JOURNAL OF BACTERIOLOGY, July 1980, p. 205-2110021-9193/80/07-0205/07$02.00/0

Vol. 143, No. 1

Agglutination of Human Erythrocytes by Fusobacteriumnucleatum: Factors Influencing Hemagglutination and Some

Characteristics of the AgglutininPHILIP DEHAZYA AND R. S. COLES, JR.*

Department ofMicrobiology, New Jersey Medical School, College ofMedicine and Dentistry ofNew Jersey,Newark, New Jersey 07103

Various aspects of agglutination of human erythrocytes by Fusobacteriumnucleatum were examined. Titration experiments done in buffered saline at pH6 to 10 showed the same agglutination endpoint. The presence of high concentra-tions of NaCl in reaction mixtures did not alter titers, but KCl in concentrationsof 0.5 to 3.6 M increased titers twofold. The agglutinin was inactivated by heat,acid, alkali, 5% Formalin, and the proteolytic enzyme subtilopeptidase A andtherefore appeared to be a protein. Treatment of bacterial cells with 2-mercap-toethanol had no effect. Hemagglutination inhibition experiments revealed thatarginine and other compounds containing a guahido group as part of theirstructure were inhibitory at low concentrations. Various hexoses, some hexosederivatives, and most common metal ions, when added to bacterium-erythrocytemixtures, had no effect. The binding of dansyl-L-arginine to bacteria, but noterythrocytes, was demonstrable by ultraviolet fluorescence microscopy.

Many examples of bacterial hemagglutinationinvolve appendages such as flagella or pili on thecell surface. Some strains of enteropathogenicEscherichia coli may attach to cells by meansof pili which have been shown to be plasmidcoded (16) and which are capable of attachmentto cell surface receptors (11, 12). Hemaggluti-nation by many strains ofStreptococcus sanguiswas almost always correlated with fimbriae pro-duction (8). Purified type I E. coli pili (15) canagglutinate guinea pig erythrocytes and trypsin-ized human erythrocytes.The hemagglutinin of Fusobacterium nuclea-

tum is not associated with pili or flagella. Elec-tron microscope examination ofwhole organisms(2) or cell fragments (5) fails to reveal the pres-ence of these structures.

In a report on fusobacterial hemagglutination,Failder and Hawley (5) have shown that F.nucleatum is able to agglutinate A, B, and 0human erythrocytes and erythrocytes fromother species. They also describe some aspectsof the agglutination of sheep erythrocytes bythis organism.The experiments reported here elucidate some

properties and characteristics of the F. nuclea-tum agglutinin and describe the effects ofvariousagents and conditions on the interaction of thisorganism with human erythrocytes.

MATERIALS AND METHODSOrganism. F. nucleatum (ATCC 10953) was main-

tained as previously described (1). Bacteria for all

experiments were grown for 18 h to a cell density of108 organisms/ml in tryptic soy broth (Difco Labora-tories, Detroit, Mich.) under an atmosphere of 95%N2-5% CO2.The bacteria were harvested by centrifugation at

10,000 x g for 15 min at 50C in a Dupont Sorvall (IvanSorvall, Inc., Norwalk, Conn.) refrigerated centrifuge.The pellets were washed twice in 0.01 M phosphate-buffered saline, pH 7.5 (PBS), by centrifugation andresuspension. Washed bacteria were suspended to ap-proximately 10'0 organisms/nil in PBS and stored at-20°C for no longer than 2 months. For the experi-ments, frozen portions were thawed at room temper-ature and diluted with cold PBS (50C) to variousdensities. Bacterial suspensions treated with enzymesand dansyl-L-arginine contained 8 x 109 and 2.5 x 107organisms/ml, respectively. All other experimentsused suspensions containing 8 x 108 organisms/ml.

Erythrocytes. Fresh human group A erythrocyteswere used to determine hemagglutination titers. Thecells were washed twice with PBS and resuspended to5 x 107 cells/ml for most experiments. Those experi-ments perforned to determine the effect of metal ionsand inhibitors used suspensions containing 108 cells/mil, whereas suspensions treated with proteolytic en-zymes contained 109 cells/ml. Erythrocyte and bacte-rial counts were determined by using a Petroff-Haus-ser counting chamber.

