Vol.
175,
March
No.
15,
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1991
Pages
713-719
Purification, Characterization and Immunolocalization of Fimbrial Protein from Porphyromonas (Bacteroides) gingivalis Hakimuddin
T. Sojar, Jin-Yong Lee, Gurrinder Moon-II Cho and Robert J. Genco
S. Bedi,
Department of Oral Biology, 3435 Main Street, 213 Foster Hall State University of New York at Buffalo, Buffalo, New York 14214 Received
February
4, 1991
Summary: Rapid and reproducible method is describedhere for the purification of the 43 kDa fimbrial protein from E gingivalis by preferential fractionation in the presenceof 1% SDS and 0.2M of a bivalent cation at pH 6.5. Homogeneity of the purified 43 kDa was confirmed by SDS-PAGE and Western blot analysisusing monoclonaland polyclonal antibodiesraisedagainstthis protein. Amino acid composition and the amino acid sequenceof the first 30 amino acid residuesof the purified fimbriae are consistent with the composition and sequencepredicted from the cloned gene of the fimbrial subunit. Circular dichroism spectrashowshigh levels of B-sheetstructure. The purified 43 kDa polymer shows fimbriae-like morphology under the electronmicroscope.Ultrastructural localization of the 43 kDa protein by the immunogoldtechniquerevealed specificlabelingof the fimbriae with a diameterof approximately 3.5 to 5.0 run. Localization of this protein suggestthat the 43 kDa component is a fimbrial subunit. 0 1991Academic Press,Inc.
Epithelial cell adherenceand colonization of the mucosalsurfaceby pathogenicmicroorganismsis the first stepwhereby bacteriaestablishresidenceon the mucosa,a necessaryprerequisiteto infections at thesesurfaces. Gram-negative bacteriamay have severaladhesinson their surfacewhich are responsible for attachment to specific host receptors (1,2,3,4,5). These adhesinsinclude proteinaceousfilamentous surface appendages called pili or fimbriae, hemagglutinins, polysaccharide capsules and other surface-bindingcomponents. In order to study the role of outer cell surfacecomponentsof I: ginaivalis in microbial adhesion,it is important to purify thesecomponentsto homogeneity in their intact native form. Fimbriae detachedfrom P. aintivalis cells by meansof shearingor mild sonication, contain two major protein componentsof 43 kDa and75 kDa, respectively. The 43 kDa componenthasbeenshown to be a major timbrilin monomersubunit of fimbriae from E. gingivalis (6,7,8,9). Although both 43 kDa fimbrial protein and 75 kDa major envelope protein from P. ainaivalis strain 381 have recently been purified (lO,ll),
the proteins purified according to previous published procedures were often
contaminatedwith trace amountsof eachother. Sincethe 75 kDa protein is very immunogenic,antibodies raisedagainst preparationsof the 43 kDa fimbrial protein often showedcrossreaction with the 75 kDa protein, which complicate studiesof fimbrial function. The intention of this study was to purify 43 kDa fimbrial protein to homogeneity in native form and free from cross-contaminationwith 75 kDa protein. In the present study, the physico-chemical and immunological properties of the purified fimbrial component are reported. In addition, the location of this component is localized on whole cells using immunogoldlabeling of the 43 kDa protein.
713
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, hc. All rights of reproduction in any form reserved.
Vol.
175, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
MATERIALS
AND METHODS
RESEARCH COMMUNICATIONS
Bacterial strains and growth conditions: P. 2561 (ATCC strain no. 33277) was grown in brain heart infused broth (Difco Chemical Co) supplemented with 5 mg of yeast extract, 5 pg of hemin and 0.2 p.g of menadione per ml, pH 7.4 at 37’C for two days in a Forma anaerobic chamber (85% N2,10% H2, and 5% C02). Purification of 43 kDa fimbriae protein from E. eingivalis: Em cells were harvested from 2 liter batches of culture using centrifugation and were washed three times with a 50 mM Tris-Cl buffer at a pH 8.0 containing 10% sucrose and 1 mM of the protease inhibitor PMSF. The cell pellet was suspended in the same buffer and the fimbriae sheared from the cells by mild sonication with a 3 mm microtip using a 20W pulse setting with a 50% duty cycle in a Vibra cell model VC 250 sonicator. After, this treatment, less than 1% of the cells appeared microscopically broken. Whole cells were removed by centrifugation at 10,000 g for 15 minutes in a Sorvall RC 5C centrifuge. The supematant was further centrifuged at 100,000 g for one hour in a Beckmann LS8-80 ultracentrifuge. The supernatant, which contained mainly the 43 kDa and 75 kDa proteins, was dialyzed overnight against a 5C mM T&-Cl buffer at pH 8.0. A IO-fold concentrated stock solution of SDS and MgC12 was added to bring final concentrations to 1% SDS and 0.2M MgC12, pH adjusted to 6.5 with 1M HCl and the mixture was stirred continuously overnight at 4°C. The precipitated proteins were collected by centrifugation at 10,000 g for 30 minutes, dissolved in a small volume of 50 mM Tris buffer at pH 8.0 and dialyzed against the same buffer. The clear supematant was readjusted to pH 6.5 with 1 M HCl and the precipitation step was repeated 3 to 4 times to obtain purified 43 kDa fimbrial protein free of the 75 kDa contaminant. Supernatants contained the 75 kDa enriched fraction. To remove SDS from the purified 43 kDa protein, the sample was passed through a column (1.5 cm x 5 cm) of extracti-gel D (Pierce Chemical Co.) previously equilibrated with 50 mM T&-Cl pH 8.0 according to the manufacturer’s directions. Preparation of antisera and Monoclonal antibodies: Male New Zealand white rabbits (5 to 6 lbs) were immunized at multiple intradermal sites with 200 pg of purified 43 kDa protein in 0.5 ml of isotonic saline emulsified with an equal volume of Freunds’ complete adjuvant. Animals were boosted at weekly intervals with 200 l.tg of the antigen emulsified with incomplete Freund’s adjuvant. Six weeks later the rabbits were bled and the sera tested for antibody by immunoblotting (12) as well as by the enzyme linked immuno-absorbent assay (13). Antibodies used for immunolocalization studies were purified by chromatography on Sepharose-Protein A column using Immuno-Pure IgG ( Pierce Chemical Company) to prepare IgG fractions of the sera. Monoclonal antibodies to the 43kDa protein were generated as described earlier (14). Analytical Procedures: Protein was determined by the Bradford method (15) using bovine serum albumin as the standard. The neutral sugars, hexosamines and amino acid analyses were performs as described earlier (16). Purified protein was subjected to automated stepwise sequencing on an Applied Biosystems Model 477A gas phase sequencer with an on line Mode1 120A PTH analyzer (14).SDS-PAGE was performed in a minigel system (Mini-Protean II cell, Bio Rad) using a 1 mm thick 7.5% running gel according to the method described by Lammeli (17). Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes in 25 mM Tris-glycine buffer pH 8.3 for Western blot analysis as described previously (14). The purified 43 kDa protein was tested for hemagglutination by the method described earlier by Slots and Genco (18). Circular Dichroism Studies: A Jasco J-600 Spectropolarimeter was used to measure the CD spectra of the 43 kDa in a 0.1 cm path length quartz cell at a protein concentration of 0.25 mg/ml in Tris-Cl buffer at a, pH of 7.5 with or without 0.1% SDS. The secondary structure of both the proteins were analysed by the method of Provencher and Glockner (19). Ultrastructure and Immunogold Labeling of the Purified 43kDa Protein: The ultrastructure of the purified 43 kDa protein, and its binding to purified antibodies were simultaneously investigated using indirect electron microscopy. For this purpose a small amount (l-3 l.tl) of the purified 43 kDa protein sample ( 1 mg/ml in 20 mM phosphate pH 7.4 ) was applied on Formvar and carbon-coated nickel grids and air dried. After washing in TBS (10 mM Tris-HCl buffer containing 150 mM NaCl, pH 7.4) for 20 min. the samples were incubated for 1 hr in the IgG against the 43 kDa protein (150-200 p.&nl). The samples were washed in the same buffer and further incubated for 1 hr with goat anti-rabbit IgG conjugated with IO-nm gold particles (1:20) (Janssen Life Sciences Products, Piscataway, N.J.). After washing in TBS and distilled water, the samples were air dried and negatively stained with 1% uranyl acetate. Controls consisted of incubation of the samples either, (i) in the absence of the rabbit antibodies, or (ii) in the presence of normal rabbit IgG, or (iii) in the presence of antibodies against the 75 kDa protein. Electron micrographs were taken with a Hitachi T-600 electron microscope at 75KV. 714
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Immunogold Labeling of the 43 kDa Fimbrial Protein on Whole t. gingivalis: A suspension (l-2 ~1) of whole cells of p. &giy& was applied to Formvar and Carbon-coated nickel grids. Excess fluid was drained using filter paper and the sample was air dried. For indirect immunogold labeling of the 43 kDa, the bacterial sample on the grid was incubated within a drop of purified IgG fraction of rabbit antibody to the 43 kDa (100 - 150 l@nl) in PBS for 2 hrs. After washing with PBS for 30 min, the sample was further incubated for 1 hr within a drop of goat anti-rabbit IgG conjugated with 5-nm or IO-nm gold particles (1:20). The sample was then washed for 30 min in the same buffer, fixed in 2% glutaraldehyde in PBS for 30 min to ensure the binding of the antibodies to the bacteria, washed in distilled water, and air dried. Visualization of the whole complex was achieved by negative staining with 1% aqueous uranyl acetate. Any non-specific labeling was assessed by evaluation of controls which consisted of incubation of the samples in the absence of the purified rabbit antibodies, the conjugate alone, or in the presence of normal rabbit IgG plus conjugate as a replacement for the purified antibodies to the 43 kDa. RESULTS
AND DISCUSSION
Fimbriae were sheared from E gineivalis strain 2561 by mild sonication, followed by ammonium sulfate precipitation at 40% concentration. SDS-PAGE of the pooled preparation under denaturing conditions (100°C for 10 min. in the presence of 5% B-mercaptoethanol)
showed two major bands with
an apparent molecular size of 75 kDa and 43 kDa and also some additional minor bands. Under partially denaturing conditions (80°C for 5 min in the absence of l3-mercaptoethanol) fimbriae appeared as a series of bands with a strong liklihood of representing polymeric forms. The 75 and 43 kDa components in the ammonium sulfate precipitated sonicate could not be adequately dissociated under conventional protein purification
procedures
which
included:
chromatography
on columns
of Sepharose
CL-6B,
DEAE-Sepharose CL-6B and FPLC on a mono-Q anion-exchange column. Furthermore, the 75 kDa and 43 kDa components could not be separated by FPLC on Suprose- column in the presence of 6 M urea or other detergents including CHAPS, digitonin, polyethylene Tween-20, However,
Tween-80,
sodium deoxycholate,
glycol, octylglucopyranoside,
sodium oxycholate,
sucrosine,
Triton X-114 and Triton X-100.
partial dissociation of these two components could be achieved with a saturated solution of
guanidine hydrochloride. The inability to completely dissociate these components prompted further study of the chemical nature of the 43 kDa and 75 kDa components.
Both components were found to stain with Coomassie
blue, Sudan black and fat red 7B, but not with PAS.
This staining pattern suggested that these
components may be either proteolipid in nature or associated with lipids. This was further confirmed when both components dissociated with the organic solvents such as phenol and dimethyl formamide. (Data not shown). Based on the lipid-like nature of these proteins we have developed a method for their differential precipitation.The
fimbrial protein (43 kDa component) was preferentially precipitated in the
presence of 1% SDS and 0.2 M MgC12 at pIi 6.5 and homogeneous preparations regularly obtained by repetitive precipitation steps. Similar preferential precipitation procedures in the presence of divalent metal ions have traditionally been used for precipitation of lipoproteins (20).
