Vol. 60, No. 11INFECrION AND IMMUNITY, Nov. 1992, p. 4528-45330019-9567/92/114528-06$02.00/0

Immunochemical and Biological Characterization of OuterMembrane Proteins of Porphyromonas endodontalis

TOMOHIKO OGAWA,1 SHIGEHIRO KURIBAYASHI,1'2 HIDETOSHI SHIMAUCHI,l TADAO TODA,2AND SHIGEYUKI HAMADAl*

Department of Oral Microbiology, Osaka University Faculty ofDentistry, Suita-Osaka 565,1 and DepartmentofEndodontics, Osaka Dental University, Chuo-ku, Osaka 540,2 Japan

Received 6 March 1992/Accepted 4 August 1992

Outer membrane proteins (OMP) of Porphyromonas endodontalis HG 370 (ATCC 35406) were preparedfrom the cell envelope fraction of the organisms. The cell envelope that had been obtained by sonication of thewhole cells was extracted in 2% lithium dodecyl sulfate and then successively chromatographed with SephacrylS-200 HR and DEAE-Sepharose Fast Flow. Two OMP fractions, OMP-I and OMP-II, were obtained, and theirimmunochemical properties and induction of specific antibodies were examined. The OMP-I preparationconsisted of a major protein with an apparent molecular mass of 31 kDa and other moderate to minor proteinsof 40.3, 51.4, 67, and 71.6 kDa, while the OMP-II preparation contained 14-, 15.5-, 27-, and 44-kDa proteinsas revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. OMP-I was found to formhydrophilic diffusion pores by incorporation into artificial liposomes composed of egg yolk phosphatidylcholineand dicetylphosphate, indicating that OMP-I exhibited significant porin activity. However, the liposomescontaining heat-denatured OMP-I were scarcely active. Spontaneous and antigen-specific immunoglobulin M(IgM)-, IgG-, and IgA-secreting spot-forming cells (SFC) enzymatically dissociated into single-cell suspensionsfrom chronically inflamed periapical tissues and were enumerated by enzyme-linked immunospot assay. Inpatients with radicular cysts or dental granulomas, the major isotype of spontaneous SFC was IgG. Inradicular cysts, the OMP-II-specific IgG SFC represented 0.13% of the total IgG SFC, while the antigen-specific IgA or IgM SFC was not observed. It was also found that none of these mononuclear cells producedantibodies specific for OMP-I or lipopolysaccharide of P. endodontalis.

The gram-negative bacterial cell envelope contains anouter membrane composed of lipopolysaccharides (LPS)and proteins (OMP), which are variably antigenic. The outermembrane serves as a selective permeability barrier and alsoexhibits a variety of immunobiological activities to the host(11, 31). Pore-forming proteins, porins, are one of the majorproteins present in the outer membrane and serve to regulatethe passage of small hydrophilic molecules of the exclusionlimit through the membrane pores. There have been manyinvestigations into porins of Escherichia coli, Salmonellatyphimurium, Pseudomonas aeruginosa, Neisseria gonor-rhoeae, Bacteroides fragilis, and some other bacterial spe-cies. Porins are generally resistant to sodium dodecyl sulfate(SDS) denaturation and are tightly but noncovalently asso-ciated with LPS and the underlying peptidoglycan (18, 26,33).Evidence that obligatory anaerobic black-pigmented Por-

phyromonas and Prevotella species are implicated in thepathogenesis of chronic inflammatory periodontal and peri-apical lesions has accumulated (17). Among these species,Porphyromonas endodontalis has frequently been isolatedfrom periapical abscesses (46) and chronic root canal inflam-mations (41), suggesting that this organism may play someetiologic role in the development of these diseases. P.endodontalis can be differentiated from either Porphyromo-nas asaccharolyticus or Porphyromonas gingivalis by DNAhomology and physiological properties such as sheep eryth-rocyte agglutination, production of phenylacetic acid, reper-toire of enzymes produced, immunochemical specificationsof cellular antigens, and so on (7, 15, 20); however, there

* Corresponding author.

have been no reports on the characterization of the OMP ofP. endodontalis.

In the present study, we have isolated two OMP fractionsfrom P. endodontalis and examined their immunochemicaland biological properties and induction of specific antibodiesin the chronic periapical lesions in humans.

MATERIALS AND METHODS

Bacterial strain and culture condition. P. endodontalisHG370 (ATCC 35406) was kindly supplied by A. J. vanWinkelhoff, Vrije University, Amsterdam, The Netherlands.The organisms were inoculated in GAM broth (Nissui Sei-yaku Co., Tokyo, Japan) and grown at 37°C for 3 days in anN2-H2-CO2 (90:5:5) atmosphere in an anaerobic chamber(model 1024; Forma Scientific Inc., Marietta, Ohio). Thisculture (4 liters) was transferred to GAM broth (16 liters) andcultivated anaerobically for an additional 30 h as describedabove. Cells were harvested by centrifugation at 26,000 x gfor 30 min, washed twice with 50 mM phosphate-bufferedsaline (PBS; pH 7.2) and once with distilled water, andlyophilized. The recovery of lyophilyzed cells was 0.93g/liter of broth culture.

