalyst.Hemagglutination assay. Bacterial cells were

harvested from 10.0-ml cultures in a bench centrifugeat 2,500 rpm, washed once in normal saline, and resus-pended in saline to an optical density of 1.0 at 550 nm(Turner model 350 spectrophotometer). Citratedhorse, sheep, and guinea pig blood were purchasedfrom Woodlyn Laboratories Ltd., Guelph, Ontario,Canada. Human type A, B, 0, and AB blood sampleswere obtained from the Canadian Red Cross, OntarioDivision. RBC were washed in 0.01 M phosphate-buffered saline (PBS) at pH 7.0 by repeated centrifu-gation at 1,000 rpm and resuspended to contain 2%(vol/vol) packed cells. Two drops of PBS and 1 dropof the bacterial and RBC suspensions were mixed inwells of plasmacrit flocculation slides (Scientific Prod-ucts) and agitated at room temperature on a TekTatorV flatbed serological rotator (American Hospital Sup-ply Corp., Evanston, Ill.) at a standard speed of 180rpm. After 5 min, the degree of hemagglutination wasscored on a 0 to 8+ scale according to the size of RBCaggregates (Fig. 1). Hemagglutination scores were gen-erally reproducible within a given batch of cells; wherescores for duplicate readings differed widely, a thirdtest was run. However, inexplicable variation in scoreswas sometimes noted between batches which weregrown for different experiments and run on separateoccasions (see Tables 1 and 2), even though inocula,media, and cultural conditions were identical.

FIG. 1. Standard scale for hemagglutination scoreafter 5 min of incubation of Actinomyces-RBC sus-pension mixtures. 8+, Maximum agglutination; 0, nohemagglutination detected.

Neuraminidase treatment of RBC. Human ABRBC were used to test the effect of neuraminidasetreatment on subsequent hemagglutination by Actin-omyces cells. RBC suspensions were incubated in ashaking water bath at 37°C for 1 h in PBS (pH 5.0)containing clostridial neuraminidase (type VI; SigmaChemical Co., St. Louis, Mo.) at a concentration of 1U/ml. Control suspensions without neuraminidasewere incubated simultaneously under similar condi-tions. The neuraminidase-treated RBC (NTRBC) andcontrol RBC were washed twice in PBS at pH 7.0 andcompared in the standard hemagglutination assay.

Effect of sugars on hemagglutination. Sincehemagglutination and other attachment-related phe-nomena of several bacterial species have been reportedto be sensitive to the introduction of specific simplesugars (4, 5, 16, 17, 18), hapten inhibition studies wereperformed with the Actinomyces-RBC system. Theseexperiments utilized both neuraminidase-treated anduntreated human AB RBC tested with each of threelaboratory strains and one freshly isolated strain (seeTable 4). The following analyzed biochemical gradesugars (D isomer unless otherwise stated) were testedfor their relative abilities to impair hemagglutinationwhen incorporated in the cell mixture: fructose, L-fucose, galactose, a-methyl-galactoside, fl-methyl-gal-actoside, glucose, a-methyl-glucoside, lactose, lyxose,mannose, mannosamine, melibiose, N-acetyl-neura-minic acid, talose, trehalose, and xylose. Two-foldserial dilutions of a maximum final concentration of50 mM were used. In addition, the abilities of thevarious sugars to disperse Actinomyces-agglutinatedRBC were compared by adding each sugar at 10 mM(final concentration) to pre-agglutinated sugar-freecell mixtures. These were rotated for an additional 5min, and the degree of dispersion was estimated as thedifference in hemagglutination scores before and afterthis period.

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A. VISCOSUS AND A. NAESLUNDII HEMAGGLUTINATION 337

Agglutination ofRBC with lectins. Soybean lec-tin and concanavalin A (ConA) were used to determinewhether terminal galactosides or mannosides or bothwere located on the RBC or bacterial cells and whetherneuraminidase treatment of RBC increased the acces-sibility of these sugars. RBC, NTRBC, and Actino-myces strains W752 and 8A06 were tested for theability to agglutinate with soybean lectin and ConA(P-L Biochemicals Inc., Milwaukee, Wis.). Slide agglu-tination tests using standard cell suspensions in PBSat pH 7.0 and containing twofold serial dilutions of thetwo lectins were performed at room temperature. Themaximum final concentrations of the lectins were 200jig of soybean lectin per ml and 200,ug of ConA per ml.