Homogeneity
of the purified
kDa on SDS-polyacrylamide
protein
: The purified proteins showed single bands of 43
gel electrophoresis
under reducing conditions when stained with either
Coomassie or with the more sensitive silver staining method (Fig.l).The
preparations were free from
cross-contamination with LPS since silver staining did not show any additional bands corresponding to LPS. Staining with “Stain all” failed to show phosphoprotein contaminants. Preparations of protein was also radiolabelled
with [1251] and electrophoresed
on SDS-PAGE.
71.5
OnIy a single radioactive band
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BIOCHEMICAL
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7
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Figure 1. SDS-PAGEand Westernblot analysisof crudefimbriae and purified 43 kDa protein. Sampleswere electrophoresed on 7.5% reducedSDS-polyacrylamidegelsand stainedwith Coomassie blueR-250. Lane1containsthefollowingmolecularweightmarkers:Phosphorylase b, 95 kDa; Glutamatedehydrogenase, 55 kDa; Gvalbumin,43 kDa; Lactatedehydogenase, 36 kDa andCarbonicanhydrase.29 kDa. Lane2, crudefimbriaein nativeform without heating. Lane3, crudefmbriae heatedat 80°Cfor 10minutes.Lane4, crudefimbriaeheatedat 100°Cfor 10minutes. Lane5, purified43kDaproteinin nativeform withoutheating.Lane6, purified43 kDa protein heatedat 80°C for 10minutes. Lane7, purified 43 kDa heatedat 100°Cfor 10 minutes.Lane8, immunoblotof puritied43 kDa usingthe43 kDa polyclonalantibody. Lane9, immunoblotof crudefimbriaeusing43 kDapolyclonalantibody.
corresponding to 43 kDa molecular mass, was observed for the purified protein preparations. The purified 43 kDa protein showed a ladder like pattern at 80°C and could be dissociated into it’s monomeric form only after heating at KKPC for 10 min. The apparent molecular weight of the oligomeric forms of 43 kDa protein were calculated to be 60,73,80,107,125,140 and higher polymers. Although each band appears to be a multiple of 43 kDa monomer, this aberrant migration pattern observed cannot be explained at this time. Homogeneity of the purified proteins was further confiied by Western blot analysis 0;ig.l). Western blot analysis using polyclonal antibodies specific for the purified 43 kDa protein reacted with both polyclonal and monoclonal antibodies raised against 43 kDa protein and did not react with monoclonal or polyclonal antibodies raised against the 75 kDa protein. Western blots of the purified 43 kDa also showed single bands when tested with antisera developed to whole cells of mgingivalis. Since E. gingivt-& possess strong haemagglutinating activity, we investigated the involvement of purified proteins in the agglutination of sheep erythrocytes. Using purified 43 kDa, we were not able to show any hemagglutinating activity. These results indicate that 43 kDa protein is not able to agglutinate sheep erythrocytes in their purified forms. Chemical Composition and Amino-terminal sequence analysis: The amino acid analysis of the purified 43 kDa protein is given in Table I. The amino acid analysis shown is an average of duplicate hydrolysate samples at 24 h. The amino acid composition of the 43 kDa protein is identical to the deduced composition obtained from the cloned fimbrilin gene (Table I). Hexosamines or neutral sugars were not detected in either proteins. The sequence of the first thirty residues of the purified 43 kDa protein waselucidatedby automatedEdtnandegradationand wasconsistentwith the elucidatedpredicted sequenceof cloned fimbrilin gene from a related strain of p. &@yt& (21).
Electron Microscopy Whole Cells of1.w:
and Immunogold Localization of the 43kDa Protein on The purified 43 kDa protein preparation showed a fimbrial structure 716
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BIOCHEMICAL
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TABLE
RESEARCH
COMMUNICATIONS
I
Amino acidcomnosition of P.ainaivalis2561Fimbrillinurotein Aminoacids
ClonedFimbrillinb
PurifiedProteina %Residues Res./Molecule
%Residues
Res./Molecule
Asx Thr Ser Glx
13.7 8.3 4.4 11.0
54.8 33.2 17.6 44.0
13.1 8.6 3.6 9.5
44 29 12 32
GUY Ala Val Met lie
10.0 3.4 12.7 6.0 1.5
7.7 3.9 12.8 7.4 ::t
:z 43 25 6
7.1 5.6
:: 19
26:: 1.5 A:;
23 5 5 3
LeU
t
Tyr
3:8
40.0 13.6 50.8 24.0 6.0 16.4 29.2 15.2
Phe LYS His ‘4% CA
2.3 7.2 1.6 1.8 0.9
2::: 6.4 7.2 3.6
pro
:
a The purified protein was hydrolysedwith 6N HCI at 1lo’C for 22 hrs. Analytical valuesareexpressed asresidues/molecule basedon molecularweight43,000daltons. b Deducedfrom the genecloningsequence of Dickinsonet al. (21). Thesevaluesare basedon the predictedsizeof sequenced protein36,CKKl daltons.