Preparation of the cell envelope fraction. The lyophilizedcells (15 g [dry weight]) were suspended in 240 ml of 50 mMPBS (pH 7.2) containing 2.7 mg of DNase I (bovine pan-creas; Sigma Chemical Co., St. Louis, Mo.) and 2.7 mg ofRNase A (type I-AS from bovine pancreas; Sigma). The cellsuspension was disrupted with an ultrasonicator (type UD-201; Tomy Seiko Co., Tokyo, Japan) at 4°C with an output of200 W for 20 min. After undisrupted cells were removed bycentrifugation at 26,000 x g for 30 min, the supernatantcontaining broken cells was centrifuged at 100,000 x g for 90

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0.3-Vo VtA B

0.2

0.1

0 50 100 150 200Tube number

FIG. 1. Elution profile of P. endodontalis OMP on SephacrylS-200 HR column. Crude cell envelope dissolved with 20 mMHEPES-LiOH buffer (pH 8.0) containing 1% LDS and 0.5 M LiClwas applied to a column (5.0 by 90 cm). Elution was done with thebuffer. Two peaks, A and B, were pooled as indicated by thebrackets. V0, void volume; V,, total bed volume.

min to obtain the cell envelope fraction as a sediment. Thecell envelope fraction was washed five times with 50 mMPBS (pH 7.2) and twice with distilled water by centrifuga-tion. Thus, 5.5 g (dry weight) of the cell envelope wasobtained at a yield of 36.7%.

Preparation ofOMP. The cell envelope fraction (3.0 g) wassuspended in 300 ml of 0.5% sodium lauroylsarcosine (SLS;Sigma) aqueous solution, and the suspension was gentlystirred at 25°C for 30 min to solubilize the inner membraneby the method of Filip et al. (14). The SLS-treated envelopefraction was centrifuged at 100,000 x g for 90 min, and thesediment was suspended in 140 ml of 2% lithium dodecylsulfate (LDS; Sigma) dissolved in 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Sigma)-lithium hydroxide (HEPES-LiOH) buffer (pH 8.0). Aftergentle stirring at 4°C for 10 min, the suspension was centri-fuged at 100,000 x g for 90 min. The supernatant containingLDS-solubilized outer membrane proteins of the cell enve-lope was applied to a Sephacryl S-200HR (Pharmacia LKBBiotechnology, Uppsala, Sweden) column (5.0 by 90 cm)that had been equilibrated with 20 mM HEPES-LiOH buffercontaining 1% LDS and 0.5 M LiCl. The same buffer wasused for elution of the applied materials. The eluate was

monitored by measuring the A280. Two peaks, A and B (Fig.1), were obtained; each peak was concentrated with Ficoll400 (Pharmacia LKB Biotechnology) and dialyzed against 5mM Tris-HCl buffer (pH 7.4). After removal of LDS fromthe dialysate with 1 M potassium phosphate buffer (pH 7.5)(42), peak A or peak B was dialyzed against 5 mM Tris-HCl-buffered solution (pH 7.4) again and applied to a column (2.6by 10 cm) of DEAE-Sepharose Fast Flow (Pharmacia LKB)which had been equilibrated with the same buffer. Thecolumn was eluted with a linear gradient of 0 to 1.0 M NaClin 5 mM Tris-HCl buffer (pH 7.4). TheA280 was continuouslyrecorded, and total hexose of each fraction was determinedcolorimetrically by the anthrone reaction (2). A major peak,i.e., OMP-I, was eluted at 0.22 M NaCl (Fig. 2A), whileOMP-II was eluted at 0.15 M NaCl from the column asindicated in Fig. 2B. These peak fractions were pooled andconcentrated with Ficoll 400. The yield of OMP-I was 9.2mg, composing 36.8% of peak A, and that of OMP-II was 10mg, composing 33.3% of peak B. SDS-polyacrylamide gelelectrophoresis (PAGE) was carried out in 1.5-mm-thick slabgels with a 4% stacking gel and a 14% separating gelessentially as described by Neville (32). These samples werepretreated in a solution containing 1% SDS, 0.05 M Tris,25% glycerol, and 1% 2-mercaptoethanol at 25 or 70°C for 20min or at 100°C for 10 min before electrophoresis. Anelectrophoresis calibration kit (Pharmacia LKB Biotechnol-ogy) was used as a molecular weight standard. Electropho-resis was done at 20 mA per gel at 4°C for 90 min, and proteinbounds were visualized by Coomassie blue staining.

