Vol. 153, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1983, p. 955-9610021-9193/83/020955-07$02.00/0Copyright © 1983, American Society for Microbiology

Localization of the Major Antigenic Determinant of EDP208Pili at the N-Terminus of the Pilus Protein

ELIZABETH A. WOROBEC,' ASHOK K. TANEJA,2 ROBERT S. HODGES,2 AND WILLIAMPARANCHYCH'*

Department ofBiochemistry' and the Medical Research Council Group in Protein Structure and Function,2University ofAlberta, Edmonton, Alberta, Canada T6G 2H7

Received 10 May 1982/Accepted 26 November 1982

Trypsin digestion of pilin monomers from EDP208 conjugative pili causes

cleavage at Lys12 to yield an N-terminal dodecapeptide, ET1 (Mr- 1,500), andthe remaining C-terminal fragment, ER (Mr 10,000). Using the amino acidsequence for ET1 provided by Frost et al. (J. Bacteriol. 153:950-954), we synthe-sized the N-terminal dodecapeptide chemically, conjugated it to bovine serum

albumin, and subjected it to immunological studies. Antisera prepared againstintact EDP208 pili as well as against the synthetic ET1-BSA conjugate were usedin experiments involving an enzyme-linked immunosorbant assay and electropho-retic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels tonitrocellulose sheets. Both experimental approaches showed strong reactivitybetween the synthetic dodecapeptide and antiserum raised against whole pili. Itwas also found that antiserum raised against the synthetic peptide was reactiveagainst intact pilus protein, indicating that the N-terminal dodecapeptide is animportant antigenic determinant of the EDP208 pilus protein. Additional studiesshowed that the C-terminal fragment, ER, may contain one or two additionalantigenic sites.

EDP208 conjugative pili are encoded by aderepressed derivative of a naturally occurringlac plasmid, Folac, originally isolated fromSalmonella typhi (7). Armstrong et al. (1) haveshown that EDP208 pili contain a single poly-peptide subunit of 11,500 daltons with a blockedN-terminus. More recently, Frost et al. (9) iden-tified the blocking group on EDP208 pilin as anN-acetyl moiety and determined the amino acidsequence of 12 residues at the N-terminus of theprotein. This dodecapeptide was the primaryproduct released when EDP208 pilin was digest-ed with trypsin. Although the remaining 10,000-dalton C-terminal fragment contained additionaltryptic cleavage sites, they were inaccessible tothe enzyme since no further cleavage was ob-served.

In the present communication, we describethe chemical synthesis of the EDP208 N-termi-nal dodecapeptide and show that (i) the synthet-ic peptide reacts strongly with antiserum raisedagainst whole pili and that (ii) antibodies raisedagainst the synthetic peptide conjugated to bo-vine serum albumin react positively with nativepili. Additional studies suggested that the10,000-dalton C-terminal fragment may containone or two additional antigenic sites. Thesefindings were taken as confirmation of the accu-racy of the EDP208 N-terminal sequence report-ed by Frost et al. (9) and as an indication that

this region of the pilus protein represents onemajor antigenic determinant of the pilus.

MATERIALS AND METHODSAbbreviations. Abbreviations used are: Boc, Ne'-

tert-butyloxycarbonyl; DCC, N,N'-dicyclohexylcar-bodiimide; BOC-ON, 2-tert-butyloxycarbonyloxyi-mino-2-phenylacetonitrile; DIEA, diisopropyl-ethylamine; BSA, bovine serum albumin; TLC, thin-layer chromatography; AB-ONSu, N-hydroxysuccinimide ester of 4-azidobenzoic acid;SDS, sodium dodecyl sulfate; ELISA, enzyme-linkedimmunosorbent assay; TPCK, tolylsulfonyl phenyla-lanyl chloromethyl ketone.

Bacteria. The EDP208 plasmid was carried in thehost strain JC6256 (Escherichia coli K-12 F- trp lac).EDP208 is the derepressed form of Fo lac (7) and waskindly donated by N. S. Willetts, Department of Mo-lecular Biology, University of Edinburgh, Edinburgh,Scotland.