Chemicals. Amino acids were obtained from Nu-tritional Biochemicals Corp., Cleveland, Ohio. Sugars,dansyl-L-arginine, poly-L-arginine, arginine bradyki-nin, benzoyl-L-arginine ethyl ester, and hippuryl-L-ar-ginine were obtained from Sigma Chemical Co., St.Louis, Mo. Formaldehyde solution (37%) was fromFisher Scientific Co., Fairlawn, N.J. Metal salts wereof reagent-grade quality and were obtained from Mal-

205

on January 31, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

206 DEHAZYA AND COLES

linckrodt Chemical Works, St. Louis, Mo. %

Determination of hemagglutination titers. Ti-ters were determined by serial twofold dilution of 0.3-ml volumes of bacterial suspension in acid-washedKimax (Kimble Products Inc., Toledo, Ohio) tubes (13by 100 mm). To each dilution tube, 0.3 ml of erythro-cyte suspension was added. The tubes were shakenand incubated at 50C for 3 h, and the titers wererecorded. The endpoint was defined as the last dilutiontube showing complete agglutination of the erythro-cytes. Titers are reported here as the reciprocal of theendpoint dilution.Hemagglutination inhibition experiments. To

screen amino acids, sugars, and other substances forinhibition of hemagglutination, the substances wereincorporated into bacterium-erythrocyte mixtures asused for hemagglutination titer determination. Aminoacids and sugars were screened at final concentrationsof 0.01 and 0.1 M, respectively. All other substanceswere initially tested at 0.01 M concentration. Whereinhibition was noted, the minimal inhibitory concen-tration was determined. The inhibitor concentrationreported is the highest dilution of the substance ableto decrease the titer twofold. Hemagglutination titersat pH 5 through 9 were determined in PBS adjustedto the appropriate pH. Titrations at pH 10 were per-formed in 0.01 M carbonate-bicarbonate-buffered sa-line.The effects of ionic strength on agglutination titers

were determined in 0.15, 0.25, 0.50, and 1.0 M NaCland KCI. Also tested were 2.5 M NaCI and 3.6 M KCI.All salt solutions contained 0.01 M phosphate buffer,pH 7.5. These titers were recorded after 16 h of incu-bation at 5C.To determine the effects of temperature, hemagglu-

tination experiments were done at 5, 22, 30, and 370C.Titers were recorded after 2 h at 220C and 1 h at 30and 370C.The effects of metal ions on hemagglutination titers

were determined by performing experiments in thepresence of 1 mM solutions of the following: MnCI2.H20, CoCl2.6H20, Na2HAsO4.7H2O, RbCl, K2Cr2O7,HgCl2, CuCl2, ZnCI2, and UO2(CH3COOH)2. Bacteriawere pretreated with 1 mM AgNO3 in distilled water.After incubation for 30 min at 00C, the bacteria werecentrifuged as described previously, suspended to theiroriginal volume with saline, and titrated.Treatment ofbacteria with physical and chem-

ical agents and their effect on hemagglutination.(i) Heat. Portions (5.0 ml) of a bacterial suspensionwere incubated at: 56, 60, 65, and 700C. At 5-, 12.5-,25-, and 45-min intervals, samples were taken fromeach portion, cooled to 5°C, and titrated. Bacteriaheated at 1000C were sampled from a 1-ml aliquotafter 1 min of heating.

(ii) Acid and alkali. Bacterial suspensions (0.9ml) were treated with 0.1 ml of either 1 N NaOH orHCO. After incubation for 30 min at 00C, the portionswere adjusted to pH 7.5 with either 1 N HCI or NaOHand then titrated.

(iii) Alkylation. Alkylation of bacteria was doneby suspending bacteria in PBS containing 5% Formalin(pH 7.5). The mixtures were incubated for 22 h at 5,22, 37, and 500C. Each sample was then washed,resuspended in PBS to its original volume, and thentitrated.

(iv) Treatment with arginine. Bacterial anderythrocyte suspensions were incubated in 5 mM L-arginine hydrochloride dissolved in PBS at pH 7.5.After 30 min at 00C, one portion was washed twicewith PBS at 50C by centrifugation and resuspensionas described previously, brought to its original 2-mlvolume with PBS, and then titrated. Another portionwas titrated after dialysis for 24 h at 50C against 1,000volumes of PBS.