Preparation of LPS. Lyophilized whole cells of P. endo-dontalis HG370 (10 g) were extracted with a hot phenol-water mixture (48), which was further treated as describedpreviously (16). This preparation was designated LPS in thisstudy.Assay for porin activity. Porin activity was measured by

the liposome-swelling method (25, 34). Briefly, egg yolkphosphatidylcholine (2.6 ,umol; Sigma) and dicetylphosphate(0.1 ,umol; Sigma) were mixed, sonicated, and dried in vacuoto form a film at the bottom of a test tube, and an indicatedamount of the test fraction was added. Liposomes wereprepared by dissolving OMP-I or OMP-1I and the phospho-lipid mixture in Tris-hydrochloride, drying the mixture undera reduced pressure, and then suspending the mixture in 0.6

OMP-I

I~~~~~~[~.~~~~~~~~~ .

50 100 150Tube number

600

400:E

200 *

x

0.3

Q 0.2

.-0

a. 0.1

0

0

A

0.4 r OMP-II

I-

z1

50 100 150Tube number

BFIG. 2. Further purification of P. endodontalis OMP (peaks A and B) on DEAE-Sepharose Fast Flow column. Peak A (A) or B (B) was

applied to a column (2.6 by 10 cm) equilibrated with 5 mM Tris-hydrochloride buffer (pH 7.4) and eluted with a linear gradient of 0 to 1.0 MNaCl in the same buffer. The main peaks, designated OMP-I and OMP-II, were pooled as indicated by the brackets.

0.2

2

Q 0.1c

4.0

IL

0

1 ,000

800 9

600 S

400 a0x

200Z

0

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TABLE 1. SFC in enzymatically dissociated MNC from chronically inflamed periapical tissues

Subject group (no. of subjects: Age Spontaneous SFC/106 MNC" (mean + SE)male/female) (yr) IgG IgA IgM

Radicular cyst (n =7/14) 14-65 91,200 ± 10,600 13,600 ± 3,200 700 ± 100Dental granuloma (n = 7/11) 18-70 71,600 - 4,900 14,600 ± 2,800 1,600 ± 280

a Total numbers of IgM, IgG, and IgA SFC responses were determined among MNC isolated from inflamed tissues by the ELISPOT assay, and the numberof SFC was enumerated under a dissecting microscope.

ml of 15% dextran T-40 (Pharmacia LKB) in 5 mM Tris-hydrochloride buffer (pH 7.4). Portions (15 RI) of theseliposomes and 0.7 ml of 30 mM solution of sugars such asarabinose, glucose, sucrose, raffinose, or stachyose (Sigma)in 5 mM Tris-hydrochloride buffer (pH 7.4) were mixed in acuvette, and the A4 was recorded. The rate of penetrationof the sugar was determined by the initial rate of swelling ofthe liposome membranes added with the isotonic solution ofthe test sugar. The swelling rate, i.e., dA4-1/dt, was calcu-lated as reported by de Gier et al. (12). In a controlexperiment, a porin fraction which had been denatured byheating in 1% SDS at 100°C for 10 min and precipitated with1M potassium phosphate buffer (pH 7.5) to remove SDS wasused for the liposome-swelling assay.

Isolation of MNC from chronically inflamed periapicaltissues. Chronically inflamed periapical tissues were surgi-cally removed from 39 patients with radicular cysts (21subjects) or dental granulomas (18 subjects) during surgeryat Osaka Dental University Hospital. The characteristics ofthese patients are summarized in Table 1. The excised tissuewas placed in sterile tubes containing RPMI 1640 mediumsupplemented with 5% fetal calf serum (FCS; HycloneLaboratories, Logan, Utah). Mononuclear cells (MNC) wereisolated from the periapical tissue specimens by enzymatictreatment with Dispase (Boehringer-Mannheim Biochemi-cals, Mannheim, Germany), followed by separation on mini-Ficoll-Hypaque (Sigma) gradients as previously described(27). Cell yields obtained by this procedure averaged 230,000± 27,000 or 210,000 + 33,000 cells per 100 mg of radicularcyst or dental granuloma and were >97% viable as deter-mined by the trypan blue exclusion test.

Determination of total spontaneous antibody-secreting cells.To determine the numbers of immunoglobulin M (IgM)-,IgG-, and IgA-secreting spot-forming cells (SFC) present inthe chronically inflamed periapical tissues, the enzyme-linked immunospot (ELISPOT) assay was employed aspreviously described (9, 37). Briefly, lids of polystyreneplates (24-well cluster plates, Mark II 3424; Costar, Cam-bridge, Mass.) were coated with affinity-purified goat F(ab')2anti-human IgM (Jackson Immunoresearch, West Grove,Pa.), anti-human IgG, or anti-human IgA (Pel-Freez Biolog-icals, Rogers, Ark.). After the plate was blocked with 10%FCS in RPMI 1640, periapical MNC were incubated oncoated plates for 4 h at 37°C in a humidified atmosphere of5% CO2. The plates were washed with PBS and PBScontaining 0.02% Tween 20 (PBS-T) and incubated foranother 18 h at 4°C with biotinylated goat F(ab')2 anti-human,u, -y, or a chain-specific antisera (Tago, Burlingame, Calif.).Plates were then washed and incubated with avidin-peroxi-dase (1 mg/ml) for 2 h at 25°C. Spots representing therespective isotype were developed with agar substrate con-taining 1% Noble agar (Difco Laboratories, Detroit, Mich.),p-phenylenediamine (Sigma), methanol, and H202. Thenumber of spots present was counted with the aid of adissecting microscope.