Effect of EDTA. Divalent cations are required inmany binding mechanisms, including those of somelectins (23). The possible role of calcium ions in Acti-nomyces-mediated hemagglutination was tested by us-ing NTRBC and fresh isolate 6-12d. Hemagglutinationmixtures containing 0.025mM ethylenedinitrilotetraa-cetic acid (EDTA) at pH 8.0 and similar control mix-tures free of EDTA were incubated for 5 min, and thedegree of hemagglutination was recorded. ExcessCaCl2 was then added to the mixtures at a final con-centration of 0.2 mM. A control experiment studiedthe effect of incorporating the excess CaCl2 in theoriginal mixture and adding EDTA (0.025 mM) afterthe 5-min hemagglutination reaction. These experi-ments were repeated substituting (10 mM) galactosefor the EDTA.RBC priming by Actinomyces cells. The enhanc-

ing effect of neuraminidase treatment and the abilityof some strains to hemagglutinate more strongly thanothers suggested that Actinomyces cells might haveneuraminidase activity themselves. This could, in ef-fect, unmask penultimate ,8-galactosides (fl-gal), whichwere found to be the most effective inhibiting sugars.To test this hypothesis, strains representing five of thenumerical taxonomy clusters (see Fig. 2) were eachmixed with RBC suspensions in PBS at pH 7.0 andincubated for 1 h at 37°C. The agglutinated RBC werethen dispersed with a PBS solution containing 5% (wt/vol) lactose, and all RBC in the suspension werewashed free of bacteria by repeated centrifugation at1,000 rpm (twice with lactose buffer and twice withPBS at pH 7.0). Samples of the washed RBC werestained and examined by light microscopy to assurecomplete removal of bacterial cells. The lactose-dis-persed RBC, designated "primed" RBC (PRBC), werecompared with untreated RBC and NTRBC for abilityto agglutinate with cells of strains W752 and 38fc(cluster 1), H21 and B236 (cluster 2), and 4A05 and12104 (cluster 5). The experiments were repeated,using 94 of the coronal dental plaque and tongue freshisolates as the primers and strains W752 and 8A06 asthe agglutinators. Strain W752 was chosen as an ex-ample of a strong agglutinator, and 8A06 was chosenas a non-agglutinator of untreated RBC. PRBC whichhad been primed by nine of the fresh isolates demon-strating diverse priming activity were tested for agglu-tination with various concentrations of soybean lectin,as described above.The amount of sialic acid liberated from the RBC

during priming (presumably due to sialidase activityof the Actinomyces cells) was measured by the thio-

barbituric acid assay of Warren (27), using variousconcentrations of N-acetyl-neuraminic acid (SigmaChemical Co.) to establish a standard curve. Bufferswith and without clostridial neuraminidase served aspositive and negative controls, respectively. Selectedstrains which demonstrated diverse abilities to primeRBC were used for these experiments (see Fig. 3).Because of the possibility that the different Actino-myces strains might metabolize liberated sialic acid tovarying degrees, the amount of sialic acid remainingon the RBC after priming was also estimated. WashedActinomyces-primed RBC were treated as describedabove with clostridial neuraminidase, and the residualremovable sialic acid was measured (27).

RESULTSAgglutination of RBC from different

mammalian species. Data describing the fre-quency and degree of hemagglutination by Ac-tinomyces cells mixed with RBC from variousanimal sources are listed in Table 1. Horse andhuman AB RBC were agglutinated most oftenand most rapidly. Only 4 of the 36 strains tested(all A. naeslundii) agglutinated guinea pig RBC,and these reactions were weak. Among the lab-oratory strains, those resembling typical A.naeslundii strains of cluster 5 demonstrated thegreatest degree of hemagglutination. The mostconsistent hemagglutinator among cluster 1strains was W752, which is the only A. naeslun-dii strain to segregate with the typical cluster 1A. viscosus strains by numerical taxonomy (10).Although the numbers of freshly isolated A.viscosus and A. naeslundii strains tested wereunequal and thus difficult to compare, fresh A.naeslundii isolates demonstrated the frequenthemagglutination typical of the laboratorystrains. Laboratory strains classified in clusters3 and 4 showed little, if any, hemagglutinationactivity when mixed with RBC from any of theanimal species.Agglutination of NTRBC. Tables 2 and 3