Chemicals. Unless otherwise stated, all chemicalsand solvents are reagent grade. Boc-amino acids werefrom the following: Protein Research Foundation,Japan; Chemical Dynamics Corporation, SouthPlainfield, N.J.; Bachem Fine Chemicals Inc., MarinaDel Rey, Calif.; Spinco Division of Beckman Instru-ments Inc., Palo Alto, Calif.; and Vega Laboratories,Tucson, Ariz. DCC was from Pierce Chemical Co.,Rockford, Ill. [1-_4C]Gly (0.25 mCi) was from NewEngland Nuclear Corp., Boston, Mass. BOC-ON andDIEA were from Aldrich Chemical Co., Milwaukee,Wis. BSA and 4-aminobenzoic acid were from SigmaChemical Co., St. Louis, Mo. Sephadex G-25 and G-

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50 (superfine particle size, 10 to 40 ,um) were fromPharmacia Fine Chemicals, Uppsala, Sweden. Anionexchange resin (AG 1 x 4, chloride form, 100 to 200mesh) was from Bio-Rad Laboratories, Richmond,Calif. Anisole was from Fisher Scientific Co., Fair-lawn, N.J. Hydrofluoric acid was from Matheson,Whitby, Ontario, Canada. Co-poly(styrene, 1% di-vinyl-benzene)-benzhydrylamine-hydrochloride resin(200 to 400 mesh; 0.56 meq of nitrogen per g) was fromBeckman Instruments, Inc., Palo Alto, Calif. Trifluor-oacetic acid was from Halocarbon Products, Hacken-sack, N.J.Dichloromethane was distilled over CaCO3 before

use. DIEA was distilled first over NaH and then overninhydrin. Pyridine was distilled over ninhydrin. Pic-ric acid was dissolved in dichloromethane and driedover MgSO4 before use.TLC. TLC was performed on precoated silica gel 60

F-254 plates (0.25-mm thick; E. Merck AG, Darm-stadt, Federal Republic of Germany). The followingsolvent systems were used: chloroform-methanol-ace-tic acid (85:10:5, vol/vol/vol; system A); and 1-buta-nol-ammonium hydroxide-water (80:10:10, vol/vol/vol; system B). Spots were visualized under UV light(254 nm) or by treatment with ninhydrin spray (1%[wt/vol] in acetone) after removal of the Boc-group byexposure of the dry TLC plates to HCI fumes in asealed tank for 10 min. The compounds were declaredhomogeneous by TLC when no impurities could bedetected in the solvent system described after applica-tion of 200 ,g to the plates.

Boc[1-'4C]Gly. Radioactive Boc-Gly was preparedby the method of Itoh et al. (15). The solution ofradioactive [1-_4C]Gly (0.25 mCi; 1.45 mg; 19 ,umol) in0.1 N HCI was evaporated to dryness and mixed withnon-radioactive Gly (66 mg; 880 ,umol). Triethylamine(245 ,ul; 1.77 mmol), water (600 1,l), and BOC-ON (280mg; 1.15 mmol) dissolved in dioxane (650 IlI) wereadded and stirred at room temperature for 6 h. Thecompletion of the reaction was indicated by the ab-sence of Gly on TLC in solvent systems A and B. Thesolvents were removed under reduced pressure andthe residue taken up in water (20 ml). The aqueouslayer, after extraction with CH2C12 (2 extractions,each time with 10 ml), was acidified with citric acid topH 3.0 and exhaustively extracted with CH2CI2 (6extractions, each time with 50 ml), which upon dryingover anhydrous sodium sulfate, filtration, and evapo-ration of the solvent gave the desired product with a98% yield based on radioactivity measurements. Thespecific activity of Boc-[1-14C]Gly was 500 cpm/nmol.AB-ONSu. AB-ONSu was prepared from 4-amino-

benzoic acid as previously described (4).Amino acid analyses. Routine amino acid analyses

were performed on a Durrum D-500 amino acid ana-lyzer. Quantities of peptides were determined fromamino acid analysis after hydrolysis in 6 N HCIcontaining 0.1% phenol in evacuated sealed tubes for24 h at 110°C, using the mean of the molar ratios of allaccurately measurable amino acids in the acid hydro-lysate to calculate the concentration. Peptide resinswere hydrolyzed similarly in a mixture of 2 ml of 12 NHCI, 1 ml of acetic acid, and 1 ml of phenol (10).