Enumeration of OMP-specific antibody-secreting cells. Inorder to determine antigen-specific IgM-, IgG- and IgA-secreting cells in the inflamed periapical tissues of patients,ELISPOT assay was done as described previously (36). Lidsof polystyrene plates (Costar) were coated with OMP-I,OMP-II, or LPS of P. endodontalis. All wells were thenblocked with 10% FCS. Periapical MNC were incubated inantigen-coated plates for 4 h at 37°C. The lids were washedand incubated with biotinylated goat anti-human ,u, y, or achain-specific antibody (Tago). After the lids were washedwith PBS-T, avidin-peroxidase and agar substrate wereadded for development of spots. In addition to cellularcomponents of P. gingivalis, Escherichia coli 055:B5 LPS(Sigma), cholera toxin (Sigma), and dinitrophenylated bo-vine serum albumin (Sigma) were used as coating antigensfor comparison.

RESULTS

Figure 1 illustrates the elution proffle obtained with anLDS extract of the SLS-treated cell envelope by gel filtrationon a Sephacryl S-200 HR column. The SLS-insoluble butLDS-soluble fraction of the envelope gave two major peakfractions (Fig. 1). The DEAE-Sepharose Fast Flow chroma-tography of peaks A and B is shown in Fig. 2. Analysis bySDS-PAGE showed that the OMP-I preparation consisted ofa 31-kDa major protein in addition to 40.3-kDa and otherminor 51- to 72-kDa proteins. The 40.3-kDa protein wasfound to be heat modifiable. An increase of 17,000 in themolecular weight was observed when the temperature ofsolubilization was shifted from 25 or 70 to 100°C (Fig. 3).Various temperature and time conditions of treatment pro-duced only minor effects on the other OMP profiles. On theother hand, it was shown that the OMP-II preparation wascomposed of 14.4-, 15.5-, 27-, and 44-kDa proteins (Fig. 3).The porin activities of the outer membrane proteins,

OMP-I and OMP-II, were examined for their abilities to formhydrophilic diffusion channels by incorporation of varioussugars into artificial liposome membranes. Liposomes con-taining the OMP-I protein exhibited a marked increase in theinitial rate of swelling because of the penetration of arabi-nose into liposomes, while the liposomes containing theheat-denatured OMP-I protein or OMP-II protein werescarcely active (Fig. 4). The swelling rates of OMP-I-con-taining liposomes after addition of mono- and oligosaccha-rides such as arabinose, glucose, sucrose, raffinose, andstachyose were then compared (Fig. 5). It was demonstratedthat arabinose and glucose with molecular weights (Mr) of150 and 180 permeated the OMP-I-containing liposomemembranes, while sucrose (Mr = 342) penetrated onlyslowly. It was noted that raffinose (Mr = 504) and stachyose(Mr = 666.7) did not permeate significantly.When IgM-, IgG-, or IgA-secreting cells in Dispase-disso-

ciated MNC derived from chronically inflamed periapicallesions were enumerated by the ELISPOT method, high

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94K _.-67K _.-

43K _-

30K _

20.AK _

14.4K _

C693.es IL %q . 96V___ -__ _ ___ _ 0 0

100'C, 10 min 70'C, 20 min 2S'C, 20 min

FIG. 3. SDS-PAGE patterns for detergent extracts and OMP-Iand OMP-II. Samples were pretreated at 25 or 70°C for 20 min or at100°C for 10 min in the sample buffer containing 1% SDS, 0.05 MTris, 25% glycerol, and 1% 2-mercaptoethanol. SLS-soluble mem-brane protein (18 ,ug), LDS extract (10 ,ug), OMP-I (7 pg), andOMP-II (7 ,ug) pretreated at 100°C for 10 min, 70°C for 20 min, and25'C for 20 min were applied for PAGE.

numbers of isotype-specific SFC were found (Table 1). IgGSFC were found to be dominant; IgA SFC were seensignificantly, while IgM SFC were only scarcely found. Asimilar profile of isotype-specific antibody-secreting cellswas noted in the order of IgG> >IgA>IgM in the MNC frompatients with radicular cyst or dental granuloma. The iso-types of P. endodontalis OMP-I-, OMP-II-, or LPS-specificSFC were assessed in MNC isolated from periapical lesionsby the ELISPOT assay. In radicular cysts, the OMP-Il-specific IgG SFC represented 120 ± 20 per 107 MNC, e.g.,0.13% of the total IgG SFC, but no OMP-II-specific IgA orIgM SFC were seen (Table 2). These MNC did not contain

0.1

v-

E

-

-

0.3

v-

L0.2

-

< 0.1la

IArabinose

100 150 180 342 504 666.7

log MrFIG. 5. Swelling of the OMP-I-embedded liposome membrane

because of various sugars. Liposomes were made of 2.6 ,umol ofphosphatidylcholine and 0.1 pmol of dicetylphosphate with 2 p,g ofOMP-I, and the rate ofA4w change induced by a 30 mM solution ofvarious sugars in 5 mM Tris-hydrochloride buffer (pH 7.4) wasdetermined. Open circles indicate the rate ofA4. change by sugars.

