Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1335-1341

November 14, 1991

IMMUNOBIOLOGICAL ACTIVITIES OF SYNTHETIC PEPTIDE SEGMENTS OF F I M B R I A L P R O T E I N F R O M PORPHYROMONAS GINGIVALIS Tomohiko Ogawa, Yutaka Kusumoto, Hiroshi Uchida, Shigeru Nagashima, Hideji Ogo and Shigeyuki Hamada* Department of Oral Microbiology, Osaka University Faculty of Dentistry, Yamadaoka, Suita-Osaka, 565 Japan Received October 3, 1991

Several oligopeptide segments of fimbrial subunit protein (fimbrilin) of Porphyromonas gingivalis strain 381 were synthesized and tested for immunobiological activities. Peptides F3(3150; amino acid residue numbers 31 to 50, based on the amino acid sequence of the fimbrilin proposed by Dickinson et al., Infect. Immun., 170, 1658, 1988), F12(212-231) and F17(312331) were found to be immunodominant epitopes of this fimbrial protein as revealed by ELISA. Furthermore, peptides F5(71-90) and F17(312-331) were demonstrated to agglutinate rabbit erythrocytes, and were mitogenic for BALB/c spleen cells but not thymocytes. These peptides enhanced the number of fimbria-specific antibody-secreting cells in BALB/c spleen cell cultures, and induced cytokines such as tumor necrosis factor-alpha and interleukin-6 production in human monocyte/macrophage cultures. The data demonstrate that these defined peptide segments are responsible for the immunostimulating portions within the fimbrial protein molecule. ®~99z Academic Press, Inc.

A variety of bacterial species possesses some surface appendages such as fimbriae or pili producing from the outermost layer of the organisms. These surface components are considered to be important in host-parasite interaction in the development of infectious diseases (1-3). Among these, the fimbriae of an anaerobic Gram-negative rod Porphyromonas gingivalis have been extensively studied in terms of purification and biochemical characterization of the fimbriae (4), cloning of the subunit protein of the fimbriae (i.e. fimbrilin) (5), interaction of the fimbriae with host cells (6) and the possible role of the fimbria in the development of chronic, localized infections such as periodontal disease, one of the most ubiquitous oral diseases in human (7-9). It has been shown that P. gingivalis fimbriae are isolated from whole cells and purified by column chromatography, and SDS-PAGE of the purified fimbriae gives a 41KDa subunit protein (fimbrilin) band (4, 10). Having demonstrated the immunological and biological activities of the purified fimbriae of P. gingivalis

strain 381, we undertook this study to determine the

immunodeterminant of the fimbriae and the molecular regions inducing biological responses in the *Corresponding author. Abbreviations: BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; ELISPOT, enzyme-linked immunospot; FBS, fetal bovine serum; IL-6, interleukin-6, LPS, lipopolysaccharide; M0, macrophages; MNL, mononuclear leukocytes; PBA, polyclonal B-cell activation; PBS, phosphate buffered saline; SDS-PAGE, sodium dodecylsulphate-polyacrylamide gel electrophoresis; TNF-a, tumor necrosis factor-alpha. 0006-291X/91 $1.50 1335

Copyright © 1991 by Academic Press, Inc. All rights" of reproduction in any form reserved.

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

host ceils, using chemically synthesized peptides representing the limited segments of the fimbrial molecule.