Erythrocytes treated with arginine were washedtwice with cold PBS by centrifugation at 2,000 x g for5 min and resuspended to their original density. Then0.3-ml volumes were added to serially diluted, un-treated bacteria, and agglutination was allowed toproceed.Treatment of bacteria with proteolytic en-

zymes and lysozyme and their effect on hemag-glutination. For attempted digestion of the agglu-tinin, the following enzymes were used: bacterial pro-nase (K & K Laboratories, Hollywood, Calif.), lyso-zyme (Calbiochem, La Jolla, Calif.), trypsin type IX,a-chymotrypsin, protease type IV from Streptomycescaespitosis, subtilopeptidase A from Bacillus subtilis,papain type IV (Sigma Chemical Co., St. Louis, Mo.),and Staphylococcus aureus V8 protease (Miles Labo-ratories, Elkhart, Ind.). Fresh enzyme solutions weremade up in 0.02 M PBS (pH 7.5) before each experi-ment. Enzyme solutions contained 2 mg/ml except forprotease type IV, which contained 20 mg/ml becauseof lower proteolytic activity.

Bacteria were suspended in 0.02 M PBS (pH 7.5) to8 x 109 organisms/ml. Two-milliliter portions of thecell suspensions were incubated at 370C for 5 min, andthen 0.1 ml of enzyme solution was added.

Control suspensions received 0.1 ml of PBS. Por-tions treated with papain contained 2 mM EDTA and5 mM cysteine hydrochloride. A control suspensionfor papain treatment also contained 2 mM EDTA and5 mM cysteine hydrochloride but no enzyme. Duringincubation, changes in turbidity of the suspensionswere determined with a Coleman Junior spectropho-tometer (Coleman Instruments, Springville, Md.) setat 660 nm. After incubation at 370C for 20 h withconstant shaking, the organisms were harvested bycentrifugation and washed once with PBS at 50C aspreviously described. After suspension of the orga-nisms to their original volume with PBS, the suspen-sion was titrated.Treatment oferythrocytes with proteolytic en-

zymes and their effect on hemagglutination. Por-tions of an erythrocyte suspension (0.25 ml) containing109 cells/ml were incubated with the enzymes as de-scribed above. After 3 h of incubation, the cells werecooled to 00C and washed with 20 volumes of PBS at50C by resuspension and centrifugation. The super-natant fluid was removed by aspiration, and the eryth-rocytes were diluted to 5.0 ml with PBS at 5°C. Theenzyme-treated cells were added to serial twofold di-lutions of bacterial suspensions of known titer.UV microscopy. One milliliter of either erythro-

cyte or F. nucleatum suspension was mixed with equalvolumes ofa 10-2M dansyl-L-arginine solution in PBS.After incubation for 30 min at 00C, a drop of eachmixture was placed on a slide and allowed to dry at220C. The slides were rinsed in distilled water untilunbound dansyl-L-arginine was removed and then

J. BACTERIOL.

on January 31, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ERYTHROCYTE AGGLUTINATION BY F. NUCLEATUM 207

blotted dry. They were examined for fluorescence witha Zeiss research microscope (Carl Zeiss, Inc., NewYork) fitted with UV objectives and an Olympus cam-

era with automatic exposure control.

RESULTSEffect of pH, ionic strength, and temper-

ature of incubation on hemagglutination.The effect of various conditions on the hemag-glutination titer of a suspension of F. nucleatumis shown in Table 1. Titers were unchanged byvariation of pH from 6 to 10. At pH 5, autoag-glutination of erythrocytes occurred. Some he-molysis was observed at pH 10 in carbonate-bicarbonate buffer, but a control titration in thisbuffer at pH 7.5 exhibited no hemolysis or effecton the agglutination endpoint.No reduction in hemagglutination titers was

brought about by increases in the ionic strengthof the reaction mixture. In the presence of up to2.5 M NaCl, titers of bacterial suspensions were

unchanged. However, concentrations of KCIfrom 0.5 to 3.6 M caused twofold titer increases.When a bacterial suspension was titrated at

various incubation temperatures, those tubeskept at 50C (control) and 22°C showed a titer of32. Tubes incubated at 30 and 37°C showed onlypartial agglutination at any dilution of bacteria.However, resuspension of agglutination mix-tures incubated at 30 and 370C and subsequentincubation at 50C showed the same titer as 50Ccontrols. Also, when tubes that had been allowedto agglutinate normally at 50C were moved to a