SFC-secreting antibodies specific for E. coli 055 B5 LPS,cholera toxin, or dinitrophenylated bovine serum albumin.Furthermore, neither OMP-I- nor LPS-specific SFC wereobserved in the MNC from radicular cyst or dental granu-loma.

DISCUSSIONWe have demonstrated here how membrane proteins ofP.

endodontalis HG370 could be extracted and separated chro-matographically. The two protein fractions obtained, I andII, were probably OMPs, because they were isolated from anSLS-insoluble fraction of the cell envelope. Although ideallya higher detergent-to-protein mass ratio should have beenused for SLS extraction, this method is known to preferen-tially extract inner membrane proteins. Moreover, fraction Ishowed porin activity, further supporting the notion that atleast this protein came from the outer membrane. Thismethod has been widely used to prepare OMP from various

0.1 r

-

E

-

0

0 2 4.. ....6..

1

0 2 4 6 8 10

Protein (hg)A

0.05p.

0 2 4 6 8 10

Protein (ig)B

FIG. 4. Swelling of the liposome membrane embedded with increasing amounts of OMP-I (A) or OMP-II (B). Liposomes were made by2.6 pmol of phosphatidylcholine and 0.1 pmol of dicetylphosphate with or without OMP, and the rate ofA4w change induced by a 30 mMsolution of arabinose in 5 mM Tris-hydrochloride buffer (pH 7.4) was determined. Symbols: *, OMP; 0, heat-denatured OMP.

. . .*a - =: . .7= . . . . .

---L- I

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TABLE 2. P. endodontalis OMP-specific SFC in enzymaticallydissociated MNC from chronically inflamed periapical tissues

P. endodontalis OMP-II-

Subject group No. of subjects specific SFC/10EMNC'Subjecgroup (male/female) (mean ±t SE)IgG IgA IgM

Radicular cyst 7/14 120 ± 20 0 ± 0 0 ± 0Dental granuloma 7/11 0 ± 0 0 ± 0 0 ± 0

a P. endodontalis OMP-specific IgM, IgG, and IgA SFC in the MNC frominflamed tissues were enumerated by the ELISPOT assay.

bacterial species, including Pseudomonas aeruginosa (47),Campylobacterjejuni (5), Eikenella corrodens (6), and Fuso-bacterium nucleatum (43).OMP-I consisted mainly of a 31-kDa protein with a lesser

amount of heat-modifiable 40.3-kDa protein as revealed bySDS-PAGE analysis (Fig. 3) and was found to facilitatepermeation of hydrophilic sugars (Fig. 4 -and 5). Figure 3clearly shows the heat-modifiable nature of the 40.3-kDaprotein. Similar proteins were described with OMP fromother bacterial species, such as C. jejuni and P. aeruginosa(4, 19). The function of these proteins has not been deter-mined. Various kinds of porins have been characterizedfrom other gram-negative bacterial species; OmpF, OmpC,and PhoE are porins ofE. coli (26), while OmpD, OmpF, andOmpC are in S. typhimurium (44), F, P, and Dl are in P.aeruginosa (1), and I is in N. gonorrhoeae (13). Further-more, a porin has been described for F. nucleatum, oneother organism thought to be associated with periodontaldisease (43). These porins generally have an apparent mo-lecular weight ranging from 30,000 to 50,000. We haveshown that OMP-I but not OMP-II embedded in liposomestransported glucose and arabinose (Fig. 4 and 5). Porins ofvarious bacterial species possessing different pore sizes wereprepared. For example, the liposome vesicles reconstitutedfrom OMP of Chlamydia trachomatis (3) and N. gonor-rhoeae (49) were permeable to 2,000- to 3,000-molecular-weight oligosaccharides. On the other hand, the molecularweight exclusion limit for OMP of Haemophilus influenzaecorresponded to approximately 1,400 (45). Pores of entericbacteria such as E. coli and Salmonella, Proteus, or Entero-bacter spp. were found to have exclusion limits of approxi-mately 700 (21, 29, 30, 35), while the pore size of Yersiniapestis porin allowed the diffusion of oligosaccharides withmolecular weights of 500 to 600 (10). It was also reportedthat the exclusion limit of the OMP of Bacteroides fragiliswas similar to the molecular weight of disaccharides, i.e., Mrof 340 to 400 (23).The content of LPS in OMP-I and -II of P. endodontalis