with a diameter of approximately 3-5 nm when observedunder the electron microscope. Their fimbrial nature was further confirmed by specific immunogold labelling with 43 lcDa fimbrial protein specific antibodies (Fig. 2a). The samplesshowed no significant labeling with either normal rabbit IgG or in controls using antibodies againstthe 75 kDa protein (Data not shown). The proceduredescribedfor the purification of fimbriae usesmild precipitation conditions. These mild conditions do not irreversibly dissociatethe fimbrial structure, sincethe isolatedfimbriae appearto be intact asdemonstratedby electron microscopic studies (Fig. 2a). Earlier procedures described in the literature required either organic solvents(22) or higher temperatures(23). The preciselocalization of both the 43 lcDaproteinson the cells of P. pingivalis wassuccessfully achieved using indirect immunogold labeling and negative staining with 1% aqueousuranyl acetate. Ultrastructural localization of the 43 kDa protein by the immunogoldtechniquerevealedthat gold particles specifically labelled fimbriae with a diameterof approximately 3 to 5 nm, especially thoseradiating from the outer cell membrane(Fig.2b). No labeling was observed when the primary antibodies were either omitted or replacedwith non-immuneimmunoglobulinsascontrols. In this study, ultrastructural study of the purified 43 lcDaprotein allowed the characterization and morphology of this protein in aqueoussolutions. In addition, immunogold labeling using monospecific antibodies to this protein provided very valuable and conclusive information on it’s localization on p. pincivalis. Ultrastructural analysis of the purified 43 kDa protein preparation after negative staining clearly demonstratedthat this protein forms elongated filamentous structures 3 to 5 nm in diameter, similar to the typical fimbriae extending from the surfaceof p. gintzivalis cells. Furthermore, specific 717
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Figure 2a. Inununogold labelling of the purified 43 kDa protein. Note typical fimbriae (arrowheads) and their specific labelling with gold particles. Figure 2b. Irnrnunogold localization of the 43 kDa protein on a whole cell of E. g&&& 2561 strain. Note a heavy labelling of fimbriae (arrowheads) with gold particles, and lack of labelling in control (Inset). immunogold labeling of both, the purified 43 kDa protein and the native fimbriae of p. gingivalis, with antibodies to the 43 lcDa protein strongly suggests that the 43 kDa protein represents a major structural protein of fimbriae. Circular Dichroism
studies:
The stability and oligomeric
nature of the purified
43 kDa
proteins prompted further investigation into the secondary structure of these components. The CD spectra between 200 to 260 nm. shows no negative peak at 208 or 222 nm, a region characteristic of alpha helical structure. The spectrum remained unchanged after boiling the samples in the presence of SDS at 100°C. The 43 lcDa protein in native form, as well as after boiling at 100°C showed no detectable alpha helical, 67% beta sheet and 37% remainder structure. Circular dichroism spectra indicated the predominant secondary structure in the 43 kDa components was a beta sheet structure. This is consistent with the predicted secondary structure of the fimbrial subunit protein sequence (24). The CD spectra of this component is similar to most other outer membrane proteins with a high content of beta sheet structure. The high percentage of &sheet and random coil probably contribute to the unusual stability of this component. Our preliminary salivary
components
pathogenicity
binding studies indicate that purified 43 l&a protein binds specifically to several and to buccal epithelial cells.
the further extensive investigation
To understand
the exact role of fimbriae
about fimbriae structure,
in
specific salivary binding
components as well as structure of epithelial cell receptor is essential. ACKNOWLEDGMENTS:
This study was support in part by U.S. Public Health Sciences grants
DE08240, DE07034, DE04898, DE06514.
The authors thank Ms. Darlene Badgett for her excellent
technical assistance and Ms. Karen Flowers for her help in typing the manuscript. REFERENCES 1. 2.
Beachy, E.H. (1981) J. Infect. Dis. m, 325345. Gibbons, R.J. and van Houte, J. (1980) Bacterial adherence: In (E.H. Beachey ed.) Series B, Vol. 6, pp 61-104 Chapman and Hall, London and New York. 718
Vol.
5. 6. 7. i: 10. II. 12. 13. 14. 1.5. 16. 17. 18. 19. 20. 21. 22. 23. 24.
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2, 1991
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Gibbons, R.J. (1984) J. Dent. Res. a, 378-385. Jones, G.W. (1977) In Microbial interactions: (J. L. Reissig ed.), Series B Vol. 3 pp 139-176 Chapman and Hall, London. Ofek, I., and Beachey, E.H. (1980) In “Bacterial adherence” (E.H. Beachey ed.), Series B Vol. 6 pp l-29, Chapman and Hall, London and New York. Okuda, K., and Takazoe, I. (1974) Arch. Oral Biol. 1p, 415416. Okuda, K., Slots, J. and Genco, R.J. (1981) Cum Microbial. 6, 7-12. Slots, J. and Gibbons, R.J. (1978) Infect. Immun. le, 254-264. Woo, D. D. L., Holt, S.C. and Leadbetter, E.R. (1979) J. Infect. Dis. 139, 534-546. Yoshimura, F., Takahashi, K., Nodasaka, Y, and Suzuki, T. (1984) J. Bacterial. m, 949-957. Yoshimura, F., Takahashi, K., Yoneyama, M., Yamaguchi, T., Shiokawa, H. and Suzuki, T. (1985)J.Bacteriol.l,730-734. Towbin, H., Staehlin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. U.S.A. %, 4350-4354. Bedi, G.S., Back, N. (1987) Hybridoma. 4, 521-526. Lee, J.Y., Sojar, H.T., Bedi, G.S. andGenco, R.J. (1991) Infect. Immun. Z, 383-389. Bradford, M.M. (1976) Anal. Biochem. 2, 248-254. Bedi, G.S., French, W.C. and Bahl, O.P. (1982) J. Biol. them 257, 4345-4355. Laemmli, U. K. (1970) Nature 227, 680-685. Slots, J. and Genco, R.J. (1979) J. of Clinical. Microb. 14(3), 371-373. Provencher, S.W. and Glockner, J. (1981) Biochem. 20, 33-37. Burstein, M. and Scholnick, H.R. (1973) Adv. Lipid. Research. 11, 68-108. Dickinson, D.P., Kubiniec, M.K., Yoshimura, F. and Genco, R.J. (1988) J. Bacterial. 170, 1658-1665. Yoshimura, F., Wantanabe, K.I., Takasawa, T., Kawanami, M. and Kato, H. (1989) Infect. Immun. s( 1 I), 3646-3652. Hoschutzky, H.F., Lottspeich, and Jann, K. (1989) Infect. Immun. Z, 76-81. Genco, R.J., Lee, J.Y., Sojar, H.T., Bedi, G.S., Loos, B.G. and Dyer, D.W. (1991) In “Periodontal Disease: Pathogen and host immune responses” (Hamada, S., Holt, SC. and McGhee, J.R. eds.). Quintessence Publishing Co., Ltd. Japan (In Press).
