Vol. 39, No. 1INFECTION AND IMMUNITY, Jan. 1983, p. 253-2610019-9567/83/010253-09$02.00/0Copyright C 1983, American Society for Microbiology

Identification and Characterization of the Major Cell EnvelopeProteins of Oral Strains of Actinobacillus

actinomycetemcomitansJOSEPH M. DI RIENZO* AND ERIC L. SPIELER

Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia,Pennsylvania 19104

Received 11 June 1982/Accepted 18 October 1982

The major cell envelope protein compositions of seven Actinobacillus actino-mycetemcomitans strains of human origin were compared by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The major envelope polypeptideswere homogeneous, in relation to molecular weight, in all of the strains that wereexamined. The characterization of the five major proteins, designated Env]through EnvS, in the leukotoxic strain Y4 revealed that proteins Env2 to -5 mayreside in the outer membrane as suggested by differential detergent extractionsand 125I-labeling experiments. The proteins did not demonstrate covalent or ionicinteractions with the peptidoglycan; however, one protein, Env2, displayed heat-modifiable properties, having apparent molecular weights of 32,000 and 45,000when heated in sodium dodecyl sulfate at 50 and 100°C, respectively. The proteincomposition of the extracellular "bleb" material, normally released by strain Y4,was determined, and proteins Envi to 4 were the predominant protein speciesfound. A comparison of the cell envelope proteins of strain Y4 with those of othermembers of the human oral flora, including species within the genera Capnocyto-phaga, Bacteroides, and Fusobacterium, revealed distinct differences on the basisof molecular size and heat-modifiable properties. However, the membraneproteins of Haemophilus aphrophilus showed a remarkable degree of homologywith those of A. actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, agram-negative, capnophilic, rod-shaped bacteri-um has attracted considerable attention as apossible periodontopathic agent in young adults.This microorganism has been recovered fromoral lesions associated with localized juvenileperiodontitis (27, 34, 38), and these isolates,when introduced into gnotobiotic animals, stim-ulated tissue destruction characteristic of thisform of periodontal disease (18, 28). In addition,A. actinomycetemcomitans has also been isolat-ed from other clinically significant infections,including brain (23) and thyroid gland (7) ab-scesses, endocarditis (12, 25), osteomyelitis(26), as well as from a urinary tract infection(40).One strain of A. actinomycetemcomitans,

designated Y4, has recently been the subject ofintensive investigations concerning the morphol-ogy, serology, and pathogenicity of this bacteri-um. Strain Y4 exhibits a typical gram-negativeultrastructure and contains extracellular poly-meric material, as well as vesicles or "blebs"(17). Multiple biological activities have beenassociated with this strain. Kiley and Holt (20)have purified the lipopolysaccharide and demon-

strated that this material stimulated bone resorp-tion, was toxic for macrophages, and inhibitedplatelet aggregation. Shenker and co-workers(B. Shenker, W. McArthur, C. C. Tsai, P.Baehni, and N. Taichman, J. Dent. Res. Spec.Issue A, vol. 60, abstr. no. 858, 1981) identifiedand partially purified a factor which suppressedthe concanavalin A-induced proliferation of hu-man peripheral blood lymphocytes. The prelimi-nary characterization of this material suggesteda 40,000- to 50,000-molecular-weight protein(33). In addition, strain Y4 contains a heat-labile, protease-sensitive component (leuko-toxin), which is cytotoxic for human peripheralblood polymorphonuclear leukocytes (1) andmonocytes (36). Although the results of theseinvestigations suggest that the leukotoxin is pro-teinaceous in nature, the composition of thismolecule has not been elucidated. Hammondand Stevens (15) found that the presence ofleukotoxic activity appears to be correlated withthe presence of a methylpentose-enriched carbo-hydrate antigen. This carbohydrate polymer ap-pears to represent one of the major antigens inthe cell; however, it does not demonstrate leu-kotoxic properties in isolated form. Interesting-