list data demonstrating the profound enhance-ment of Actinomyces-mediated hemagglutina-tion when the RBC were treated with clostridialneuraminidase. All of the 17 laboratory strainsagglutinated NTRBC more strongly than RBC(Table 2). However, the degree of augmentation(difference between hemagglutination scores forN'RBC and RBC) differed among taxonomicclusters. Neuraminidase treatment of RBC aug-mented hemagglutination with most strains by+3 to +8, whereas hemagglutination by the twocluster 3 strains tested was only increasedslightly (+1).Neuraminidase-enhanced hemagglutination

was also evident among 100 of 110 freshly iso-lated strains (Table 3). Only 13 of these strainsagglutinated untreated RBC strongly enough tobe scored greater than +2. In contrast, most

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TABLE 1. Hemagglutination ofRBC from different mammalian species by strains ofA. viscosus and A.naeslundii

Hemagglutination score

Species Numerical tax- Strain(s) Horse Sheep Guinea Human RBConomy cluster Horse___Sheep__pig

RBC RBC RBC 0 A B AB

Laboratory strainsA. viscosus 1 Be64 2+ 2+ 0 0 0 0 0

1 11B2 2+ 0 0 0 0 0 +1 WVU627 2+ 0 0 0 0 0 02 H21 3+ 2+ 0 0 0 0 2+2 B236 0 0 0 0 0 0 04 Be32 0 0 0 0 0 0 06 B25 2+ + 0 0 0 0 +6 W1053 0 0 0 0 0 0 0

Unclustered' P2, M100, T14B, 0 0 0 0 0 0 0WVU845, 38fc

A. naeslundii 1 W752 4+ 4+ 0 0 0 0 3+3 B74, 820 0 0 0 0 0 0 03 B120 + 0 0 0 0 0 05 398A 3+ 0 + 2+ 2+ 2+ 2+5 TF11 0 0 0 0 0 0 +5 4A05 + 0 0 0 0 0 +5 ATCC12104 0 0 0 0 0 0 4+

Unclusteredb 631 4+ 0 + 2+ 2+ 2+ 3+Unclusteredb N9 3+ 0 0 + + + 3+Unclusteredb C10 2+ 0 0 + + + 3+Unclusteredb C2 + 0 0 + + + +

Fresh isolatesA. viscosus 3-67fc 4+ 2+ 0 0 0 0 0

3-37fc 3+ 2+ 0 0 0 0 3+4-65a 3+ 3+ 0 + + + 3+4-16a 2+ 0 0 0 0 0 +4-19a + 0 0 0 0 0 +4-65a, 4-54a, 3-64fd 0 0 0 0 0 0 0

A. naeslundii 4-60b 3+ 0 + 2+ 2+ 2+ 3+4-71b 2+ 0 0 + + 0 2+3-66fc 3+ 0 + + + + +

a Not included in original numerical taxonomy study (10) but serologically similar to cluster 1 strains.Serologically similar to cluster 5 strains.

strains, even those which did not agglutinateuntreated RBC, agglutinated NTRBC rapidly toachieve scores of 5+ or greater. Among the 110fresh isolates, the augmentation of the hemag-glutination score was 5.13 ± 0.21 (mean ± stand-ard error of the mean).Sugar and EDTA inhibition experiments.

Among the sugars tested, hemagglutination ofboth RBC and NTRBC was most sensitive togalactose and fl-gal (Table 4). The strongestinhibitors (those active at the lowest concentra-tions) were the 13-gal lactose and ,B-methyl-gal-actoside. In general, inhibition ofNTRBC agglu-tination required higher concentrations of gal-actosides than inhibition of RBC agglutination.The only nongalactoside to demonstrate anydegree of hemagglutination inhibition below aconcentration of 50 mM was D-talose. Hemag-glutination of RBC and NTRBC was reversible.