MATERIALS AND METHODS

Microorganisms and preparation of fimbriae and LPS: P. gingivalis strain 381 was grown anaerobically in GAM broth (Nissui, Tokyo) supplemented with hemin and menadione at 37°C for 26 h, and fimbriae were prepared as described previously (10). The basic structure of fimbriae (fimbrilin) was identified as a single band of the 41,000-molecular-weight protein by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE)(10). P. gingivalis LPS were extracted from lyophilized cells by the hot phenol/water method (11). Synthesis of peptides: Seven peptides were synthesized with a model 9050 peptide synthesizer (Millipore Corporation) by the solid-phase methods of Merrifield (12) and were based on the amino acid sequence of the native f'mabrilin of P. gingivalis 381 derived from the nucleotide sequence (5) (Table 1). After cleavage of the protecting groups with trifluoroacetic acid/mcrezol/ethanedithiol/methanesulfonic acid (90 : 2.5 : 5 : 2.5), the peptides were purified by high performance liquid chromatography on a reversed phase column (Capcell Pak C18 SG120, 1.5 x 15 cm; Shiseido, Tokyo, Japan) with a linear gradient (6 to 60%) of acetonitrile in 0.1% acetic acid at a flow rate of 23 ml per min. The major peak fraction of each peptide was collected by monitoring the absorbance at 220 nm. The purified peptides were analyzed for composition by quantitative amino acid analysis and sequenced by automated Edman degradation with a model 6400/6600 protein sequencer (Millipore Corporation). Analysis of immunodominant epitopes: ELISA inhibition assay was done using synthetic peptides as soluble inhibitors in order to determine the immunodominant epitope(s). Rabbit antisera specific for P. gingivalis fimbriae were prepared as reported previously (10). Increasing concentrations of synthetic peptides of purified fimbriae were preincubated with a constant dilution of antiserum at 37°C for 2 h. Then, ELISA of the adsorbed antisera was carried out to immobilized purified fimbriae. The inhibition by the synthetic peptides was expressed as % control without additions. Hemagglutinating assay: Hemagglutination assay was performed with a 96-well round bottm Coming 25850 microtiter plate. Samples (25 ~tl) diluted serially twofold in phosphate buffered saline (pH 7.4; PBS) were added to an equal volume of a 2% rabbit erythrocyte suspension in PBS. After gentle shaking, the microtiter plate was incubated at 37°C for 2 h, and the hemagglutination titer was shown as the concentration of samples giving a positive reaction by visual inspection. Stimulation of murine lymphocytes. (i) Thymidine uptake: Spleen cells or thymocytes (5 x 105 cells) of BALB/c mice (male, 8 weeks old) obtained from Charles River Japan (Atsugi, Japan) were cultured with increasing concentrations of test samples in 0.2 ml of RPMI1640 medium (Biken, Osaka, Japan) supplemented with 5% fetal bovine serum (FBS) in a 96-well flat bottom Falcon 3040 microtiter plate for 48 h in 5% CO2 and 95% air. During the final 16 h of cultivation, the cultures were pulsed with 1.0 ~Ci of [3H]thymidine (185 MBq/mmol; ICN Radiochemicals) per well and were harvested onto glass fiber filter strips. Thymidine uptake was measured in an LKB model 1215 liquid scintillation counter. (ii) Polyclonal B-ceU activation (PBA): Measurements for PBA were made by using an enzyme-linked immunospot (ELISPOT) assay as previously described (13). Spleen cells (2.5 x 106 cells) of BALB/c mice (male, 6 weeks old) were cultured with graded doses of test materials in RPMI 1640 medium containing 5% FBS at 37°C for 72 h in 5% CO2 and 95% air. After washing, antibody-secreting cells were quantitated by the ELISPOT assay (13). In brief, spleen cells were added to the goat anti-mouse immunoglobulin (Southern Biotechnology Associates Inc.)-coated plate which had been pretreated with 5% FBS, and were incubated for 4 h. After washing, the plates were incubated with biotinylated goat anti-mouse co, 7, or i1 chain-specific antisera (Southern Biotechnology Associates Inc.) overnight at 25°C, washed with PBS, and treated with horseradish peroxidase-conjugated streptavidin (Zymed Laboratories) for the development of spots for antibody-secreting cells. The number of spots was enumerated with the aid of a dissecting microscope. 1336

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Preparation of monocyte/macrophage (Me) cultures and cytokine assays. (i) Monocyte/M¢ culture: Human peripheral blood monocytes were isolated by Ficoll Hypaque separation of mononuclear leukocytes (MNL), followed by removal of nonadherent cells. MNL suspensions containing 1.5 x 106 cells per ml of Eagle's minimum essential medium (MEM; Biken) containing 10% FBS were placed in individual wells of a 96-well flat bottom Falcon 3072 microtiter plate, to which various concentrations of test samples were added. The plate was incubated for 24 h at 37°C in 5% CO 2 and 95% air, and the culture supemantant was collected by centrifugation. Monocytes were found to comprise 85%-90% of the cells as determined by morphology and non-specific esterase staining. (ii) TNF-ct assay: TNF-ct levels were determined by measuring cytotoxity against murine fibroblastic L-929 ceils as described previously (14). Aliquots (100 ~tl) of L-929 cell suspension (5 x 105/ml) were pipetted into a 96-well flat bottom Falcon 3072 microtiter plate and incubated for 24 h. Actinomycin D (2 ~tg/ml, Sigma Chemical Co.) and 100 ~ of each dilution of the culture supernatant of monocytes/M¢ or various concentrations of human rTNF-ct were added to L-929 cells in MEM containing 10% FBS to obtain a standard curve. After incubation at 37°C for 24 h, the supernatant was discarded and the cells were fixed in 5% formaldehyde, followed by staining with 0.2% crystal violet. The stained plate was washed and dried. Since viable cells retained the dye, A595 of each well was read with a Titertek Multiskan MC photometer. Values obtained from triplicate wells were averaged to obtain the mean values. The titer of TNF-ct in monocytes/M¢ supernatants was determined by comparison with a set of standards made by adding human rTNF-ct (3x106 units/mg; Dainippon Pharmaceutical Co., Osaka) and was expressed as TNF-ct units + standard error. (iii) IL-6 assay: IL-6 assay was performed by enzyme-linked immunosorbent assay (ELISA) (15). Briefly, 96-well ELISA plates (M129; Dynatech Laboratories Ltd., Billingshurst, UK) were coated with 100 lal of monoclonal antibody (mAb) specific for human IL-6 (1 gg/ml; kindly provided by Drs. T. Kishimoto and T. Hirano, Osaka University, Suita-Osaka, Japan) at 4°C overnight in 0.1 M carbonate buffer (pH 9.6). The wells were then blocked with 100 ~tl of 1% BSA-PBS at 4°C overnight and washed with PBS containing 0.05% Tween 20 (PBS-T), and test samples were placed in each well. After overnight incubation at 4°C, the wells were washed with PBS-T and rabbit anti-IL-6 antibodies (Genzyme Corporation) biotinylated using Biotin-X-NHS (Calbiochem) (10 ng in 100 ~tl) were added. The plates were incubated at 4°C overnight and washed with PBS-T. Then the Vector ABC-AP kit reagent (Vector Laboratories) was added to each well and A405 was read. Determination of IL-6 titers in a culture supernatant was done in comparison with an IL-6 standard curve under the assay condition and titers were expressed as units + standard error (SE) of each subject group. Statistics: Each assay was done in triplicate, and the means and standard errors were calculated. Comparisons between the test and control groups were done by Student's t test for independent samples. RESULTS Peptides mimicking segments of the subunit structure of P. gingivalis fimbriae were synthesized (Table 1) based on the amino acid sequence derived from the nucleotide sequence of