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254 DI RIENZO AND SPIELER

ly, the extracellular blebs of strain Y4 containlipopolysaccharide (17), the methylpentose-en-riched antigen, and another non-lipopolysaccha-ride, heat-sensitive bone-resorbing factor; andexhibit leukotoxic activity (15). Taubman andco-workers (39) have reported that almost 90%of the localized juvenile periodontitis patientswhom they examined had detectable immuno-globulin G (IgG) antibodies against strain Y4, asmeasured by the enzyme-linked immunosorbentassay, and 67 to 71% of these patients respondedspecifically to known cell surface components,that is, the leukotoxin, methylpentose-enrichedantigen, and lipopolysaccharide (39).Based upon the preponderance of biological

activities expressed by A. actinomycetemcomi-tans Y4, a thorough understanding of the cellsurface architecture of this bacterium is essen-tial to elucidate the mechanisms of pathogen-host interactions. Several of these biologicalactivities, such as the leukotoxic and secondarybone-resorbing factors, as well as a significantproportion of the antigenic response in localizedjuvenile periodontitis patients, implicate the in-volvement of bacterial surface proteins. Theouter membrane proteins of gram-negative bac-teria function as cell surface antigens (9, 19) andmitogens (8, 24). In view of the potential impor-tance of cell surface proteins in the biologicalinteractions of A. actinomycetemcomitans Y4and as indicators, on the basis of antigenicactivity, ofjuvenile periodontitis, this study wasinitiated to identify and characterize the cellenvelope proteins of this microorganism and toexamine the distribution of these proteins inother strains of this bacterium. In addition, wecompare the membrane proteins of A. actinomy-cetemcomitans with those of other oral gram-negative bacteria.

(This work was presented, in part, at the 59thGeneral Session of the International Associationfor Dental Research [E. L. Spieler and J. M. DiRienzo, J. Dent. Res. Spec. Issue A, vol. 60,abstr. no. 853, 1981].)

MATERIALS AND METHODSBacterial strains and culturing conditions. The bacte-

rial strains that were used in this study and theirsources are listed in Table 1. Of the seven strains of A.actinomycetemcomitans examined, strain 653 is aviru-lent, based upon leukocidal properties, and strainATCC 29523 is variable (37). A. actinomycetemcomi-tans strains were grown in either thioglycolate mediumcontaining 0.5% yeast extract (Difco Laboratories),1.5% Trypticase (BBL Microbiology Systems), 0.75%glucose, 0.25% NaCl, 0.075% L-cysteine, 0.05% sodi-um thioglycolate, and 0.5% freshly prepared sodiumbicarbonate; brain heart infusion broth (Difco) supple-mented with 0.2% yeast extract, 0.05% L-cysteine, and0.5% sodium bicarbonate; or peptone-yeast extractmedium (16), supplemented with various carbohy-

TABLE 1. List of strainsStrain designation Sourcea

A. actinomycetemcomitans Y4 B. F. HammondA. actinomycetemcomitans 511A. actinomycetemcomitans 2112A. actinomycetemcomitans 653A. actinomycetemcomitansATCCb 29522

A. actinomycetemcomitansATCC 29523

A. actinomycetemcomitansATCC 29524

H. aphrophilus 80 S. C. HoltH. aphrophilus 81H. aphrophilus 626Capnocytophaga gingivalis 27Capnocytophaga 2010 V. J. IaconoCapnocytophaga ochracea 25 S. C. HoltBacteroides ochraceus 2228 D. G. GuineyB. melaninogenicus subsp. B. F. Hammond

melaninogenicus ATCC 15930F. nucleatum ATCC 10953 B. F. HammondF. nucleatum ATCC 25586 ATCCF. nucleatum FDCc 364 S. S. Socransky

a B. F. Hammond, Department of Microbiology,School of Dental Medicine, University of Pennsylva-nia, Philadelphia; S. C. Holt, Department of Microbi-ology, University of Massachusetts, Amherst; V. J.Iacono, Department of Periodontics, School of DentalMedicine, State University of New York at StonyBrook; D. G. Guiney, Department of Medicine,School of Medicine, University of California at SanDiego; S. S. Socransky, Forsyth Dental Center, Bos-ton, Mass.

b ATCC, American Type Culture Collection.c FDC, Forsyth Dental Center.

drates at a final concentration of 1%, depending uponthe individual experiment. All of the remaining strainswere grown in the brain heart infusion broth-yeastextract medium. For Bacteroides sp. this medium wasalso supplemented with 5 ,ug of hemin per ml and 0.5,ug of menadione per ml. All of the strains werecultured anaerobically in a hydrogen-carbon dioxideatmosphere (GasPak; BBL) at 37°C for 48 h.