High-voltage paper electrophoresis. High-voltage pa-per electrophoresis was performed at pH 6.5 (pyridine-acetic acid-water, 100:3:900, vol/vol/vol) and pH 1.8(formic acid-acetic acid-water, 1:4:45, vol/vol/vol).

Electrophoresis was carried out at 60 V/cm for 45 minon Whatman paper no. 1.

Preparation of whole pili and pilin monomers. Thepurification of EDP208 pili was as described previous-ly by Armstrong et al. (1). Dissociation of intact piliinto pilin and gel filtration through SDS-Sephadex G-200 columns to remove tightly associated contami-nants such as lipopolysaccharide were as describedpreviously by Armstrong et al. (2). The resulting SDS-treated pilin was subjected to acetone precipitation (2)to remove detergent and phospholipids.

Preparation of N-terminal dodecapeptide (ET1) and10,000-dalton C-terminal fragment (ER) from EDP208pilin. Purified EDP208 pilin was digested with trypsin(TPCK) to yield ET1 and ER as described by Frost etal. (9). We are grateful to L. S. Frost for providing uswith the preparations of ET1 and ER used in thesestudies.Chemical synthesis of ETI tNa-acetyl-pilin(1-12)am-

ide]. The 12-residue peptide Na-acetyl-pilin(1-12)am-ide was synthesized by the general procedures forsolid-phase peptide synthesis on a Beckman peptidesynthesizer (model 990) (5). Coupling of a Boc-Lys(2-chlorobenzyloxycarbonyl) (0.35 mmol/g) to the benz-hydrylamine resin resulted in a substitution of 0.28mmol of amino acid per g as determined by picratemonitoring (11). The remaining free amino groups onthe resin were terminated by washing with an acetylat-ing mixture (pyridine-acetic anhydride-benzene, 3:1:1by volume, 12.5 ml per g of resin) for 5 min, followedby acetylation with the same mixture for 60 min. Thepicrate monitoring established complete acetylation ofall the available amino groups.

All amino acids were protected at the a-aminoposition with Boc groups, and the following side chain-protecting groups were used: for Lys, 2-chlorobenzyl-oxycarbonyl, for Asp, o-benzyl (OBzl), and for Thr,benzyl (Bzl). The Boc groups were removed at eachcycle of the synthesis by treatment for 30 min with 50ml of 50% trifluoroacetic acid-CH2Cl2 (vol/vol). Aftereach deprotection step, neutralization action was car-ried out with 50 ml of 5% DIEA-CH2CI2 (vol/vol).Boc-amino acids (0.84 mmol; 3 equivalents) in 8 ml ofCH2Cl2 were added to the peptide-resin followed by a5 ml solution of DCC (0.84 mmol) in CH2Cl2. Doublecouplings of 90 min each were performed at each stepof the synthesis. The program used for attachment ofeach amino acid and the picrate monitoring procedurewere as previously described (13).The radioactive peptide was prepared by incorporat-

ing [1-14C]Gly into the peptide at position 7. Afterdeprotection and neutralization of protected peptideresin (Boc-Lys[2C1,Z]-Asp[OBzl]-Val-Asp[OBzl]-Lys[2Cl,Z] resin), Boc-l1-'4C]Gly (0.31 mmol; 1.1equivalents) and DCC (0.31 mmol) were added andmixed for 90 min. The second coupling was carried outwith non-radioactive Boc-Gly as described above.The cleavage of the peptide from its resin support

was accomplished with hydrofluoric acid at 0°C for 45min with 10o anisole (vol/vol) as a cation scavenger(12). The hydrofluoric acid and most of the anisolewere removed under reduced pressure at 0°C, fol-lowed by removal of the residual anisole at roomtemperature overnight. The resin was washed withether, and the peptide was extracted with trifluoroace-tic acid (4 extractions, each with 5 ml). The trifluoroa-cetic acid was removed by evaporation, and the resi-

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due dissolved in 1% ammonium bicarbonate andlyophilized. The peptide was obtained with an 80%yield, as determined by amino acid analysis and radio-activity measurements.