was calculated by the Limulus test by using a chromogenicLimulus amoebocyte lysate assay kit. The minimum gelationdoses of OMP-I and OMP-II were 248 endotoxin units (EU)(15.4%, weight basis) and 261 EU (16.2%), respectively (datanot shown). It has been shown that OMP exhibit a variety ofbiological activities, including B-cell mitogenicity, poly-clonal B-cell activation, and macrophage stimulation (22, 28,40, 43). However, attention should be paid to the coexis-tence of LPS even in chromatographically isolated OMPsuch as reported in this study. In this regard, liposomescontaining P. endodontalis LPS in their bilayer were notactive in terms of permeability ability (data not shown).

Neutrophils, macrophages, T and B lymphocytes, andplasma cells infiltrate in chronically inflamed periapicallesions (24, 39). Immunohistological observations demon-

strated that IgG-, IgA-, IgM-, and IgE-positive cells ap-peared in the periapical lesions (38), and helper and suppres-sor T lymphocytes were also found in these lesions (8). Inthis study, we have enumerated the total antibody-secretingcells specific for P. endodontalis cellular components inMNC prepared from periapical lesions of patients withradicular cysts or dental granuloms (Tables 1 and 2). Thenumbers of MNC isolated from enzymatic digests of theperiapical inflamed tissues were 2.1 x 105 to 2.3 x 105/100mg of tissue, and cell viability after the enzyme treatmentwas >97%, while similar enzymatical treatments of theinflamed gingival tissues of patients with adult periodontitisyielded an average of 5 x 105 cells per 100 mg of tissue (27).These results indicate that a considerable infiltration ofMNC occurs in chronic, inflamed tissues. The total immu-noglobulin-secreting cells represented approximately 10% ofMNC from radicular cysts and dental granulomas (Table 1).Among these, IgG-, IgA-, or IgM-secreting cells composed82 to 86%, 13 to 17%, and 0.1 to 1.8%, respectively (Table 1).ELISPOT analysis showed that OMP-II- but not OMP-I- andLPS-specific antibody-secreting cells represented 0.13% ofthe total IgG SFC in MNC from radicular cysts (Table 2).Pulver et al. (38) demonstrated that the majority of immu-noglobulin-containing cells in periapical lesions were of theIgG isotype with significant numbers of IgA but few IgM-and IgE-positive cells also being detected by using animmunofluorescence technique. These findings are concor-dant with our findings by the ELISPOT assay.The emergence of antibody-secreting cells specific for an

important periodontal disease-associated pathogen, P. gin-givalis, was also demonstrated in MNC isolated from in-flamed gingival tissues of different stages of adult periodon-titis (36). We have further found that different IgG or IgAsubclass response patterns may be seen at various diseasestages as well as in different types of bacterial antigens interms of their chemical nature, i.e., protein or polysaccha-ride. Further studies will be required to clarify the relation-ship between the types and stages of the periapical diseasesand immunoglobulin subclass response patterns.

REFERENCES1. Angus, B. L., and R. E. W. Hancock. 1983. Outer membrane

porin proteins F, P, and Dl of Pseudomonas aeruginosa andPheE of Escherichia coli: chemical cross-linking to reveal nativeoligomers. J. Bacteriol. 155:1042-1051.

2. Ashwell, G. 1957. Colorimetric analysis of sugars. MethodsEnzymol. 3:84-85.

3. Bavoil, P., A. Ohlin, and J. Schachter. 1984. Role of disulfidebonding in outer membrane structure and permeability in Chla-mydia trachomatis. Infect. Immun. 44:479-485.

4. Blaser, M. J., J. A. Hopkins, R. M. Berka, M. L. Vasil, andW.-L. L. Wang. 1983. Identification and characterization ofCampylobacterjejuni outer membrane proteins. Infect. Immun.42:276-284.

5. Blaser, M. J., J. A. Hopkins, and M. L. Vasil. 1984. Campylo-bacterjejuni outer membrane proteins are antigenic for humans.Infect. Immun. 43:986-993.

6. Chen, C.-K. C., and M. E. Wilson. 1990. Outer membraneprotein and lipopolysaccharide heterogeneity among Eikenellacorrodens isolates. J. Infect. Dis. 162:664-671.

7. Coykendall, A. L., F. S. Kaczmarek, and J. Slots. 1980. Geneticheterogeneity in Bacteroides asaccharolyticus (Holdeman andMoore 1970) Finegold and Barnes 1977 (Approved Lists, 1980)and proposal of Bacteroides gingivalis sp. nov. and Bacteroidesmacacae (Slots and Genco) comb. nov. Int. J. Syst. Bacteriol.30:559-564.

8. Cymerman, J. J., D. H. Cymerman, J. Walter, and A. J. Nevins.1984. Human T lymphocyte subpopulations in chronic periapi-cal lesions. J. Endodon. 10:9-11.