Cell fractionation procedures. Cell envelope andsoluble protein fractions were isolated as describedpreviously (J. M. Di Rienzo, submitted for publica-tion). Briefly, cells harvested from 20-ml cultures werewashed and suspended in 10 ml of 10 mM Tris-hydrochloride (pH 8.0) and disrupted by sonication.Unbroken cells were removed by centrifugation at12,000 x g for 10 min (Spinco SS-34 rotor), and thecell envelope fraction was sedimented by centrifuga-tion at 85,000 x g for 60 min (Spinco 50 Ti rotor). Theprotein that remained in suspension after the high-speed centrifugation step was designated the solubleprotein fraction. The pellet containing the cell enve-lope fraction was extracted twice with 5 ml of 2%Triton X-100 and 2 mM MgCl2 in 10 mM Tris-hydro-chloride (pH 8.0) buffer for 30 min at room tempera-ture to remove cytoplasmic membrane protein (32).The detergent-insoluble membrane fraction was re-moved by centrifugation and reextracted twice with

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MEMBRANE PROTEINS OF A. ACTINOMYCETEMCOMITANS 255

2% Triton X-100-5 mM EDTA (disodium salt) in 10mM Tris-hydrochloride (pH 8.0).The peptidoglycan fraction was recovered from the

total cell envelope fraction from 20 ml of cells. Amixture of 2% sodium dodecyl sulfate (SDS), 10%glycerol and 10 mM Tris-hydrochloride (pH 7.4) wasadded, and the preparation was heated for 45 min at55°C (31). The extraction mixture was centrifuged at85,000 x g for 30 min, and the pellet was reextractedwith the same volume of buffer. The resulting pelletrepresented peptidoglycan and any complexed pro-tein.

Bleb material, isolated from A. actinomycetemcomi-tans, was kindly supplied by B. F. Hammond and wasprepared by the differential centrifugation of spentgrowth medium as previously reported (15).

Trypsin treatment of whole cells. A 100-ml culture ofA. actinomycetemcomitans Y4 was washed, suspend-ed in 5.7 ml of 10 mM Tris-hydrochloride (pH 8.0), anddivided into three equal portions. Each portion of cellsreceived 0.1 ml of either 100 mM EDTA, 100 mMEDTA-400 jig of trypsin, or 500 mM MgCl2-400 ,ug oftrypsin. The reaction mixtures were incubated at 37°Cfor 30 min, the cells were harvested and washed, andthe envelope fractions were prepared.

Radiolabeling methods. Whole cells of A. actinomy-cetemcomitans were washed three times in phosphate-buffered saline (10 mM sodium phosphate [pH 7.2]-0.85% NaCI) and suspended in phosphate-bufferedsaline at 750 Klett units (Klett-Summerson colorim-eter, filter number 42). Portions of this suspension (200,ul) were labeled with 100 pLCi of 1251 (16.2 mCi/jig;Amersham Corp.) by the lactoperoxidase method asdescribed by Bjorck and Kronvall (4).

Polyacrylamide gel electrophoresis. SDS-polyacryl-amide gel electrophoresis and autoradiography wereperformed on 17.5% slab gels, and samples containing30 to 50 jLg of protein were solubilized before electro-phoresis in a solution containing 0.08 M Tris-hydro-chloride (pH 6.8)-2% SDS-10% glycerol (10). Gelswere stained with Coomassie brilliant blue R (SigmaChemical Co.) as described previously (10).

Analytical procedures. Protein concentraton was de-termined by the method of Lowry and co-workers(22), employing bovine serum albumin as a standard.Protein was recovered from the bleb material byprecipitation with trichloroacetic acid (5%, final con-centration) in an ice bath for 30 min. The precipitatewas washed twice with 1 ml of 95% ethanol and driedunder a stream of nitrogen.