370C water bath, the mat of agglutinated eryth-rocytes collapsed and partial agglutination was

seen.It was observed that when control tubes con-

taining only buffer and erythrocytes were incu-bated at 370C, buttoning of the erythrocytesoccurred within 30 min. This was about fourtimes faster than at 50C. Since agglutination can

TABLE 1. Effect of various conditions onfusobacterial hemagglutination titers'

Reaction mixture conditions Titer, reciprocal

Control, PBS, pH 7.5 32pH 6-10 32NaCl, 0.15-2.5 M 32KCl

0.15-0.25 M 320.5-3.6 M 64

Hg2+ ions 128Other metal ions 32Temp of incubation (°C)

5, 22 3230,37 0

'All reaction mixtures contained 5 x 107 erythro-cytes/ml. The total volume was 0.6 ml. All experi-ments were done at least twice.

take place at 30 and 370C, a possible explanationfor lack of complete agglutination at these tem-peratures is that a reduction in density of thesuspending medium at elevated temperaturesallowed the collapse or prevented the formationof the agglutinated mat of erythrocytes.Effect ofmetal ions on hemagglutination.

Upon addition of various metal ions to the bac-terium-erythrocyte reaction mixture, severalchanges were noted. Hg2+ caused a fourfold in-crease in titer, from 32 (control level) to 128(Table 1). Bacteria pretreated with Hg2+ beforethe addition of erythrocytes did not exhibit al-tered agglutinating ability, but erythrocytes pre-treated with Hg2+ and then added to bacteriainvariably lysed. Due to the incompatibility ofAg+ with other ions, agglutination experimentscould not be carried out in its presence. Exposureof bacteria to Ag+ for 30 min in distilled waterand their subsequent suspension in saline causedno change in agglutinating ability. Finally, theaddition of Cu2" or Zn2+ to erythrocytes causedthem to autoagglutinate so that any effects on

agglutination by fusobacteria could not be de-termined.

Inhibition of agglutination by variouscompounds. Attempts were made to detect spe-cific inhibitors of hemagglutination. Aminoacids, sugars, amino sugars, and other sub-stances were screened for inhibitory activity bytheir incorporation into agglutinating mixtures.Those tested and found active are reported inTable 2 with their miniimal inhibitory concentra-tions.As can be seen, the most potent inhibitors

were arginine and polyarginine (average molec-ular weight, 60,000). Ornithine, however, al-

TABLE 2. Inhibition offusobacterialhemagglutination by amino acids and derivatives'

Miniimal molar inhibitory

Compound concn

10-6 10-5 10-4 10 10-2

Arginine XPoly-L-arginine XCanavanine XBenzoyl-L-arginine ethyl

ester XHippuryl-L-arginine XArginine bradykinin XArginyl glutamine XDansyl-L-arginine XGuanidine hydrochloride XCreatine XLysine X

a The compounds were able to decrease hemagglu-tination titers when present in reaction mixtures atthe levels indicated. Concentrations reported are av-erages of at least five experiments.

VOL. 143, 1980

on January 31, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

208 DEHAZYA AND COLES

though structurally resembling arginine butlacking a guanido group, showed no activity evenwhen present in a final concentration of 0.1 M.Furthermore, guanidine and several guanido-containing compounds were relatively potent in-hibitors. Lysine inhibited agglutination at a con-centration of 102 M, possibly because of itspositively charged epsilon amino group (pKa10.2). Neither the positively charged alcohol pol-yvinyl pyrrolidone (0.25%; average molecularweight, 25,000) nor protamine sulfate (1 mg/ml)interfered with agglutination. The pharmacolog-ical agent bradykinin, having arginine residuesat both amino and carboxy terminals, was alsoinhibitory.The sugars glucose, galactose, mannose, fu-

cose, N-acetyl-D-glucosamine, and N-acetyl-D-galactosamine exhibited no inhibitory activity at0.1 M concentration. N-acetylneuraminic acidagglutinated erythrocytes at concentrations of10' M and higher. The effects of this sugar onhemagglutination by F. nucleatum could not bedetermined at these concentrations. Lower con-centrations of this substance had no effect onagglutination titers.Binding of arginine to cell surfaces. We

attempted to determine whether arginine boundto either bacteria or erythrocytes or both toexert its inhibitory effect. Suspensions of F. nu-cleatum (titer, 32) and erythrocytes were eachincubated in the presence of 5 mM argininehydrochloride. This concentration is over 500times the amount necessary to inhibit hemagglu-