Cell aggregates which formed during the stand-ard 5-min assay in buffer free of added sugardispersed when each of the inhibiting galacto-sides was added. Other sugars which could notinhibit hemagglutination at low concentrationsalso failed to disperse RBC-Actinomyces aggre-gates.

In the experiments with fresh isolate 6-12d,hemagglutination was also sensitive to the ad-dition of EDTA (Table 5). Mixtures of bacteria,NTRBC, and EDTA yielded no detectable ag-glutination unless excess CaCl2 was added sub-sequently. EDTA addition after hemagglutina-tion in the presence of excess calcium could notreverse the reaction. Galactose added to theagglutinated Actinomyces cells and NTRBC,even in the presence of excess CaCl2, dispersedthe aggregates. If galactose was incorporated inthe original bacteria-NTRBC mixture, no he-

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A. VISCOSUS AND A. NAESLUNDII HEMAGGLUTINATION 339

TABLE 2. Hemagglutination ofhuman AB NTRBCby laboratory strains ofA. viscous and A.

naeslundiiHemagglutination

Taxonomy . scoreBacterial straincluster

RBC NTRBC

1 W752 3+ 8+P2 0 8+WVU627 0 6+11B2 0 4+Be64 0 4+

2 H21 0 4+B236 0 4+

3 B120 0 +B102 0 +

4 Be32 0 3+8A06 0 3+

5 4A05 3+ 7+TF11 2+ 5+12104 1+ 8+C10 0 6+N9 + 6+

6 W1053 0 4+

magglutination occurred, even if excess CaCl2was added subsequently. These experimentssuggest a role for divalent cations in Actino-myces-mediated hemagglutination.RBC priming experiments. Lactose revers-

ibility allowed for the recycling of RBC whichhad been exposed to, and agglutinated by, Actin-omyces cells. Priming RBC before their exposureto agglutinator strains of Actinomyces greatlyaugmented the degree of hemagglutination (Fig.2). However, the ability to prime RBC for sub-sequent agglutination differed among strains.Priming RBC by strains classified in clusters 1,2, 3, and 4 augmented hemagglutination signifi-cantly regardless of which agglutinator strainwas utilized. The cluster 5 strains were poorprimers, increasing hemagglutination by thesame indicator strains only slightly.

Figure 3 illustrates the amount of sialic acidreleased from PRBC and the amount retainedafter priming by Actinomyces strains of diversepriming abilities. Clostridial neuraminidase wasboth the strongest primer and the most efficientliberator of sialic acid. Conversely, neuramini-dase-free buffer was the poorest primer and re-leased no measurable sialic acid. Without ac-

counting for possible metabolism of liberatedsialic acid, strains considered to be strongprimers generally released more sialic acid fromthe RBC during priming than strains known tobe weak primers. Moreover, the amount of sialicacid remaining on the RBC after priming by thevarious strains was in exact order of their de-creasing priming ability. The retention of most

sialic acid on the RBC was especially evident forweakly priming cluster 5 strains.

Like the laboratory strains, most fresh isolateswere able to prime RBC for subsequent hemag-glutination by two strains (W752 and 8A06) ofdissimilar RBC-agglutinating activity (Table 6).Augmentation was greater for hemagglutinationby strain W752 (mean ± standard error, 3.12 +0.11) than by strain 8A06 (mean ± standarddeviation, 2.02 ± 0.18). Priming by only 3 of 94strains failed to augment W752 hemagglutina-tion; 33 strains failed to augment strain 8A06.These results were similar to the relative aug-mentation of the hemagglutination of the twostrains when the RBC were primed with neur-aminidase (Table 2).Lectin agglutination of RBC. Neuramini-

dase treatment made RBC more sensitive thanuntreated control RBC to agglutination with thegalactoside- and N-acetyl-galactosaminide-spe-cific soybean lectin (Table 7). The concentrationof soybean lectin needed to agglutinate un-treated RBC was 125-fold greater than the con-centration needed to agglutinate NTRBC. Incontrast, mannose- and glucose-specific ConA

TABLE 3. Distribution offreshly isolated strainsaccording to degree of hemagglutination ofNTRBC

Hemagglutination scoreNo. of strains'

NTRBC RBC

8+ Strongb 5Weak 120 20

7+ Strong 1Weak 10 1

6+ Strong 2Weak 10 5

5+ Strong 1Weak 90 36

4+ Strong 4Weak 00 0

3+ Strong 0Weak 00 1

2+ Strong 0Weak 00 0

+ Strong 0Weak 00 1

0 Strong 0Weak 00 10

'The total number of strains was 110.b Strong, = >2+ but c4+; weak, >0 but c2+.