RESULTSIdentification and strain distribution of the

major cell envelope polypeptides. Those strainsof A. actinomycetemcomitans listed in Table 1were grown for 48 h in thioglycolate medium,the soluble protein and cell envelope fractionswere recovered, and the polypeptide composi-tion of each of these fractions was then com-pared by SDS-polyacrylamide gel electrophore-sis. The polypeptide profiles of the solublefractions are shown in Fig. 1. Complex polypep-tide profiles were obtained for each strain; how-ever, on the basis of apparent molecular weight,a high degree of homology was observed. The

A B C D Elv _ "" '-

F

ab

d

eFIG. 1. SDS-polyacrylamide gel electrophoresis of

the soluble protein fractions of strains of A. actinomy-cetemcomitans. Lane A, Strain Y4; lane B, strain29522; lane C, strain 29524; lane D, strain 29523; laneE, strain 511; and lane F, strain 2112. The arrowsdepict the molecular weight standards: a, bovine se-rum albumin (68,000); b, ovalbumin (43,000); c, henegg white lysozyme (14,300); d, cytochrome c(11,700); and e, insulin (6,000).

same results were obtained when the cell enve-lope fractions were analyzed (Fig. 2). The cellenvelope polypeptides appeared to be highlyconserved in the limited number of strains thatwere examined. Some qualitative variationamong the minor proteins was observed; howev-er, only quantitative differences were evidentamong the three major proteins, designated Env(envelope) -1, -2, and -3. The most notabledifference was a smaller relative amount ofprotein Env3 in strains 2112 and 511 (Fig. 2,lanes A and B, respectively). Based upon thevisualization of stained gels, the Env2 proteinconsistently appeared as the most abundant pro-tein in strains of A. actinomycetemcomitansgrown in thioglycolate medium.

In initial experiments, samples were preparedfor analysis by heating at 100°C in the presenceof SDS. However, when the cell envelope frac-tions were heated at 50°C before electrophore-sis, to determine whether any polypeptides dem-onstrated heat-modifiable properties (30), theEnv2 protein demonstrated an increased electro-phoretic mobility (Fig. 3, lanes B, D, and F,large arrow). This heat-modifiable property waslimited to the Env2 protein and was observed inall of the strains, including a previously unexam-ined strain 653 (Fig. 3, lanes E and F). Theapparent molecular weights of proteins Env] and-3 were 56,000 and 34,000, respectively. ProteinEnv2 had apparent molecular weights of 45,000

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256 DI RIENZO AND SPIELER

A B C D E FI.4*-W

a

env2-env3

FIG. 2. SDS-polyacrylamide gel electrophoresis ofthe cell envelope polypeptides of various strains of A.actinomycetemcomitans. Lane A, Strain 2112; lane Bstrain 511; lane C, strain ATCC 29523; lane D, strainATCC 29524; lane E, strain ATCC 29522; and lane F,strain Y4. Molecular weight standards a through e aredescribed in the legend to Fig. 1.

and 32,000 after solubilization at 100 and 50°C,respectively.The relative abundance, molecular size, and

heat-modifiable property of the Env2 proteinsuggested that it could be similar to the integralouter membrane proteins found in other gram-negative bacteria (11). Some of these proteins,most notably the matrix or porin proteins (31)and the lipoprotein (5) of Escherichia coli, arebound to the peptidoglycan, an interactionwhich promotes the functions of these proteins.The possibility that the major envelope polypep-tides of A. actinomycetemcomitans were alsopeptidoglycan associated was investigated. Cellswere extracted with 2% SDS-10 mM Tris-hydro-chloride (pH 7.4)-10% glycerol, and the peptido-glycan fraction was recovered. This materialwas examined on SDS gels, with and withoutprior digestion with lysozyme, and the resultssuggested that peptidoglycan-bound proteinswere lacking in these strains.

Since the three major envelope proteins werecommon to all seven strains of A. actinomy-cetemcomitans and due to the recent interestgenerated by one of these strains, in relation toits periopathogenic properties (1), strain Y4 wasselected for the characterization of the majorenvelope proteins.

Localization of the major envelope proteins.The cell envelope fraction from A. actinomy-cetemcomitans Y4 was extracted with the non-ionic detergent Triton X-100, containing MgCl2,in an attempt to separate the cytoplasmic andouter membrane proteins (32). The resultingdetergent-insoluble and -soluble fractions were

A B C D E F

env --: ."_wma I.< :..a ._Rmwenlv2 - *_ amI -Aenv3 - _

FIG. 3. Identification of heat-modifiable mem-brane proteins. Cell envelope fractions were divided,and each portion was heated in the presence of SDS at50 or 100°C and then examined by SDS-polyacryl-amide gel electrophoresis. Samples A, C, and E wereheated at 100°C for 10 min, and samples, B, D, and Fwere treated at 50°C for 20 min before electrophoresis.Lanes A and B, Strain Y4; lanes C and D, strain 29523;lanes E and F, strain 653. The large arrow indicates theposition of the heat-modifiable form of the Env2 pro-tein.