719
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1991
Pages
713-719
Purification, Characterization and Immunolocalization of Fimbrial Protein from Porphyromonas (Bacteroides) gingivalis Hakimuddin
T. Sojar, Jin-Yong Lee, Gurrinder Moon-II Cho and Robert J. Genco
S. Bedi,
Department of Oral Biology, 3435 Main Street, 213 Foster Hall State University of New York at Buffalo, Buffalo, New York 14214 Received
February
4, 1991
Summary: Rapid and reproducible method is describedhere for the purification of the 43 kDa fimbrial protein from E gingivalis by preferential fractionation in the presenceof 1% SDS and 0.2M of a bivalent cation at pH 6.5. Homogeneity of the purified 43 kDa was confirmed by SDS-PAGE and Western blot analysisusing monoclonaland polyclonal antibodiesraisedagainstthis protein. Amino acid composition and the amino acid sequenceof the first 30 amino acid residuesof the purified fimbriae are consistent with the composition and sequencepredicted from the cloned gene of the fimbrial subunit. Circular dichroism spectrashowshigh levels of B-sheetstructure. The purified 43 kDa polymer shows fimbriae-like morphology under the electronmicroscope.Ultrastructural localization of the 43 kDa protein by the immunogoldtechniquerevealed specificlabelingof the fimbriae with a diameterof approximately 3.5 to 5.0 run. Localization of this protein suggestthat the 43 kDa component is a fimbrial subunit. 0 1991Academic Press,Inc.
Epithelial cell adherenceand colonization of the mucosalsurfaceby pathogenicmicroorganismsis the first stepwhereby bacteriaestablishresidenceon the mucosa,a necessaryprerequisiteto infections at thesesurfaces. Gram-negative bacteriamay have severaladhesinson their surfacewhich are responsible for attachment to specific host receptors (1,2,3,4,5). These adhesinsinclude proteinaceousfilamentous surface appendages called pili or fimbriae, hemagglutinins, polysaccharide capsules and other surface-bindingcomponents. In order to study the role of outer cell surfacecomponentsof I: ginaivalis in microbial adhesion,it is important to purify thesecomponentsto homogeneity in their intact native form. Fimbriae detachedfrom P. aintivalis cells by meansof shearingor mild sonication, contain two major protein componentsof 43 kDa and75 kDa, respectively. The 43 kDa componenthasbeenshown to be a major timbrilin monomersubunit of fimbriae from E. gingivalis (6,7,8,9). Although both 43 kDa fimbrial protein and 75 kDa major envelope protein from P. ainaivalis strain 381 have recently been purified (lO,ll),
the proteins purified according to previous published procedures were often
contaminatedwith trace amountsof eachother. Sincethe 75 kDa protein is very immunogenic,antibodies raisedagainst preparationsof the 43 kDa fimbrial protein often showedcrossreaction with the 75 kDa protein, which complicate studiesof fimbrial function. The intention of this study was to purify 43 kDa fimbrial protein to homogeneity in native form and free from cross-contaminationwith 75 kDa protein. In the present study, the physico-chemical and immunological properties of the purified fimbrial component are reported. In addition, the location of this component is localized on whole cells using immunogoldlabeling of the 43 kDa protein.
713
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, hc. All rights of reproduction in any form reserved.
Vol.
175, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
MATERIALS
AND METHODS
RESEARCH COMMUNICATIONS
Bacterial strains and growth conditions: P. 2561 (ATCC strain no. 33277) was grown in brain heart infused broth (Difco Chemical Co) supplemented with 5 mg of yeast extract, 5 pg of hemin and 0.2 p.g of menadione per ml, pH 7.4 at 37’C for two days in a Forma anaerobic chamber (85% N2,10% H2, and 5% C02). Purification of 43 kDa fimbriae protein from E. eingivalis: Em cells were harvested from 2 liter batches of culture using centrifugation and were washed three times with a 50 mM Tris-Cl buffer at a pH 8.0 containing 10% sucrose and 1 mM of the protease inhibitor PMSF. The cell pellet was suspended in the same buffer and the fimbriae sheared from the cells by mild sonication with a 3 mm microtip using a 20W pulse setting with a 50% duty cycle in a Vibra cell model VC 250 sonicator. After, this treatment, less than 1% of the cells appeared microscopically broken. Whole cells were removed by centrifugation at 10,000 g for 15 minutes in a Sorvall RC 5C centrifuge. The supematant was further centrifuged at 100,000 g for one hour in a Beckmann LS8-80 ultracentrifuge. The supernatant, which contained mainly the 43 kDa and 75 kDa proteins, was dialyzed overnight against a 5C mM T&-Cl buffer at pH 8.0. A IO-fold concentrated stock solution of SDS and MgC12 was added to bring final concentrations to 1% SDS and 0.2M MgC12, pH adjusted to 6.5 with 1M HCl and the mixture was stirred continuously overnight at 4°C. The precipitated proteins were collected by centrifugation at 10,000 g for 30 minutes, dissolved in a small volume of 50 mM Tris buffer at pH 8.0 and dialyzed against the same buffer. The clear supematant was readjusted to pH 6.5 with 1 M HCl and the precipitation step was repeated 3 to 4 times to obtain purified 43 kDa fimbrial protein free of the 75 kDa contaminant. Supernatants contained the 75 kDa enriched fraction. To remove SDS from the purified 43 kDa protein, the sample was passed through a column (1.5 cm x 5 cm) of extracti-gel D (Pierce Chemical Co.) previously equilibrated with 50 mM T&-Cl pH 8.0 according to the manufacturer’s directions. Preparation of antisera and Monoclonal antibodies: Male New Zealand white rabbits (5 to 6 lbs) were immunized at multiple intradermal sites with 200 pg of purified 43 kDa protein in 0.5 ml of isotonic saline emulsified with an equal volume of Freunds’ complete adjuvant. Animals were boosted at weekly intervals with 200 l.tg of the antigen emulsified with incomplete Freund’s adjuvant. Six weeks later the rabbits were bled and the sera tested for antibody by immunoblotting (12) as well as by the enzyme linked immuno-absorbent assay (13). Antibodies used for immunolocalization studies were purified by chromatography on Sepharose-Protein A column using Immuno-Pure IgG ( Pierce Chemical Company) to prepare IgG fractions of the sera. Monoclonal antibodies to the 43kDa protein were generated as described earlier (14). Analytical Procedures: Protein was determined by the Bradford method (15) using bovine serum albumin as the standard. The neutral sugars, hexosamines and amino acid analyses were performs as described earlier (16). Purified protein was subjected to automated stepwise sequencing on an Applied Biosystems Model 477A gas phase sequencer with an on line Mode1 120A PTH analyzer (14).SDS-PAGE was performed in a minigel system (Mini-Protean II cell, Bio Rad) using a 1 mm thick 7.5% running gel according to the method described by Lammeli (17). Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes in 25 mM Tris-glycine buffer pH 8.3 for Western blot analysis as described previously (14). The purified 43 kDa protein was tested for hemagglutination by the method described earlier by Slots and Genco (18). Circular Dichroism Studies: A Jasco J-600 Spectropolarimeter was used to measure the CD spectra of the 43 kDa in a 0.1 cm path length quartz cell at a protein concentration of 0.25 mg/ml in Tris-Cl buffer at a, pH of 7.5 with or without 0.1% SDS. The secondary structure of both the proteins were analysed by the method of Provencher and Glockner (19). Ultrastructure and Immunogold Labeling of the Purified 43kDa Protein: The ultrastructure of the purified 43 kDa protein, and its binding to purified antibodies were simultaneously investigated using indirect electron microscopy. For this purpose a small amount (l-3 l.tl) of the purified 43 kDa protein sample ( 1 mg/ml in 20 mM phosphate pH 7.4 ) was applied on Formvar and carbon-coated nickel grids and air dried. After washing in TBS (10 mM Tris-HCl buffer containing 150 mM NaCl, pH 7.4) for 20 min. the samples were incubated for 1 hr in the IgG against the 43 kDa protein (150-200 p.&nl). The samples were washed in the same buffer and further incubated for 1 hr with goat anti-rabbit IgG conjugated with IO-nm gold particles (1:20) (Janssen Life Sciences Products, Piscataway, N.J.). After washing in TBS and distilled water, the samples were air dried and negatively stained with 1% uranyl acetate. Controls consisted of incubation of the samples either, (i) in the absence of the rabbit antibodies, or (ii) in the presence of normal rabbit IgG, or (iii) in the presence of antibodies against the 75 kDa protein. Electron micrographs were taken with a Hitachi T-600 electron microscope at 75KV. 714
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Immunogold Labeling of the 43 kDa Fimbrial Protein on Whole t. gingivalis: A suspension (l-2 ~1) of whole cells of p. &giy& was applied to Formvar and Carbon-coated nickel grids. Excess fluid was drained using filter paper and the sample was air dried. For indirect immunogold labeling of the 43 kDa, the bacterial sample on the grid was incubated within a drop of purified IgG fraction of rabbit antibody to the 43 kDa (100 - 150 l@nl) in PBS for 2 hrs. After washing with PBS for 30 min, the sample was further incubated for 1 hr within a drop of goat anti-rabbit IgG conjugated with 5-nm or IO-nm gold particles (1:20). The sample was then washed for 30 min in the same buffer, fixed in 2% glutaraldehyde in PBS for 30 min to ensure the binding of the antibodies to the bacteria, washed in distilled water, and air dried. Visualization of the whole complex was achieved by negative staining with 1% aqueous uranyl acetate. Any non-specific labeling was assessed by evaluation of controls which consisted of incubation of the samples in the absence of the purified rabbit antibodies, the conjugate alone, or in the presence of normal rabbit IgG plus conjugate as a replacement for the purified antibodies to the 43 kDa. RESULTS
AND DISCUSSION
Fimbriae were sheared from E gineivalis strain 2561 by mild sonication, followed by ammonium sulfate precipitation at 40% concentration. SDS-PAGE of the pooled preparation under denaturing conditions (100°C for 10 min. in the presence of 5% B-mercaptoethanol)
showed two major bands with
an apparent molecular size of 75 kDa and 43 kDa and also some additional minor bands. Under partially denaturing conditions (80°C for 5 min in the absence of l3-mercaptoethanol) fimbriae appeared as a series of bands with a strong liklihood of representing polymeric forms. The 75 and 43 kDa components in the ammonium sulfate precipitated sonicate could not be adequately dissociated under conventional protein purification
procedures
which
included:
chromatography
on columns
of Sepharose
CL-6B,
DEAE-Sepharose CL-6B and FPLC on a mono-Q anion-exchange column. Furthermore, the 75 kDa and 43 kDa components could not be separated by FPLC on Suprose- column in the presence of 6 M urea or other detergents including CHAPS, digitonin, polyethylene Tween-20, However,
Tween-80,
sodium deoxycholate,
glycol, octylglucopyranoside,
sodium oxycholate,
sucrosine,
Triton X-114 and Triton X-100.
partial dissociation of these two components could be achieved with a saturated solution of
guanidine hydrochloride. The inability to completely dissociate these components prompted further study of the chemical nature of the 43 kDa and 75 kDa components.
Both components were found to stain with Coomassie
blue, Sudan black and fat red 7B, but not with PAS.
This staining pattern suggested that these
components may be either proteolipid in nature or associated with lipids. This was further confirmed when both components dissociated with the organic solvents such as phenol and dimethyl formamide. (Data not shown). Based on the lipid-like nature of these proteins we have developed a method for their differential precipitation.The
fimbrial protein (43 kDa component) was preferentially precipitated in the
presence of 1% SDS and 0.2 M MgC12 at pIi 6.5 and homogeneous preparations regularly obtained by repetitive precipitation steps. Similar preferential precipitation procedures in the presence of divalent metal ions have traditionally been used for precipitation of lipoproteins (20).