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TABLE 4. Effect of sugars on hemagglutination by A. viscosus and A. naeslundii strainsLowest inhibitory concn (mM)a

Sugar RBCh NTRBC"

W752 N9 W752 N9 627 6-52c

Lactose 0.1 0.1 0.8 0.4 0.8 0.8,8-Methyl-galactoside 0.2 0.2 1.6 1.6 1.6 1.6Galactose 1.6 0.8 3.0 3.0 3.0 3.0Galactosamine 1.6 0.8 3.0 3.0 3.0 3.0Talose 1.6 0.8 3.0 3.0 3.0 3.0a-Methyl-galactoside 6.25 12.5 >50 25 25 25Others >50 >50 >50 >50 >50 >50

a Endpoint concentration causing clearly detectable inhibition compared with control suspension (at least 1+difference).

'Untreated human AB RBC.'Human AB RBC treated with clostridial neuraminidase.d Fructose, L-fucose, glucose, a-methyl-glucoside, lyxose, mannose, mannosamine, melibiose, N-acetyl-neu-

raminic acid, trehalose, xylose.

TABLE 5. Effect ofEDTA, CaCl2, and galactose (insequence) on the hemagglutination ofNTRBC by

strain 6-12d0He-mag-

Original mixture Addition' glutin-ationscore

Bacteria + NTRBC + EDTA None 0Bacteria + NTRBC + EDTA CaCl2 4+Bacteria + NTRBC + CaCl2 None 4+Bacteria + NTRBC + CaCl2 EDTA 4+Bacteria + NTRBC + CaCl2 None 4+Bacteria + NTRBC + CaC12 Galactose 1+Bacteria + NTRBC + galactose None 0Bacteria + NTRBC + galactose CaCl2 0

a Human AB NTRBC were used.hThe concentrations used were as follows: CaC12, 0.2 mM;

EDTA, 0.025 mM; galactose, 10 mM.

did not agglutinate either RBC or NTRBC be-low a concentration of 50 jig/ml. MinimumPRBC-agglutinating concentrations of soybeanlectin were between those for RBC and NTRBC.PRBC showing greater degrees of hemaggluti-nation augmentations were agglutinated bylower concentrations of soybean lectin. The bac-terial cells were agglutinated by neither lectin,implying that their surfaces contained little, ifany, galactose, mannose, or glucose accessiblefor lectin binding.

DISCUSSIONAttachment phenomena are often considered

prerequisite for both colonization of mucosalpathogens and their subsequent stimulation ofbiological activity in inflammatory cells. It islikely that many of the molecular interactionsbetween surface components of microorganismsand mammalian cells involve terminal glycosideson mammalian membrane glycoproteins and

NEURAMINIDASE. ..:-1. _. . . ....

.'D---2. B:I

Wm 1f Imm627 381c 2I Xt112 n P2 .;,

H21_

B120 _

181

1 2 3 4 5 6MEAN AUGMENTATION OF HEMAGGLUTINATION SCORE

FIG. 2. RBC priming by Actinomyces strains rep-resenting five of the numerical taxonomy clusters ofFillery et al. (10). Strains used as primers are listednext to their respective positions in the taxonomyscheme on the left. Data are expressed as averagesfor two agglutinator strains representing cluster 1, 2,and 5 in the graph on the right. Data for priming byclostridial neuraminidase are shown at the top.

glycolipids (22). For example, reversible lectin-like sugar specificity has been proposed to ex-plain mucosal receptor selectivity among someenteropathogenic gram-negative species (16, 17).The investigations reported here have extendedthese observations to the gram-positive patho-gens A. viscosus and A. naeslundii. Data pre-sented suggest a two-step mechanism for Actin-omyces-mediated hemagglutination, based onthe sialidase-facilitated exposure of fl-gal siteson the RBC.Although strains ofActinomyces agglutinated

RBC frequently, few strains were found to bestrong hemagglutinators unless the RBC werepretreated with neuraminidase. Neuraminidase

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A. VISCOSUS AND A. NAESLUNDII HEMAGGLUTINATION 341

Strongest I@Nraminkins

1,eI

TusononeyClusbr Strain

2 H21

2 B2363 B1i23 B1206 W10531 W7525 TFII5 IAI

SufferControl

F........