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OMP OF P. ENDODONTALIS 4533

9. Czerkinsky, C. C., L.-A. Nilsson, H. Nygren, 0. Ouchterlony,and A. Tarkowski. 1983. A solid-phase enzyme-linked immuno-spot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65:109-121.

10. Darveau, R. P., W. T. Chaenetzky, R. E. Huribert, and R. E. W.Hancock 1983. Effects of growth temperature, 47-megadaltonplasmid, and calcium deficiency on the outer membrane proteinporin and lipopolysaccharide composition of Yersinia pestisEV76. Infect. Immun. 42:1092-1101.

11. Davies, J. A., G. K. Anderson, T. J. Beveridge, and H. C. ClarkL1983. Chemical mechanism of the Gram stain and synthesis of anew electron-opaque marker for electron microscopy which re-places the iodine mordant of the strain. J. Bacteriol. 156:837-845.

12. de Gier, J., J. G. Mandersloot, and L. L. M. van Deenen. 1968.Lipid composition and permeability of liposomes. Biochim.Biophys. Acta 150:666-675.

13. Douglas, J. T., M. D. Lee, and H. Nikaido. 1981. Protein I ofNeisseria gonorrhoeae outer membrane is a porin. FEMSMicrobiol. Lett. 12:305-309.

14. Filip, C., G. Fletcher, J. L. Wulff, and C. F. Earhart. 1973.Solubilization of the cytoplasmic membrane of Escherichia coliby the ionic detergent sodium-lauryl sarcosinate. J. Bacteriol.115:717-722.

15. Finegold, S. M., and E. M. Barnes. 1977. Report of the ICSBtaxonomic subcommittee on gram-negative anaerobic rods. Pro-posal that the saccharolytic and asaccharolytic strains at presentclassified in the species Bacteroides melaninogenicus (Oliverand Wherry) be reclassified in two species as Bacteroidesmelaninogenicus and Bacteroides asaccharolyticus. Int. J.Syst. Bacteriol. 27:388-391.

16. Fujiwara, T., T. Ogawa, S. Sobue, and S. Hamada. 1990.Chemical, immunobiological and antigenic characterizations oflipopolysaccharides from Bacteroides gingivalis strains. J. Gen.Microbiol. 136:319-326.

17. Hamada, S., S. C. Holt, and J. R. McGhee (ed.). 1991. Periodon-tal disease: pathogens and host immune response, p. 13-405.Quintessence, Tokyo.

18. Hancock, R. E. W. 1987. Role of porins in outer membranepermeability. J. Bacteriol. 169:929-933.

19. Hancock, R. E. W., and A. M. Carey. 1979. Outer membrane ofPseudomonas aeruginosa: heat- and 2-mercaptoethanol-modifi-able proteins. J. Bacteriol. 140:902-910.

20. Holdman, L. V., and R W. Kelley. 1984. Anaerobic gram-negative straight, curved and helical rods, p. 602-662. In N. R.Krieg and J. G. Holt (ed.), Bergey's manual of systematicbacteriology. The Williams & Wilkins Co., Baltimore.

21. Kaneko, M., A. Yamaguchi, and T. Sawai. 1984. Purification andcharacterization of two kinds of porins from the Enterobactercloacae outer membrane. J. Bacteriol. 158:1179-1181.

22. Kato, K., S. Kokeguchi, H. Ishihara, Y. Murayama, M. Tsqii-moto, H. Takada, T. Ogawa, and S. Kotani. 1987. Chemicalcomposition and immunobiological activities of sodium dodecylsulphate extracts from the cell envelopes of Actinobacillusactinomycetemcomitans, Bacteroides gingivalis and Fusobac-terium nucleatum. J. Gen. Microbiol. 133:1033-1043.

23. Kobayashi, Y., and T. Nakae. 1986. The permeability propertyof the outer membrane of Bacteroides fragilis, a strictly anaer-obic opportunistic pathogen. Biochem. Biophys. Res. Commun.141:292-298.

24. Kontiainen, S., H. Ranta, and I. Lautenschlager. 1986. Cellsinfiltrating human periapical inflammatory lesions. J. OralPathol. 15:544-546.

25. Luckey, M., and H. Nikaido. 1980. Specificity of diffusionchannels produced by X phage receptor protein of Escherichiacoli. Proc. Natl. Acad. Sci. USA 77:167-171.

26. Lugtenberg, B., and L. van Alphen. 1983. Molecular architec-ture and functioning of the outer membrane of Eschenchia coliand other gram-negative bacteria. Biochim. Biophys. Acta737:51-115.

27. McGhee, M. L., T. Ogawa, A. M. Pitts, Z. Moldoveanu, J.Mestecky, J. R. McGhee, and H. Kiyono. 1989. Cellular analysisof functional mononuclear cells from chronically inflamed gin-gival tissue. Reg. Immunol. 2:103-110.