Homogeneity
of the purified
kDa on SDS-polyacrylamide
protein
: The purified proteins showed single bands of 43
gel electrophoresis
under reducing conditions when stained with either
Coomassie or with the more sensitive silver staining method (Fig.l).The
preparations were free from
cross-contamination with LPS since silver staining did not show any additional bands corresponding to LPS. Staining with “Stain all” failed to show phosphoprotein contaminants. Preparations of protein was also radiolabelled
with [1251] and electrophoresed
on SDS-PAGE.
71.5
OnIy a single radioactive band
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Figure 1. SDS-PAGEand Westernblot analysisof crudefimbriae and purified 43 kDa protein. Sampleswere electrophoresed on 7.5% reducedSDS-polyacrylamidegelsand stainedwith Coomassie blueR-250. Lane1containsthefollowingmolecularweightmarkers:Phosphorylase b, 95 kDa; Glutamatedehydrogenase, 55 kDa; Gvalbumin,43 kDa; Lactatedehydogenase, 36 kDa andCarbonicanhydrase.29 kDa. Lane2, crudefimbriaein nativeform without heating. Lane3, crudefmbriae heatedat 80°Cfor 10minutes.Lane4, crudefimbriaeheatedat 100°Cfor 10minutes. Lane5, purified43kDaproteinin nativeform withoutheating.Lane6, purified43 kDa protein heatedat 80°C for 10minutes. Lane7, purified 43 kDa heatedat 100°Cfor 10 minutes.Lane8, immunoblotof puritied43 kDa usingthe43 kDa polyclonalantibody. Lane9, immunoblotof crudefimbriaeusing43 kDapolyclonalantibody.
corresponding to 43 kDa molecular mass, was observed for the purified protein preparations. The purified 43 kDa protein showed a ladder like pattern at 80°C and could be dissociated into it’s monomeric form only after heating at KKPC for 10 min. The apparent molecular weight of the oligomeric forms of 43 kDa protein were calculated to be 60,73,80,107,125,140 and higher polymers. Although each band appears to be a multiple of 43 kDa monomer, this aberrant migration pattern observed cannot be explained at this time. Homogeneity of the purified proteins was further confiied by Western blot analysis 0;ig.l). Western blot analysis using polyclonal antibodies specific for the purified 43 kDa protein reacted with both polyclonal and monoclonal antibodies raised against 43 kDa protein and did not react with monoclonal or polyclonal antibodies raised against the 75 kDa protein. Western blots of the purified 43 kDa also showed single bands when tested with antisera developed to whole cells of mgingivalis. Since E. gingivt-& possess strong haemagglutinating activity, we investigated the involvement of purified proteins in the agglutination of sheep erythrocytes. Using purified 43 kDa, we were not able to show any hemagglutinating activity. These results indicate that 43 kDa protein is not able to agglutinate sheep erythrocytes in their purified forms. Chemical Composition and Amino-terminal sequence analysis: The amino acid analysis of the purified 43 kDa protein is given in Table I. The amino acid analysis shown is an average of duplicate hydrolysate samples at 24 h. The amino acid composition of the 43 kDa protein is identical to the deduced composition obtained from the cloned fimbrilin gene (Table I). Hexosamines or neutral sugars were not detected in either proteins. The sequence of the first thirty residues of the purified 43 kDa protein waselucidatedby automatedEdtnandegradationand wasconsistentwith the elucidatedpredicted sequenceof cloned fimbrilin gene from a related strain of p. &@yt& (21).
Electron Microscopy Whole Cells of1.w:
and Immunogold Localization of the 43kDa Protein on The purified 43 kDa protein preparation showed a fimbrial structure 716
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I
Amino acidcomnosition of P.ainaivalis2561Fimbrillinurotein Aminoacids
ClonedFimbrillinb
PurifiedProteina %Residues Res./Molecule
%Residues
Res./Molecule
Asx Thr Ser Glx
13.7 8.3 4.4 11.0
54.8 33.2 17.6 44.0
13.1 8.6 3.6 9.5
44 29 12 32
GUY Ala Val Met lie
10.0 3.4 12.7 6.0 1.5
7.7 3.9 12.8 7.4 ::t
:z 43 25 6
7.1 5.6
:: 19
26:: 1.5 A:;
23 5 5 3
LeU
t
Tyr
3:8
40.0 13.6 50.8 24.0 6.0 16.4 29.2 15.2
Phe LYS His ‘4% CA
2.3 7.2 1.6 1.8 0.9
2::: 6.4 7.2 3.6
pro
:
a The purified protein was hydrolysedwith 6N HCI at 1lo’C for 22 hrs. Analytical valuesareexpressed asresidues/molecule basedon molecularweight43,000daltons. b Deducedfrom the genecloningsequence of Dickinsonet al. (21). Thesevaluesare basedon the predictedsizeof sequenced protein36,CKKl daltons.