3 =a

0~~~~~

UL

0

tination. After bacteria were either washed withor dialyzed against arginine-free buffer, theyshowed no differences in their ability to agglu-tinate erythrocytes when used in titration exper-iments. Similarly, when mixed with untreatedbacterial suspensions, arginine-treated erythro-cytes exhibited no changes in their agglutina-bility after washing. These results demonstratedthat arginine could not be bound and retainedby bacteria or erythrocytes in inhibitory quan-tities after washing or dialysis.

Since residual arginine binding could not bedetected by inhibition of hemagglutination, weattempted to visualize the binding of dansyl-L-arginine by UV microscope examination of bac-terial cells and erythrocytes. This fluorescentanalog was shown to be inhibitory at a concen-tration of lo-4M by hemagglutination inhibitionexperiments (Table 2).

After incubation of bacteria and erythrocytesin 5 mM dansyl-L-arginine, samples of each sus-pension were put on slides, dried, and examinedby UV microscopy. It was observed that bacteriawere highly fluorescent, whereas erythrocytesdid not fluoresce.Heat sensitivity of the agglutinin. The

effects of incubation at various temperatures onthe hemagglutination titer of a suspension of F.nucleatum are shown in Fig. 1. The agglutininwas rapidly inactivated at 1000C, whereas inac-tivation at lower temperatures was slower andtime dependent. A notable feature was the sta-bility of the agglutinin at 560C. After 24 h of

6* 24 h56"

70 0S 0

Hooting time (min)FIG. 1. Heat inactivation of the F. nucleatum hemagglutinin. Bacterial suspensions containing 8 x 1ic

organisms per ml were heated at various temperatures for the times indicated. Portions were removed afterheating, allowed to cool to room temperature, and titrated.

J. BACTERIOL.

on January 31, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ERYTHROCYTE AGGLUTINATION BY F. NUCLEATUM 209

exposure, titers of bacterial suspensions wereunchanged.

Sensitivity of the agglutinin to chemicaltreatment. Bacteria alkylated with 5% formal-dehyde at various temperatures showed a tem-perature-dependent inactivation of agglutina-ting ability, complete loss occurring at 37 and500C (Table 3). Inactivation at 500C was not theresult of heat denaturation since heat inactiva-tion experiments (Fig. 1) showed that the agglu-tinin was stable for a similar length of time at ahigher temperature (560C).The agglutinin was more sensitive to alkali

than acid. A complete loss of agglutinating abil-ity was seen after exposure to 0.1 N NaOH,whereas a similar concentration of HCI causedonly a fourfold drop in the titer. Treatment with2-mercaptoethanol even at 500C had no effecton titers.

Ethanol, methanol, ethyl acetate, toluene, andacetone were screened for their ability to renderbacteria non-agglutinating. After bacteria weresuspended in the solvents for 30 min, harvestedby centrifugation, and dialyzed against PBS,they still retained agglutinating ability (data notshown).

Effect of enzyme treatment of bacteriaand erythrocytes on hemagglutination. Theeffects of enzyme treatment on the agglutinatingability of F. nucleatum suspensions and the ag-glutinability of erythrocytes are shown in Table4. Of the enzymes used to treat bacteria, onlysubtilopeptidase A reduced the agglutination ti-ter. This reduction was not the result of a de-crease in the number of bacterial cells in thesuspension. Cell counts done before and afterthe incubation period were within 10% of controlvalues. However, all the enzymes were able tomarkedly reduce the turbidity of the suspen-sions. When enzyme-treated cells were stainedby Gram stain and observed microscopically,they were generally coiled or flexed. Controlcells retained their straight, spindle-shaped ap-pearance.