Purification of synthetic peptide. The crude, cleavedpeptide (165 mg; 130 ,umol) was dissolved in 3.0 ml ofpyridine acetate buffer (0.1 M in pyridine), pH 5.0, andthen applied to an AG 1 x 4 column (1.8 by 50 cm) andeluted with the same buffer at a flow rate of 60 ml/h at55°C. The column effluent was stream divided with anAuto-Analyzer (Techicon Corp., Inc., Tarrytown,N.Y.), and a rate of 10 ml/h was used for the ninhydrinanalytical system. Fractions of 2.5 ml were collected.Each fraction in the major peak was subjected to high-voltage paper electrophoresis at pH 6.5, and thehomogeneous fractions were pooled and lyophilized(yield, 65 mg [40%]). The peptide was also homoge-neous on paper electrophoresis at pH 1.8. Amino acidanalysis gave the following results: Asp (2.97), Thr(0.79), Gly (2.08), Ala (1.03), Val (1.08), Leu (0.99),Lys (2.08).

N'~-Acetyl-[bis-N-(4-azidobenzo 1-s521 iinl12)amide. To a solution of the 1 C-labeled N"-acetylpilin(1-12)amide (1.27 mg; 1 ,umol) in aqueousNaHCO3 (0.4 mg of NaHCO3 in 250 ,dl of water) at0OC, we added dropwise AB-ONSu (2.6 mg; 10 ,umol)dissolved in 250 ,ul of dioxane over a period of 10 minwith constant stirring. The reaction was allowed toproceed for 1 h at 0°C and then at room temperaturefor 24 h. The mixture was applied to a Sephadex G-25column (1.6 by 100 cm) equilibrated with 0.1 MNH4HCO3 and eluted at a flow rate of 10 ml/h.Fractions of 3.3 ml were collected. The column efflu-ent was monitored at 270 nm for detection of AB-ONSu and its reaction products and for radioactivity(Fig. 1). The radioactive peptide fractions were pooledand combined with BSA (6.8 mg; 0.1 ,umol) andlyophilized. The reaction of the Lys residues of thepeptide with AB-ONSu was shown to go to completionby the absence of ninhydrin-positive spots upon elec-trophoresis at pH 6.5.

Preparation of covalently linked peptide-BSA com-plex. The freeze-dried photoaffinity-labeled peptide-BSA mixture was dissolved in 100 ,ul of a 0.1 M

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FIG. 1. Purification of N-acetyl-[bis-N-(4-azido-benzoyl)-Lys8"2] pilin(1-12)amide by Sephadex G-25column chromatography. See text for further details.

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Sephadex G-50 column chromatography. See text fordetails.

phosphate buffer, pH 6.8, which had been previouslydegassed and saturated with nitrogen. This solutionwas photolyzed for 1 h in the cold room (4°C), using anRPR 208 preparative reactor (Rayonet; The SouthernNew England Ultraviolet Co., Middletown, Conn.)equipped with 3,500-A lamps. The reaction mixturewas applied to a Sephadex G-50 column (1.6 by 100cm) equilibrated with 1 mM HCI and eluted at a flowrate of 10 ml/h. Fractions of 3.3 ml were collected. Thecolumn effluent was monitored at 230 nm and forradioactivity (Fig. 2). Peak 1, containing the covalentpeptide-BSA complex, was pooled and lyophilized.Radioactivity measurements indicated a peptide/BSAratio of 2.8:1 in the covalent complex. A ratio ofpeptide to BSA as high as 72:1 could be obtained bycombining peptide (25 mg; 20 p.mol) with BSA (6.8 mg;0.1 p.mol) in 600 ,ul of buffer and photolyzing for 90 minas described above. Under these conditions, no freepeptide was observed, as seen in Fig. 2, peak 2.

Antisera preparation. To obtain antipilus antiserum,New Zealand White rabbits were injected subscapular-ly and intramuscularly in the gluteal area with a total of100 p.g of purified EDP208 pili dissolved in equalvolumes of sterile saline and Freund complete adju-vant. The rabbits were given booster injections 4 and 8weeks later with 100 jig of purified EDP208 pilidissolved in sterile saline and Freund incomplete adju-vant. The animals were bled 2 weeks after the finalinjection. The antiserum against ET1-BSA was pre-pared by initially injecting rabbits subcutaneously atseveral sites with a total of 600 jig of ET1-BSA (72:1)dissolved in sterile saline mixed with an equal volumeof Freund complete adjuvant. At 7 and 14 days, therabbits were given booster injections with 600 jig ofET1-BSA dissolved in sterile saline mixed withFreund incomplete adjuvant. The animals were bledapproximately 2 weeks after the last injection.