then subjected to SDS-polyacrylamide gel elec-trophoresis, and the polypeptide profiles of eachfraction are shown in Fig. 4 compared with theinitial cell envelope preparation (Fig. 4, lanes Aand B). The Env2 protein was readily identifiedon the basis of the temperature-dependent mo-lecular weight shift (Fig. 4, lanes B and D).Numerous polypeptides were extracted by thedetergent (Fig. 4, lanes E and F); however, thedetergent-insoluble fraction (Fig. 4, lanes C andD) was indistinguishable from the original cellenvelope preparation. When the detergent-insol-uble fraction was reextracted with Triton-Mg2+and Triton-EDTA, no additional proteins werereleased (data not shown). At the time of theseexperiments, several other observations weremade. When the cell envelope fractions wereelectrophoresed on longer gels, the presence oftwo additional polypeptides (Env4 and -5) wasclearly observed (Fig. 4, lane A). Also, bandEnv2 appeared to split into two polypeptides,which could be visualized more readily when theconcentration of protein Env2 was reduced byapplying smaller amounts of the cell envelope orTriton-insoluble membrane fractions on the gel(data not shown). These results suggested thatthe polypeptide composition of the cell enve-lope, as viewed on SDS gels, may be repre-sentative of the outer membrane. This finding isconsistent with those results obtained when theouter membrane proteins of other gram-negativebacteria were characterized (11). The outermembrane proteins of gram-negative bacteria

e

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MEMBRANE PROTEINS OF A. ACTINOMYCETEMCOMITANS 257

AB C D E F

env 2 _ _env2-q3_|env 3- ib

env4-env5-

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.*-... . a

_-w 1- )2-_3 ----

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FIG. 4. Localization of the major cell envelopeproteins of A. actinomycetemcomitans Y4. Cell frac-tions were isolated as described in the text and sub-jected to SDS-polyacrylamide gel electrophoresis.Samples A, C, and E were solubilized at 100°C for 10min, and samples B, D, and F were solubilized at 50°Cfor 20 min before electrophoresis. Lanes A and B,Untreated cell envelope fraction; lanes C and D,Triton X-100_Mg2+-insoluble (outer membrane) mate-rial; lanes E and F, Triton X-100_Mg2+-soluble frac-tion.

FIG. 5. Exposure of the major, Triton-insolubleproteins on the cell surface. A 100-mi culture of A.actinomycetemcomitans Y4 was washed, suspendedin 10 mM Tris-hydrochloride (pH 8.0), and dividedinto three portions. The cells were treated with EDTA,trypsin-EDTA, or trypsin-Mg2' at 37°C, the cell enve-lope fractions were prepared, and the resulting poly-peptide profiles were examined on SDS gels. SamplesA through C and D through F were solubilized at 100and 50°C, respectively. Lanes A and D, EDTA-treatedmembrane; lanes B and E, trypsin-EDTA-treatedmembranes; lanes C and F, trypsin-Mg2+-treatedmembrane fraction. The large arrow indicates theposition of the heat-modifiable form of the Env2 pro-tein.

are the most abundant proteins in the cell and,therefore, mask the appearance of the minorcytoplasmic membrane proteins in the cell enve-lope fraction. Since these results suggested thatproteins Env] to -5 were associated with theouter membrane, more definitive experimentswere performed to confirm their location.

In an attempt to identify those proteins thatwere exposed on the cell surface, whole cells ofstrain Y4 were treated with trypsin-EDTA ortrypsin-Mg2+. Presumably, trypsin cannot pene-trate the outer membrane when it is stabilized bydivalent cations, such as Mg2+, and can onlypenetrate to the level of the cytoplasmic mem-brane when cells are permeabilized with EDTA(10). The results of the trypsin experiment areshown in Fig. 5. Proteins Env2 and Env3 weresensitive to trypsin digestion in the presence ofMg2+ (Fig. 5, lanes C and F), suggesting thatthese proteins were exposed on the cell surface.Protein Env], however, was not affected even inthe presence of EDTA (Fig. 5, lanes B and E),which can be interpreted in either of two ways.This protein could be intrinsically resistant totrypsin or localized in the cytoplasmic mem-brane, a result which was inconsistent with the