TABLE 3. Effect of chemical treatment ofF.nucleatum on hemagglutinationa

Treatment Titer, reciprocal

Control 32HCI, 0.1 N 8NaOH, 0.1 N 05% Formaldehyde, 22 h

5, 220C 3237,500C 0

1% 2-mercaptoethanol 32

a Bacterial cells treated with acid or alkali were keptat 00C for 30 min; mercaptoethanol treatment tookplace at the same temperatures as formaldehyde treat-ment. Results are from at least two experiments.

TABLE 4. Effect of enzymatic digestion of F.nucleatum and erythrocytes on agglutinationa

TiterEnzyme

F. nucleatum ErythrocytesControl, no enzyme 160 16Trypsin 160 128Chymotrypsin 160 Incomplete

AgglutinationS. aureus V8 protease 160 16Papain 160 Incomplete

AgglutinationSubtilopeptidase A 0 Incomplete

AgglutinationBacterial pronase 160 Incomplete

AgglutinationLysozyme 160 Not done

a Bacterial suspensions containing 8 x 109 anderythrocyte suspensions containing 109 cells/mnl weredigested with 100,ug of the listed enzymes per ml for22 and 3 h, respectively. Enzyme-treated bacteria wereallowed to agglutinate untreated erythrocytes. En-zyme-treated erythrocytes were mixed with untreatedbacterial suspensions. Experiments were repeated atleast six times.

Fusobacterial suspensions containing 8 x 108organisms per ml which were incubated withlysozyme (100 ,ug/ml) usually displayed two- tofourfold titer decreases as compared with un-treated suspensions of the same cell density.Such changes were not detected when lysozymewas used to treat bacterial suspensions contain-ing 8 x 109 organisms/ml, even when the lyso-zyme concentration was also raised 10-fold (1mg/ml). It was subsequently found that whenlysozyme-treated cell suspensions of low turbid-ity (8 x 10' organisms/ml) displaying titer de-creases were washed several times after enzymetreatment, hemagglutination titers were re-stored to control levels. Furthermore, hemagglu-tination mixtures containing 25 ,ug of lysozymeper mi failed to react. Therefore, we concludedthat residual lysozyme was blocking the hemag-glutination reaction.When erythrocytes that had been treated with

various proteases were used in titration experi-ments, those treated with trypsin displayed en-hanced agglutinability. Hemagglutination titersof bacterial suspensions were increased fourfoldover the same suspensions titrated by using un-treated erythrocytes. Erythrocytes treated withother proteases all exhibited decreased aggluti-nability (Table 4), resulting in dilution tubesdisplaying only partial or incomplete agglutina-tion.

DISCUSSIONAgglutination of human erythrocytes by F.

nucleatum can occur under a wide variety of

VOL. 143, 1980

on January 31, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

210 DEHAZYA AND COLES

conditions. Bacteria suspended in buffered sa-line at pH 6 through 10 or in high concentrationsof NaCl did not exhibit changes in ability toagglutinate erythrocytes. However, addition ofKCl to concentrations of 0.5 M and above in-creased titers twofold. It has been reported thataddition of neutral salts to antigen-antibodymixtures can enhance antibody binding by de-hydration of the binding sites and antigenic de-terminants (18). A similar mechanism could beoperating during KCl-induced titer enhance-ment, but the lack ofenhancement after additionof NaCl would seem to contradict this explana-tion.The results presented here suggest that agglu-

tination ofhuman erythrocytes by F. nucleatumis mediated by a protein moiety on the bacterialcell surface. This conclusion is supported byexperiments demonstrating inactivation of theagglutinin by heat, Formalin, acid, alkali, andsubtilopeptidase A. The agglutinin is probablycell wall associated since no evidence has beenfound for the presence of flagella, pili, or fimbriae(2, 5), structures which have been implicated inhemagglutination by other organisms. The li-popolysaccharide of F. nucleatum is probablynot involved in hemagglutination since phenol-extracted lipopolysaccharide failed to aggluti-nate sheep (5) and human (R. S. Coles, Jr.,unpublished observations) erythrocytes.The altered morphology observed when fuso-

bacterial cells were treated with proteolytic en-zymes and lysozyme was not always correlatedwith loss of agglutinating ability. All enzyme-treated cells were distorted, yet only subtilopep-tidase A inactivated the agglutinin. Similarly,the magnitude of the decrease in the turbidityofthe suspensions was not related to inactivationof the agglutinin.