2 4 6 YoI4Slailc Acid Releasd

(ng x 102 )

* * I *% I. I *itSilic Acid Roeined

Ing x103l

FIG. 3. Comparison of free sialic acid releasedfrom RBC duringpriming (left graph) and sialic acidremaining on PRBC after priming (right graph) byActinomyces strains of differing priming abilities.Clostridial neuraminidase and buffer free of neur-aminidase were used as positive and negative con-trols, respectively.

TABLE 6. Distribution offreshly isolated strainsaccording to RBCpriming ability for subsequent

hemagglutination by agglutinator strains W752 and8A06

Augmentation of he-No. of priming strains'

HI,65UiLJHiULUI W752 8A06

+5 0 1+4 43 30+3 32 12+2 9 11+1 7 70 3 33

a Augmentation score equals hemagglutination scorefor PRBC minus hemagglutination score for RBC.Hemagglutination scores for RBC were as follows:W752, 4+; 8A06, 0.

b The total number of priming strains was 94.

treatment augmented hemagglutination for alllaboratory strains and 100 of 110 freshly isolatedstrains tested. However, the degree of NTRBC-agglutinating activity differed among the strainstested. It is interesting to note that the poorestNTRBC agglutinators among the representativelaboratory strains tested were strains of numer-ical taxonomy cluster 3. Although they are cat-alase negative and thus usually designated A.naeslundii, they are known to be serologicallydistinct from typical cluster 5 A. naeslundiistrains and to contain some agglutinating anti-gens in common with cluster 1 A. viscosus (E.D. Fillery, R. P. Ellen, K. H. Chan, and D. A.Grove, Int. Assoc. Dent. Res., Abstr. no. 991,1979).Since removal of sialic acid from RBC mem-

branes by a commercial source of bacterial neur-aminidase enhanced hemagglutination pro-

foundry, the elaboration of neuraminidases byActinomyces strains would be expected to facil-itate their surface reactions with RBC and per-haps with other cells as well. This ability wasdemonstrated clearly in this research by com-paring the RBC priming activities of severalstrains. With the exception of two primingstrains, strains with a superior ability to primeRBC for subsequent agglutination released moremeasurable sialic acid from the RBC duringpriming than did strains with feeble primingabilities. An even closer association was foundbetween increasing amounts of sialic acid re-tained on the RBC and decreasing priming abil-ity of Actinomyces strains. The difference be-tween results measuring the amount of sialicacid released and the amount of sialic acid re-tained by the RBC during priming may be ex-plained by a strain-dependent ability to rapidlyconsume liberated sialic acid (unpublisheddata). Together, these findings indicate thatwashed Actinomyces cells contain sialidase-likeenzymes which can function to augment hemag-glutination. Using different Actinomyces strainsand somewhat different methods, Costello et al.(A. H. Costello, J. 0. Cisar, P. E. Kolenbrander,and 0. Gabriel, Int. Assoc. Dent. Res., Abstr. no.1000, 1979) reported similar preliminary find-ings. Our results further suggest that the extentof this activity may differ among taxonomicclusters. However, it is significant to note thathemagglutination potential among Actinomycesstrains was not solely dependent on primingactivity. Strains belonging to the taxonomy clus-ter with the poorest RBC priming ability (cluster5) were excellent hemagglutinators of RBCprimed by other strains or by clostridial neur-aminidase. Conversely, cluster 3 strains wereadequate primers but poor hemagglutinators.Degrees of hemagglutinating activity among

strains, especially for NTRBC and PRBC, areprobably also influenced by the number and/oraccessibility of their RBC-seeking sites. Data

TABLE 7. Agglutination ofRBC, NTRBC, andActinomyces strains with soybean lectin and ConA

Lowest agglutinating concn (,ug/ml)

Compound ActinomycesRBC NTRBC PRBC strain

W752 8A06

Soybean lec- 25 0.2 6.3a >100 >100tin

ConA >200 50 NDb >200 >200

aMean value for PRBC primed by each of nine freshisolates (range, 0.4 for best primer to 12.5 for poorestprimer).bND, Not determined.