Table 1 The amino acid sequence of synthetic peptides correspondingto the segments of P. gingivalis 381 fimbrilin Peptide

Positiona

F3 (31-50) F5 (71-90) F6(91-111) F11 (192-211) F12(212-231) F16 (292-311) F17 (312-331)

31-50 71-90 91-111 192-211 212-231 292-311 312-331

Amino acid sequencea GEQQEAIKSAENATKVEDIK GKTLAEVKALlq'ELTAENQE AAGLIMTAEPKTIVLKAGKNY FNGAYTPANYANVPWLSRNY VAPAADAPQGFYVLENDYSA NYTPKNKIERNHKYDIKLTI TGPGTNNPENPITESAHLNV

a According to Dickinsonet a1.5) 1337

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Preparation

(gffml)

F3(31-50)

0.01

F5(71-90)

5

F6(91-111)

5

Fl1(192-211)

5

F12(212-231)

0.01

F16(292-311)

5

F17(312-331)

0.01

Fimbna

0.01

E. coli O55:B5

5

20

%Inhibition of E L I S A 40 60

80

100

LPS

Figure 1. ELISA inhibition of P. gingivalis fimbria-specific antibody by fimbriae and synthetic peptides mimicking segments of fimbrial subunit protein. Inhibition assay was done by incubating a constant dilution (1:106) of antibody with increasing concenlxations of synthetic peptides or fimbria as soluble inhibitors at 37°C for 2 h before assay with fimbria-coated plate for an ELISA. The inhibitory effect was expressed as %inhibition. the fimbrilin gene. Inhibition by the peptides of the ELISA reaction between rabbit IgG specific for P. gingivalis fimbriae and the purified fimbrial antigen was examined. We found that peptides F3(31-50), F12(212-231) and F17(312-331) significantly consumed IgG specific for fimbriae, resulting in reduced ELISA reactivities; however, the ELISA inhibition activity by these peptides (50-68%) was clearly lower than that of native fimbriae (Fig. 1). As shown in Table 2, P. gingivalis fimbriae strongly agglutinated rabbit erythrocytes at a concentration of 0.5 ~tg/ml. LPS from this organism also induced hemagglutination at a minimum concentration o f 7.8 ~tg/ml, while the LPS from Escherichia coli

O55:B5 did not cause

hemagglutinin up to a concentration of 250 ~tg/ml. Further, F5(71-90) and F17(312-331) were found to induce agglutination of rabbit erythrocytes at low concentrations of 15.6 and 62.5 ~tg/ml, respectively. These results indicate that P. gingivalis native fimbriae and some peptide segments exhibit hemagglutinating activity. Table 2

Hemagglutinating activity of the synthetic peptides mimicking the fimbrial segments Minimal hemagglutinating Preperation concentration (I.tg/ml) a F3 (31-50) F5 (71-90) F6(91-111) F11 (192-211) F12 (212-231) F16 (292-311) F17 (312-331) Firnbriae P. gingivaris 381 LPS E. coli 055:B5 LPS

250 15.6 250 250 250 250 62.5 0.5 7.8 --b

a Rabbit erythrocytes were used. b Nonagglutinatedup to a concentration of 250 Ixg/ml. 1338

Vol. 180, No. 3, 1991

Preparation

(gg/rnl) 5

F3 (31-50)

5~

F5 (71-9O)

5

Immunobiological activities of synthetic peptide segments of fimbrial protein from Porphyromonas gingivalis.

Several oligopeptide segments of fimbrial subunit protein (fimbrilin) of Porphyromonas gingivalis strain 381 were synthesized and tested for immunobio...
495KB Sizes 0 Downloads 0 Views