Detection of antigenic species with ELISA. The basicprinciples of the ELISA were as described previouslyby Voller et al. (21). The appropriate antigens weredissolved in 0.5 M bicarbonate coating buffer (pH 9.6)

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at a concentration of 4 ,ug/ml, and 0.2-mi aliquots wereapplied to wells in microtiter plates (Dynatech Labora-tories, Inc., Alexandria, Va.). After incubation for 16h at 4°C, the antigen-saturated wells were washed andthen allowed to react with appropriate dilutions ofantipilus or anti-ET1-BSA antiserum. The resultingantigen-antibody complexes were quantitated colori-metrically with goat anti-rabbit immunoglobulin Gconjugated to alkaline phosphatase (1:2,000) pur-chased from Boehringer Mannheim Corp. (New York,N.Y.). The alkaline phosphatase substrate used was p-nitrophenyl phosphate (Sigma 104 phosphatase sub-strate tablets). Absorbance at 405 nm was determinedwith a Titertek multiscan ELISA plate reader.

Transfer of protein from SDS-polyacrylamide gels tonitrocellulose paper. SDS-polyacrylamide gel electro-phoresis was carried out as described by Armstrong etal. (2), except that the thickness of the gels was 0.75mm. Samples were boiled for 10 to 20 s in SDS beforeapplication to the polyacrylamide gel. After electro-phoresis of the proteins in the polyacrylamide gel, theywere transferred to nitrocellulose paper with an Elec-troblot apparatus (E-C apparatus) according to theprocedure described by Towbin et al. (20). The trans-fers were carried out for 3 h. Duplicate samples werealso stained directly with Coomassie blue, using themethod of Fairbanks et al. (6).Immunological detection of proteins on nitrocellulose.

Immunological detection of proteins on nitrocellulosewas exactly as described previously by Towbin et al.(20), except that '25I-labeled protein A was used ratherthan 125I-labeled sheep immunoglobulin G.

RESULTSPreliminary studies. As shown by Frost et al.

(9), treatment of EDP208 pilin with trypsincauses cleavage at Lys12 to yield an N-terminaldodecapeptide, ET1 (Mr 1,500), and the re-maining C-terminal fragment, ER (Mr -10,000).Partial cleavage also occurs at Lys8 to yield thesmaller fragments ET2 (residues 1 to 8) and ET3(residues 9 to 12). To determine whether any ofthese cleavage products were antigenic towardsanti-EDP208 pilus antiserum, all four peptideswere purified according to the method of Frostet al. (9) and subjected to an ELISA as describedabove. A moderately positive result was ob-tained in the microtiter wells coated with ET1and ER, whereas ET2 and ET3 failed to showany significant reaction with the antipilus anti-serum. On this basis, it was concluded that theN-terminal region of EDP208 pilin may containone of the antigenic determinants of the pilus.Chemical synthesis of ET1 and conjugation of

synthetic ET1 to BSA. Since relatively largeamounts of peptide were required for additionalcharacterization of the antigenic properties ofET1, and since it was difficult to obtain thesequantities using native pili, we turned to solid-phase peptide synthesis as a means of preparinglarge quantities of ET1. Both free peptide andET1-BSA conjugate were prepared as describedin detail above. The final product had the se-

quence N-acetyl-Thr-Asp-Leu-Leu-Ala-Gly-Gly-Lys-Asp-Val-Asp-Lys-amide.