with a diameter of approximately 3-5 nm when observedunder the electron microscope. Their fimbrial nature was further confirmed by specific immunogold labelling with 43 lcDa fimbrial protein specific antibodies (Fig. 2a). The samplesshowed no significant labeling with either normal rabbit IgG or in controls using antibodies againstthe 75 kDa protein (Data not shown). The proceduredescribedfor the purification of fimbriae usesmild precipitation conditions. These mild conditions do not irreversibly dissociatethe fimbrial structure, sincethe isolatedfimbriae appearto be intact asdemonstratedby electron microscopic studies (Fig. 2a). Earlier procedures described in the literature required either organic solvents(22) or higher temperatures(23). The preciselocalization of both the 43 lcDaproteinson the cells of P. pingivalis wassuccessfully achieved using indirect immunogold labeling and negative staining with 1% aqueousuranyl acetate. Ultrastructural localization of the 43 kDa protein by the immunogoldtechniquerevealedthat gold particles specifically labelled fimbriae with a diameterof approximately 3 to 5 nm, especially thoseradiating from the outer cell membrane(Fig.2b). No labeling was observed when the primary antibodies were either omitted or replacedwith non-immuneimmunoglobulinsascontrols. In this study, ultrastructural study of the purified 43 lcDaprotein allowed the characterization and morphology of this protein in aqueoussolutions. In addition, immunogold labeling using monospecific antibodies to this protein provided very valuable and conclusive information on it’s localization on p. pincivalis. Ultrastructural analysis of the purified 43 kDa protein preparation after negative staining clearly demonstratedthat this protein forms elongated filamentous structures 3 to 5 nm in diameter, similar to the typical fimbriae extending from the surfaceof p. gintzivalis cells. Furthermore, specific 717
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Figure 2a. Inununogold labelling of the purified 43 kDa protein. Note typical fimbriae (arrowheads) and their specific labelling with gold particles. Figure 2b. Irnrnunogold localization of the 43 kDa protein on a whole cell of E. g&&& 2561 strain. Note a heavy labelling of fimbriae (arrowheads) with gold particles, and lack of labelling in control (Inset). immunogold labeling of both, the purified 43 kDa protein and the native fimbriae of p. gingivalis, with antibodies to the 43 lcDa protein strongly suggests that the 43 kDa protein represents a major structural protein of fimbriae. Circular Dichroism
studies:
The stability and oligomeric
nature of the purified
43 kDa
proteins prompted further investigation into the secondary structure of these components. The CD spectra between 200 to 260 nm. shows no negative peak at 208 or 222 nm, a region characteristic of alpha helical structure. The spectrum remained unchanged after boiling the samples in the presence of SDS at 100°C. The 43 lcDa protein in native form, as well as after boiling at 100°C showed no detectable alpha helical, 67% beta sheet and 37% remainder structure. Circular dichroism spectra indicated the predominant secondary structure in the 43 kDa components was a beta sheet structure. This is consistent with the predicted secondary structure of the fimbrial subunit protein sequence (24). The CD spectra of this component is similar to most other outer membrane proteins with a high content of beta sheet structure. The high percentage of &sheet and random coil probably contribute to the unusual stability of this component. Our preliminary salivary
components
pathogenicity
binding studies indicate that purified 43 l&a protein binds specifically to several and to buccal epithelial cells.
the further extensive investigation
To understand
the exact role of fimbriae
about fimbriae structure,
in
specific salivary binding
components as well as structure of epithelial cell receptor is essential. ACKNOWLEDGMENTS:
This study was support in part by U.S. Public Health Sciences grants
DE08240, DE07034, DE04898, DE06514.
The authors thank Ms. Darlene Badgett for her excellent
technical assistance and Ms. Karen Flowers for her help in typing the manuscript. REFERENCES 1. 2.
Beachy, E.H. (1981) J. Infect. Dis. m, 325345. Gibbons, R.J. and van Houte, J. (1980) Bacterial adherence: In (E.H. Beachey ed.) Series B, Vol. 6, pp 61-104 Chapman and Hall, London and New York. 718
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Gibbons, R.J. (1984) J. Dent. Res. a, 378-385. Jones, G.W. (1977) In Microbial interactions: (J. L. Reissig ed.), Series B Vol. 3 pp 139-176 Chapman and Hall, London. Ofek, I., and Beachey, E.H. (1980) In “Bacterial adherence” (E.H. Beachey ed.), Series B Vol. 6 pp l-29, Chapman and Hall, London and New York. Okuda, K., and Takazoe, I. (1974) Arch. Oral Biol. 1p, 415416. Okuda, K., Slots, J. and Genco, R.J. (1981) Cum Microbial. 6, 7-12. Slots, J. and Gibbons, R.J. (1978) Infect. Immun. le, 254-264. Woo, D. D. L., Holt, S.C. and Leadbetter, E.R. (1979) J. Infect. Dis. 139, 534-546. Yoshimura, F., Takahashi, K., Nodasaka, Y, and Suzuki, T. (1984) J. Bacterial. m, 949-957. Yoshimura, F., Takahashi, K., Yoneyama, M., Yamaguchi, T., Shiokawa, H. and Suzuki, T. (1985)J.Bacteriol.l,730-734. Towbin, H., Staehlin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. U.S.A. %, 4350-4354. Bedi, G.S., Back, N. (1987) Hybridoma. 4, 521-526. Lee, J.Y., Sojar, H.T., Bedi, G.S. andGenco, R.J. (1991) Infect. Immun. Z, 383-389. Bradford, M.M. (1976) Anal. Biochem. 2, 248-254. Bedi, G.S., French, W.C. and Bahl, O.P. (1982) J. Biol. them 257, 4345-4355. Laemmli, U. K. (1970) Nature 227, 680-685. Slots, J. and Genco, R.J. (1979) J. of Clinical. Microb. 14(3), 371-373. Provencher, S.W. and Glockner, J. (1981) Biochem. 20, 33-37. Burstein, M. and Scholnick, H.R. (1973) Adv. Lipid. Research. 11, 68-108. Dickinson, D.P., Kubiniec, M.K., Yoshimura, F. and Genco, R.J. (1988) J. Bacterial. 170, 1658-1665. Yoshimura, F., Wantanabe, K.I., Takasawa, T., Kawanami, M. and Kato, H. (1989) Infect. Immun. s( 1 I), 3646-3652. Hoschutzky, H.F., Lottspeich, and Jann, K. (1989) Infect. Immun. Z, 76-81. Genco, R.J., Lee, J.Y., Sojar, H.T., Bedi, G.S., Loos, B.G. and Dyer, D.W. (1991) In “Periodontal Disease: Pathogen and host immune responses” (Hamada, S., Holt, SC. and McGhee, J.R. eds.). Quintessence Publishing Co., Ltd. Japan (In Press).
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