data presented in the experiment shown in Fig.4, which suggested that protein Envi was local-ized in the outer membrane. To clarify theseconflicting findings, whole cells were labeledwith '25I, washed, and solubilized in SDS inpreparation for gel electrophoresis and autoradi-ography. Multiple polypeptides were isotopical-ly labeled (Fig. 6, lanes D and G) and appearedto correspond to proteins Env2, -3, 4, and -5 inthe cell envelope fraction (Fig. 6, lanes A andE). Interestingly, protein Envi was poorly la-beled; thus, the results support the conclusionthat this protein may indeed be associated withthe cytoplasmic membrane. Also, several addi-tional polypeptides were labeled (Fig. 6, lanes Dand G, bands I1 through I5) which were notdetected in stained cell envelope preparations(Fig. 6, lanes A and E).

Effect of growth conditions. A. actinomy-cetemcomitans Y4 was cultured in 20 ml ofthioglycolate medium, brain heart infusion-yeastextract medium, and peptone-yeast extract me-dium, all containing glucose as the carbohydratesource. The cell envelope fractions were pre-pared, and the proteins were analyzed by SDS-

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258 DI RIENZO AND SPIELER

A B C D E F G

etiv ~ ~

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env4- .

etC--- vk

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FIG. 6. Radioactive labeling of the cell surfaceproteins of A. actinomycetemcomitans Y4. Wholecells were labeled with 1251, solubilized, and examinedby SDS-polyacrylamide gel electrophoresis and auto-radiography. Samples A through C were solubilized at50°C, and samples D through G were solubilized at100°C. Lanes A and E, cell envelope fraction; lanes Band F, Coomassie blue-stained total cellular protein;lanes C and G, autoradiograms of B and F, respective-ly (6.5 x 105 cpm applied); lane D, soluble proteinfraction.

gel electrophoresis. As shown previously forthioglycolate-grown cells, the major envelopeproteins demonstrated quantitative, but no qual-itative differences. A marked increase in theamount of Env] protein and a decrease in thelevel of the Env2 protein were obtained whencells were cultured in either the brain heartinfusion broth or the peptone medium (data notshown). Cells grown in these two media alsoexhibited a significant increase in a polypeptidethat comigrated with the 50°C form of the Env2protein.To determine whether any of the major cell

envelope proteins were inducible, strain Y4 wasgrown in the peptone-yeast extract medium sup-plemented with 1% of either glucose, fructose,mannose, maltose, xylose, or mannitol-sugarsused to classify A. actinomycetemcomitans intothe various biotypes (35). Although cell yieldswere increased in cultures supplemented withfructose, xylose, and maltose, no significantquantitative or qualitative differences among theenvelope proteins were observed (data notshown).

Protein composition of extracellular bleb mate-rial. A. actinomycetemcomitans Y4, like manyother gram-negative bacteria, produces small

A BC D E F G

env 1-2- v3-

4-5-

FIG. 7. Identification of the major proteins foundin extracellular bleb material. Bleb material, recoveredfrom the spent growth medium of A. actinomycetem-comitans Y4, was solubilized in SDS, and the polypep-tide composition was compared with that of the solu-ble and cell envelope fractions by SDS-gelelectrophoresis. Samples A through D and E throughG were solubilized at 100 and 50°C, respectively. LaneA, Soluble protein fraction; lanes B and E, cell enve-lope fraction; lanes C and F, bleb material; lanes D andG, TCA-precipitated bleb material. The large arrowindicates the position of the heat-modifiable form ofthe Env2 protein.

vesicles (blebs) which have been located both onthe surface of the cell and free in the spentmedium (17). Usually the composition of extra-cellular bleb material mimics that of the cellenvelope (14). Since the blebs which have beenisolated from strain Y4 have been implicated inthe virulence of this microorganism (15), theprotein composition of the bleb material wascharacterized by SDS-polyacrylamide gel elec-trophoresis. Figure 7 shows that the proteincomposition of the blebs (Fig. 7, lanes C and F)was remarkably similar to that of the cell enve-lope (Fig. 7, lanes B and E). The major detect-able proteins were Env2, identifiable by thetemperature-dependent mobility shift (Fig. 7,lanes C and F), and protein Env4-both proteinswhich are exposed on the cell surface. Otherenvelope proteins (including Env], -3, and -5)appeared to be present in minor concentrations.Only small quantities of bleb material (50 jig [dryweight]) could be applied to the SDS-gel sincethe electrophoresis of larger amounts resulted in