It is to be noted that the N-terminus of thesynthetic peptide was acetylated whereas the C-terminus was an amide group generated directlyupon cleavage of the peptide from the benzhy-drylamine resin. To facilitate detection duringchromatographic purifications, [1-14C]Gly wasintroduced into the peptide at position 7. Thepeptide was coupled to BSA through the e-amino groups of Lys8 and Lys12, using thecoupling reagent N-(4-azidobenzoyl)-succini-mide and photolyzing with 350 nm light. ET1-BSA preparations having ET1/BSA molar ratiosof 2.8:1 and 72:1 were used in these studies.Immunization of rabbits was carried out with the72:1 preparation, whereas both preparationswere used in studies on antigenicity.ELISA studies with antipilus and anti-ET1-

BSA antisera. Antisera obtained from rabbitsimmunized with either highly purified EDP208pili or synthetic ET1-BSA conjugate were pre-pared as described above. The antisera werethen used in an ELISA experiment to determinethe extent of reaction with intact pili, free ET1,ER, and ET1-BSA conjugate. The ELISA in-volved coating the surfaces of microtiter wellswith saturating amounts of antigen, incubatingwith the appropriate rabbit antiserum, and final-ly using goat anti-rabbit antibodies conjugated toalkaline phosphatase to quantitate the antigen-antibody complexes in each well. The resultsobtained are shown in Fig. 3.

It may be seen that the ET1-BSA conjugatesreacted very strongly with both antisera, indicat-ing that the N-terminal dodecapeptide is anantigenic determinant of EDP208 pilin. The lowvalues obtained with free ET1 peptide relative tothe ET1-BSA conjugate are probably due tosteric effects caused by the direct binding of thesmall peptide to the solid plastic surface. In thecase of the ET1-BSA conjugate, the peptide ispresumably more accessible to antibody mole-cules in the surrounding solution and therefore isable to bind to ET1 antibodies more efficiently.

Similar arguments can be used to explain thefact that the 72:1 ET1-BSA conjugate yieldedonly slightly higher values than the 2.8:1 prepa-ration. The more highly conjugated preparationpresumably contains extensive cross-linking be-tween peptides, resulting in the masking of allbut a small fraction of the ET1 on the BSA.

It is of interest that the antipilus antiserumreacted with intact pili and ER more stronglythan did the anti-ET1-BSA antiserum. This maybe due to the existence of a second antigenicdeterminant in the C-terminal portion of thepilus. Thus, antiserum raised against whole piliwould contain antibodies against this secondantigenic determinant, whereas the anti-ET1-

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FIG. 3. ELISA studies with anti-EDP208 pilus and anti-ET1-BSA antisera. The ELISA was performed bycoating microtiter wells with a solution containing 4 ,ug/ml of the indicated antigens, followed by incubation with(A) a 1/1,000 dilution of antipilus antiserum in phosphate-buffered saline-Tween 20 containing 1% (wt/vol) BSA(PBST-BSA) or (B) a 1/100 dilution of anti-ET1-BSA antiserum in PBST-BSA. Antigen-antibody complexationwas quantitated at 405 nm, using the goat anti-rabbit immunoglobulin G alkaline phosphatase enzyme reaction.The results represent the averages of eight experimental observations. Error bars represent the standard error ofthe mean.

BSA antiserum would not. The low level reac-

tion of anti-ET1-BSA antiserum against ERcould be due to a small amount of uncleavedpilin present as contaminant in the ER prepara-

tion.Immunological studies on EDP208 peptides

transferred to nitrocellulose from SDS-polyacryl-amide gels. A second approach used to evaluatethe antigenicity of ET1 and ER peptides in-volved the electrophoretic transfer of peptidesand conjugates to nitrocellulose paper fromSDS-polyacrylamide gels. The nitrocellulose pa-per was then treated with pilus-specific or ET1-specific antiserum followed by 15I-labeled pro-tein A from Staphylococcus aureus. Theresulting antigen-antibody-'25I-protein A com-plexes were visualized by autoradiography. Du-plicate SDS-polyacrylamide gels were alsostained with Coomassie blue to allow directvisualization of the proteins electrophoresedinto each gel.As shown in Fig. 4A, 5-,ug amounts of pili,

pilin, and ER were easily detected by Coomassieblue staining. Both pilin and ER usually migrat-ed primarily in the monomeric state, but a smallfraction of the material also migrated as a dimer.Only the 2.8:1 ET1-BSA preparation was used inthis experiment, since the 72:1 preparation was

unable to penetrate the polyacrylamide gel. Fig.4C shows that the electrophoretic transfer ofproteins from SDS-polyacrylamide gels to nitro-cellulose paper was highly efficient. It is worthnoting that the ET1-BSA conjugate had slightlydifferent mobilities than free BSA. The reason

for this is unclear but presumably reflects themodification of the BSA by the conjugationprocess.