In some experiments (not shown), lysozymetreatment of F. nucleatum suspensions (8 x 108organisms/ml) of low titer (32) for 22 h resultedin up to a fourfold decrease in titer. Since lyso-zyme treatment resulted in significant loss ofsuspension turbidity as compared with controls,and cell counts before and after treatment wereunchanged, the titer decrease was interpreted tobe the result of a modification of the "nativity"of the agglutinin by murein hydrolysis. Thisconclusion implies that the agglutinin is some-how associated with the murein layer. However,agglutination of erythrocytes was completely in-hibited when lysozyme was present in the reac-tion mixture at 40C in a concentration of 25 ug/ml. Inhibition was not detected in experimentsusing lysozyme-treated bacterial suspensions ofhigh (160) titer. Since complex formation be-tween lysozyme and acidic proteins does occur

J. BACTERIOL.

(3, 17), lysozyme, a basic protein (pl 10.9), mayhave been bound to bacterial cells and retainedafter the suspensions were centrifuged and re-suspended. In one experiment, lysozyme-treatedfusobacteria which had exhibited a twofold titerdrop were restored to control titer levels byfurther washing. The mechanism by which ly-sozyme inhibits hemagglutination is unknown,but its amino-terminal residue, arginine, may beavailable for inhibition.The finding that almost all inhibitors of he-

magglutination had a guanido group as part oftheir structure suggests that this group plays acentral role in agglutinin-receptor binding. Theguanido group gives arginine a uniquely cationiccharacter at physiological pH. This group willremain positively charged under conditions ofhigh alkalinity, being neutralized at pH 12.5.Arginine has (i) been implicated in substraterecognition by enzymes of the glycolytic path-way (14), (ii) been suggested to function as acontact residue in the hapten binding site ofmyeloma protein 315 (9), and (iii) been impli-cated in defining the interactions of oligonucle-otides with arginine-containing peptides (13).Our data demonstrating binding of dansyl-L-ar-ginine to bacterial cells and inhibition of agglu-tination in the presence of low concentrations ofarginine imply that this amino acid may functionas a contact residue between bacteria and eryth-rocytes during agglutination. This conclusion isfurther strengthened by the observation thataddition of arginine to previously agglutinatederythrocytes to a concentration of 1 mM cancompletely disrupt the interaction.We were not able to demonstrate the retention

of arginine by bacteria or erythrocytes duringwashing in quantities sufficient to inhibit he-magglutination or reduce erythrocyte aggluti-nability. If arginine was binding to either celltype during inhibition, the washing proceduresused probably removed it. Dansyl-L-argininewas seen to bind exclusively to bacterial cells.Since this arginine analog was a potent inhibitorof hemagglutination, we feel that bacterial cellswere the target for molecules containing guanidogroups which were capable of interrupting agglu-tination.The monosaccharides glucose, galactose, man-

nose, fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosanine, and N-acetylneuraminic acid,some of which are components of erythrocyteglycoproteins (4) and F. nucleatum lipopolysac-charide (6, 10), did not inhibit agglutinationwhen present in 0.1 M concentration. However,these data do not exclude the involvement ofsugar moieties in the cell-cell interaction, sincedi- or trisaccharides could be required to cause

on January 31, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ERYTHROCYTE AGGLUTINATION BY F. NUCLEATUM 211

inhibition. However, a "lectin-like" interaction,inhibitable by simple sugar moieties, is probablynot operating in this system.The increased agglutination titers seen when

erythrocytes were trypsinized probably reflectsmore efficient cross-linking of erythrocytes bybacterial cells. These observations are probablyanalogous to enhanced lectin agglutinability (7)observed after trypsinization of erythrocytes.Decreased agglutinability of erythrocytes

after treatment with a-chymotrypsin, papain,bacterial pronase, and subtilopeptidase A sug-gested that the receptor was a protein. However,to identify the receptor will probably requireisolation of receptor activity from erythrocytemembranes and comparison of its characteristicswith those proteins of known identity.