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MEMBRANE PROTEINS OF A. ACTINOMYCETEMCOMITANS 259

dramatically skewed banding patterns. Such pat-terns are typical of carbohydrate-containingsamples, which often complicate protein identi-fication. This was thought to be due to the largeamount of lipopolysaccharide or group carbohy-drate or both which has been reported to bepresent in the bleb material (15, 17). Therefore,the bleb proteins were precipitated with tri-chloroacetic acid (TCA) before electrophoresis(Fig. 7, lanes D and G). Although a more repro-ducible banding pattern was obtained, the totalamount of protein that was applied to the gel stillcould not be significantly increased. The proteinpattern obtained with the TCA-precipitated sam-ple was identical to that of the untreated blebmaterial except that TCA treatment abolishedthe heat-modifiable property of the Env2 protein(Fig. 7, lane G).

Comparison of the cell envelope proteins of oralgram-negative bacteria. The cell envelope frac-tions were isolated from the various oral bacte-ria listed in Table 1. All of the strains werecultured anaerobically in brain heart infusion-yeast extract medium. The envelope proteincompositions were compared by SDS-polyacryl-amide gel electrophoresis (Fig. 8A and B). Char-acteristic envelope protein patterns were ob-served, at the genus level, with the limitednumber of strains that were examined. Themajor cell envelope proteins of Haemophilusaphrophilus were remarkably similar to those ofA. actinomycetemcomitans (Fig. 8A, lanes Athrough H). Heat-modifiable proteins were de-tected in all genera examined with the exceptionof Haemophilus. The major heat-modifiable en-velope protein found in the three Fusobacteriumnucleatum strains (Fig. 8B, lanes C through H)has been purified and characterized and will bereported elsewhere (Di Rienzo, submitted forpublication). No explanation could be given forthe repeatedly poor extraction of envelope pro-teins from Bacteroides melaninogenicus subspe-cies melaninogenicus.

DISCUSSIONIn this study, the major cell envelope proteins

of A. actinomycetemcomitans were identified,and their distribution among various oral strainsof this species was determined. The limitednumber of strains that were examined in thisstudy included virulent (leukotoxic) and aviru-lent cells which represented members of thevarious biotypes and serotypes (35, 37). Theresults indicated that the major envelope pro-teins are highly conserved in this species sincethey were detected in all of the strains that wereexamined. Human clinical isolates of gram-nega-tive bacteria typically display a significant de-gree of cell envelope protein heterogeneity, ashas been demonstrated in isolates of E. coli (29),

A ABC DE FGH J KLMNOPQ R

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2 _nbvl -

334-5-..

B A BC DE F GH

_ _I_.3-MA.~~~S iSY.

5-_

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Cd

- e

FIG. 8. Comparison of the cell envelope polypep-tides ofA. actinomycetemcomitans with those of otheroral gram-negative bacteria. The cell envelope frac-tions of the various strains were collected and subject-ed to SDS-polyacrylamide gel electrophoresis. (A)Lanes A and B, A. actinomycetemcomitans Y4; lanesC and D, H. aphrophilus 80; lanes E and F, H.aphrophilus 81; lanes G and H, H. aphrophilus 626;lanes I and J, C. gingivalis 27; lanes K and L,Capnocytophaga sp. strain 2010; lanes M and N, C.ochracea 25; lanes 0 and P, B. ochraceus 2228; lanesQ and R, B. melaninogenicus subsp. melaninogenicusATCC 15930. (B) Lanes A and B, A. actinomycetem-comitans Y4; lanes C and D, F. nucleatum 10953;lanes E and F, F. nucleatum 25586; lanes G and H, F.nucleatum 364. The first sample of every pair wassolubilized at 100°C, and the second sample wastreated at 50°C. The dots indicate the positions of theheat-modifiable proteins.