28. Murayama, Y., K. Muranishi, H. Okuda, K. Kato, S. Kotani, H.Takada, M. Tsujimoto, A. Kawasaki, and T. Ogawa. 1982.Immunological activities of Capnocytophaga cellular compo-nents. Infect. Immun. 36:876-884.

29. Nakae, T. 1975. Outer membrane of Salmonella typhimurium:reconstitution of sucrose-permeable membrane vesicles. Bio-chem. Biophys. Res. Commun. 64:1224-1230.

30. Nakae, T. 1976. Identification of the outer membrane protein ofE. coli that produces transmembrane channels in reconstitutedvesicle membranes. Biochem. Biophys. Res. Commun. 71:877-884.

31. Nakae, T. 1986. Outer-membrane permeability of bacteria. Crit.Rev. Microbiol. 13:1-62.

32. Nevilie, D. M., Jr. 1971. Molecular weight determination ofprotein-dodecyl sulfate complexes by gel electrophoresis in adiscontinuous buffer system. J. Biol. Chem. 246:6328-6334.

33. Nikaido, H., and T. Nakae. 1979. The outer membrane ofgram-negative bacteria. Adv. Microbiol. Physiol. 20:163-250.

34. Nikaido, H., and E. Y. Rosenberg. 1983. Porin chanels inEscherichia coli: studies with liposomes reconstituted frompurified proteins. J. Bacteriol. 153:241-252.

35. Nixdorff, K., H. Fltzer, J. Gmeiner, and H. H. Martin. 1977.Reconstitution of model membranes from phospholipid andouter membrane proteins ofProteus mirabilis. Eur. J. Biochem.81:63-69.

36. Opwa, T., M. L. McGhee, Z. Moldoveanu, S. Hamada, J.Mestecky, J. R. McGhee, and H. Kiyono. 1989. Bacteroides-specific IgG and IgA subclass antibody-secreting cells isolatedfrom chronically inflamed gingival tissues. Clin. Exp. Immunol.76:103-110.

37. Ogawa, T., A. Tarkowski, M. L. McGhee, Z. Moldoveanu, J.Mestecky, H. R. Hirsch, W. J. Koopman, S. Hamada, J. R.McGhee, and H. Kiyono. 1989. Analysis of human IgG and IgAsubclass antibody-secreting cells from localized chronic inflam-matory tissue. J. Immunol. 142:1150-1158.

38. Pulver, W. H., M. A. Taubman, and D. J. Smith. 1978. Immunecomponents in human dental periapical lesions. Arch. Oral Biol.23:435-443.

39. Stern, M. H., S. Dreizen, B. F. Mackler, A. G. Selbst, and B. M.Levy. 1981. Quantitative analysis of cellular composition ofhuman periapical granuloma. J. Endodon. 7:117-122.

40. Sultzer, B. M., and G. W. Goodman. 1976. Endotoxin protein: aB-cell mitogen and polyclonal activator of C3H/HeJ lympho-cytes. J. Exp. Med. 144:821-827.

41. Sundqvist, G., E. Johansson, and U. Sj6gren. 1989. Prevalenceof black-pigmented Bacteroides species in root canal infections.J. Endodon. 15:13-19.

42. Suzuki, H., and T. Terada. 1988. Removal of dodecyl sulfatefrom protein solution. Anal. Biochem. 172:259-263.

43. Takada, H., T. Ogawa, F. Yoshimura, K. Otsuka, S. Kokeguchi,K. Kato, T. Umemoto, and S. Kotani. 1988. Immunobiologicalactivities of a porin fraction isolated from Fusobacterium nu-cleatum ATCC 10953. Infect. Immun. 56:855-863.

44. Tokunaga, H., M. Tokunaga, and T. Nakae. 1979. Characteri-zation of porins from the outer membrane of Salnwnella typhi-murium. 1. Chemical analysis. Eur. J. Biochem. 95:433-439.

45. Vachon, V., D. J. Lyew, and C. W. Coulton. 1985. Transmem-brane permeability channels across the outer membrane ofHaemophilus influenzae type b. J. Bacteriol. 162:918-924.

46. van Wimkelhoff, A. J., A. W. Carlee, and J. de Gnaif. 1985.Bacteroides endodontalis and other black-pigmented Bacteroidesspecies in odontogenic abscesses. Infect. Immun. 49:494-497.

47. Ward, K. H., H. Anwar, M. R. W. Brown, J. Wale, and J.Gowar. 1988. Antibody response to outer-membrane antigens ofPseudomonas aeruginosa in human burn wound infection. J.Med. Microbiol. 27:179-190.

48. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharides.Extraction with phenol-water and further applications of theprocedure. Methods Carbohydr. Chem. 5:83-91.

49. Young, J. D.-E., M. Blake, A. Mauro, and Z. A. Cohn. 1983.Properties of the major outer membrane protein from Neisseriagonorrhoeae incorporated into model lipid membranes. Proc.Natl. Acad. Sci. USA 80:3831-3835.

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