Cells of diverse origin such as erythrocytes,mycoplasma, cultured cells, and E. coli sphero-plasts are capable of interaction with F. nuclea-tum (R. S. Coles, Jr., unpublished observations).This suggests that a common moiety exists onthe surface of these cells. If this is so, the agglu-tinin may be used as a tool much like an anti-body to probe the features of various mem-branes.

LMRATURE CITED1. Coles, R. S., Jr. 1973. The effect of glutathione on the

growth ofFusobacteriumpolymorphum. J. Gen. Micro-biol. 75:75-81.

2. Dahlen, G., H. Nygren, and H. A. Hansson. 1978.Immunoelectron microscopic localization of lipopoly-saccharides in the cell wall of Bacteroides oralis andFusobacterium nucleatum. Infect. Immun. 19:265-271.

3. Ehrempreis, S., and R. S. Warner. 1956. The interac-tion of conalbumin and lysozyme. Arch. Biochem. Bio-phys. 62:38-50.

4. Emerson, W. A., and S. Kornfeld. 1976. Characteriza-tion of the oligosaccharide units of bovine erythrocytemembrane glycoprotein. Biochemistry 15:1697-1703.

5. Falkler, W. A., Jr., and C. E. Hawley. 1977. Hemagglu-

tinating activity of Fusobacterium nucleatum. Infect.Immun. 15:230-238.

6. Fredriksen, G., and T. Hofstad. 1978. Chemotypes ofFusobacterium nucleatum lipopolysaccharides. ActaPathol. Microbiol. Scand. Sect. B 86:41-45.

7. Gordon, J. A., and C. A. Keuttner. 1978. Enhancedagglutination of all the erythrocytes when only half aretrypsinized. Nature (London) 272:636-638.

8. Henriksen, H. D., and E. Lov8tad. 1976. Hemaggluti-nation of twitching Streptococcus sanguis. Acta Pathol.Microbiol. Scand. Sect. B 84:437-440.

9. Klostergaard, J., L. M. Krasz, A. L. Grossberg, andD. Pressman. 1977. Arginine as a contact residue inthe hapten binding site of protein 315. Immunochem-istry 14:107-110.

10. Kristoffersen, T., and T. Hofstad. 1970. Chemical com-position of lipopolysaccharide endotoxins from humanoral fusobacteria. Arch. Oral Biol. 15:909-916.

11. Moon, H. W., B. Nagy, R. E. Isaacson, and I. Orskov.1977. Occurrence of K99 antigen on Escherichia coliisolated from pigs and colonization of pig ileum by K99+enterotoxigenic E. coli from calves and pigs. Infect.Immun. 15:614-620.

12. Ofek, I., D. Mirelman, and N. Sharon. 1977. Adherenceof Escherichia coli to human mucosal cells mediatedby mannose receptors. Nature (London) 265:623-625.

13. Porschke, D. 1978. Thermodynamic and kinetic param-eters of oligonucleotide-oligopeptide interactions: spec-ificity of arginine-inosine association. Eur. J. Biochem.86:291-299.

14. Riorden, J. F., K. D. McElvany, and C. L. Borders,Jr. 1977. Arginyl residues: anion recognition sites inenzymes. Science 195:884-886.

15. Salit, L. E., and E. C. Gotschlick. 1977. Hemagglutina-tion by purified type I Escherichia coli pili. J. Exp.Med. 146:1169-1181.

16. Smith, H. W., and M. A. Linggood. 1971. Further ob-servations on Escherichia coli enterotoxins with partic-ular regard to those produced by atypical piglet strainsand by calf and lamb strains: the transmissible natureof these enterotoxins and of a K antigen possessed bycalf and lamb strains. J. Med. Microbiol. 5:243-250.

17. Steiner, R. F. 1953. Reversible association processes ofglobular proteins. II. Electrostatic complexes of plasmaalbumin and lysozyme. Arch. Biochem. Biophys. 47:56-75.

18. Van Oss, C. J., J. F. Mohn, and R. K. Cunningham.1978. Influence of various physiochemical factors onhemagglutination. Vox Sang. 34:351-361.

VOL. 143, 1980

on January 31, 2020 by guesthttp://jb.asm

.org/D

ownloaded from