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342 ELLEN ET AL.

from the sugar inhibition experiments indicatea likely mechanism to be a high affinity of Ac-tinomyces cells for fl-gal-associated receptors onthe RBC surface. Lactose, galactose, ,8-methyl-galactoside, galactosamine, and talose (whichhas a chemical structure from carbon atoms 3 to6 similar to that of galactose) impaired hemag-glutination at very low concentrations whencompared with other sugars. The receptor con-

taining accessible f8-gal appears to be on theRBC rather than on the bacteria because RBC,NTRBC, and PRBC agglutinated rapidly withsoybean lectin and the Actinomyces cells didnot. Neuraminidase treatment, including prim-ing, probably exposed more of these receptorsbecause higher fl-gal concentrations were re-

quired to inhibit agglutination of NTRBC thanRBC and both NTRBC and PRBC agglutinatedwith much lower concentrations of soybean lec-tin than did RBC. fl-gal inhibition of agglutina-tion, reversibility of aggregation after the addi-tion of the same galactosides, and sensitivity toEDTA suggest that A. viscosus and A. naeslun-dii elaborate surface ligands which function dur-ing hemagglutination like lectins in their affinityfor fl-gal-containing receptors. This contentionis in strong agreement with previous work byMcIntire et al. and Cisar et al., who implicateda surface protein with a f-gal-sensitive lectin-like mechanism in the coaggregation of A. vis-cosus with oral streptococcal strains (4, 15).

fl-gal inhibition of Actinomyces-mediated he-magglutination contrasts with the common find-ings that hemagglutination among gram-nega-tive bacteria and the attachment of these bac-teria in other mammalian tissue systems are

often selectively impaired by D-mannose (1, 5,16, 18, 20, 21). This is especially evident in thefrequent and strong hemagglutination of guineapig RBC whole cells or by type I pili of gram-negative bacteria (18, 20) but their infrequentagglutination by the Actinomyces strains testedin this study. Hemagglutination of some Esche-richia coli strains is mannose resistant and ap-

parently sensitive to oligosaccharides containingterminal fl-gal (11), but as of this report man-

nose-sensitive hemagglutination has not beennoted among anyActinomyces strains. However,we have reported recently that most strains ofA. viscosus and A. naeslundii themselves are

selectively agglutinated by a lectin-like contam-inant in solutions of D-mannose (7). It is inter-esting to note that the mannose contaminant,although causing aggregation of the Actino-myces cells, had no inhibitory effect on the he-magglutination reaction reported here. More-over, hemagglutinating activity of Actinomycesstrains does not correlate well with their agglu-

INFECT. IMMUN.

tination by the mannose contaminant (unpub-lished data). It is likely, therefore, that the Ac-tinomyces surface ligands for the mannose-con-taminating agglutinin and for the RBC fl-gal-associated site are distinct and located farenough apart to avoid steric interference.Within the environment where A. viscosus

and A. naeslundii colonize, contact with com-plex molecules rich in terminal N-acetyl-neura-minic acid and ,8-gal is probably frequent. Someprominent examples include the salivary glyco-proteins with which many strains are known toagglutinate (12, 13), epithelial cells on variousmucosal surfaces, and inflammatory cells whichmigrate into gingival crevices. Neuraminidaseactivity among other oral bacteria is also knownto be common (19, 25, 26). It is unlikely that thesimple two-step mechanism of sialidase-facili-tated ,8-gal binding proposed for hemagglutina-tion could account for all the selective interac-tions in such a complex environment. However,the frequency with which these functions appearamong so many strains of A. viscosus and A.naeslundii suggests more than circumstantialcomplicity in determining their ecology and/orvirulence.

ACKNOWLEDGMENTS

This investigation was supported by grant MA-5619 fromthe Canadian Medical Research Council and by Public HealthService grant DE-04464 from the National Institute of DentalResearch.

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