Haemophilus influenzae (2, 21), Neisseria men-ingitidis (13), and F. nucleatum (Di Rienzo,submitted for publication). The extensive pro-tein homology exhibited by strains of A. actino-mycetemcomitans precludes the use of the enve-lope proteins as a method of typing clinicalisolates at the strain level but could be used togroup these organisms. The use of membraneproteins as a diagnostic criterion was previouslyestablished in epidemiological studies of variouspathogenic bacterial species (2, 13, 21). At the

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260 DI RIENZO AND SPIELER

genus level, the cell envelope proteins of A.actinomycetemcomitans were distinct fromthose of other oral genera, including Capnocyto-phaga, Bacteroides, and Fusobacterium; how-ever, these proteins were remarkably similar tothose of H. aphrophilus. This was undoubtedly adirect reflection of the genetic relatedness ofthese two microorganisms (6) and suggests acommon evolutionary development.The major cell envelope proteins, Env] to

EnvS, were localized and characterized in A.actinomycetemcomitans Y4. Proteins Env2 to -5appeared to reside in the outer membrane on thebasis of (i) their resistance to extraction with thenonionic detergent Triton X-100, a procedurewhich is routinely used to separate cytoplasmicand outer membrane proteins in other gram-negative bacteria (32); (ii) the fact that they wereiodinated when whole cells were labeled with1251; and (iii) the fact that they were sensitive totrypsin digestion when whole cells were treatedwith the enzyme. Alternatively, the Envi pro-tein presented an anomaly since it was notsolubilized with Triton X-100, was resistant totrypsin degradation when intact cells were treat-ed with the enzyme and EDTA, and was poorlylabeled with 12)I. These data suggest that theEnv] protein either represented an extremelyhydrophobic cytoplasmic membrane protein orwas buried within the structure of the outermembrane. It is also possible, but unlikely, thattyrosine residues were unexposed or absent inEnv]. The Env2 protein demonstrated heat-mod-ifiable properties, that is, the apparent molecularweight of the protein shifted from 32,000 to45,000 when heated in SDS at 50 and 100°C,respectively. Heat modifiability is a commoncharacteristic of many major outer membraneproteins present in various genera of gram-negative bacteria (3), including members of theoral flora (Fig. 8).As discussed earlier, A. actinomycetemcomi-

tans contains extracellular membranous struc-tures commonly referred to as blebs (17). It hasbeen reported that these extracellular blebs con-tain lipopolysaccharide (17), as well as the meth-ylpentose-enriched antigen and the leukotoxin(15). In the present study, the protein composi-tion of the bleb material is reported for the firsttime. Not surprisingly, the predominant blebproteins were identified as the Env], -2, -3, and -4 proteins. Extracellular vesicular material isfrequently found on gram-negative bacteria andusually represents secreted fragments of cellmembrane (14). Since the bleb material isolatedfrom strain Y4 has been shown to possess signif-icant leukotoxic activity, it is tempting to specu-late that a major bleb protein could serve as apossible candidate for the leukotoxin. Eitherprotein Env2 or Env3 would be a likely choice

since both proteins were exposed on the cellsurface and were protease sensitive in wholecells. However, each of these proteins has beendetected in strain 653, which is nonleukotoxic.There is not yet enough molecular informationavailable concerning these two proteins in leu-kotoxic and nonleukotoxic strains to confirmthis possibility since additional factors may beimportant for the expression of leukotoxic activ-ity. It is not unreasonable to suspect that pro-teins Env2 or Env3 or both could be masked oraltered in nonleukotoxic strains. Of course, itremains possible that a highly potent proteintoxin was present in levels that were undetect-able by procedures employed in this study. Thisconcept is supported by the observation thatseveral polypeptides, not detected in the cellenvelope fraction by staining techniques, wereintensely labeled with 125I in whole cells (Fig. 6,lanes D and G, bands I1 through 15). Whetherthese polypeptides are present only in leuko-toxic cells remains to be established. Even if themajor cell envelope proteins Env2 to -S are notassociated with leukotoxic activity, they mostlikely function as cell surface antigens and, assuch, they would be good subjects on which tobase a preventive immunotherapy.

ACKNOWLEDGMENTS

We thank S. C. Holt, V. J. Iacono, D. G. Guiney, S. S.Socransky, and B. F. Hammond for kindly supplying bacterialstrains. We are also indebted to B. Appelbaum for performingthe iodinations, B. F. Hammond for supplying extracellularbleb material, and B. Rosan for critical reading of the manu-script.

This work was supported by Biomedical Research SupportGrant RR-05337-19, awarded to the School of Dental Medi-cine, University of Pennsylvania, from the National Institutesof Health and by a University of Pennsylvania Faculty Grantawarded to J.M.D. E.L.S. was supported by a stipend fromthe School of Dental Medicine, University of Pennsylvania.

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