Figures 4B and D show the autoradiogramsobtained from the corresponding nitrocelluloseelectroblot sheets treated with antipilus and anti-ET1-BSA antisera, respectively. It may be seenthat immunological detection of pilin bands wassignificantly more sensitive to Coomassie bluestaining and thus showed up small amounts ofpilin and ER dimer which were not detectable inFig. 4A. As expected on the basis of the ELISAresults in Fig. 3, the antipilus antiserum (Fig.4B) reacted approximately twice as stronglywith pilin as with ER. Reaction with the ET1-BSA conjugate (lane 1) was also strong, confirm-ing that antipilus antibodies react with the syn-thetic peptide. Figure 4D shows the expectedinteraction of anti-ET1-BSA antiserum with theET1-BSA conjugate (lane 1). In addition, theantibodies reacted strongly with intact pilin andweakly with ER. As mentioned earlier, the weakreaction of anti-ET1-BSA antiserum with ERmay be due to contamination of ER with incom-pletely cleaved pilus protein.

It is worth noting that both antisera reactedmore intensely with pilin than with the ET1-BSAconjugate in the immunoblot system. In theELISA system, on the other hand, the reactionof each antiserum with conjugate and pilin was

approximately equivalent. The reason for thesedifferences between the two techniques is notclear but may reflect different levels of accessi-bility of the ET1 peptide to antibodies in the twodifferent physical environments employed inthese assay systems. It is also to be noted thatthe anti-ET1-BSA antiserum did not react withfree BSA either in the ELISA (Fig. 3) or theimmunoblot (Fig. 4) experiments. This is notsurprising since the anti-BSA antibodies were

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FIG. 4. Electrophoretic transferpolyacrylamide gels to nitrocellulosmassie blue-stained pilus proteins arSDS-polyacrylamide gel electrophoiof ET1-BSA (2.8:1); lane 2, 5 ,ug of3, 5 ,ug of ER; lane 4, 5 ,ug of BSApanels). The apparent molecular medaltons) are indicated at the side (Autoradiograph of proteins transfeicate of gel A to nitrocellulose papeiantipilus antiserum (1/1,000) in antiser containing 1% (wt/vol) BSA and'A. (C) Coomassie blue-stained duplelectrophoretic transfer of protein,paper. (D) Autoradiograph of pr,from a duplicate of gel A to nitrocreacted with anti-ET1-BSA antiseruum dilution buffer containing 1%"251-labeled protein A.

complexed by large amountsserum dilution buffer.On the basis of the foregoing,

ed that the N-terminal dodecapthe major antigenic determinantpilus protein but that one or moimay exist in the C-terminal fraf

DISCUSSIONSome bacterial pili play a cruc

rial pathogenesis as adherence 4enable bacteria to colonize thspecific host organs. Other typxgative) play a critical role inbacterial conjugation, a processjugative plasmid is transferred fial cell to another. The bacteriencode the conjugative processa wide range of other phenol

antibiotic resistance and production of colicins,-68 enterotoxins, hemolysin, and surface antigens.

Although it is well known that intact pili arehighly immunogenic (17), few investigators haveattempted to identify the antigenically reactiveregions of the pilus protein. Klemm (16) hasnoted that sequence differences among K88 pili

,,0 variants are all located in the central part of theP W ° 264-residue protein. On the basis of specific2 3 4 amino acid differences between ab and ad/ade

variants of K88 pili, he suggested that the anti-genic determinant may reside in the region con-

-68 taining residues 147 to 153. On the other hand,Schoolnik et al. (19) have shown that the immu-nodominant region of gonococcal pili residesnear the C-terminal end of the 160-amino acidpilin monomer, whereas the present studyshows that one major antigenic site of EDP208conjugative pili is at the N-terminus of the

* -10 protein. Thus, there appears to be little similar-2 3 4 ity in the location of antigenic sites among

different types of pili, and it is likely that noof proteins from general pattern will emerge as additional pilus

se paper. (A) Coo- types are characterized.nd conjugates after Identification of antigenic determinants inresis. Lane 1, 5 tLg bacterial pili is important for at least two rea-r

(same lanes in all sons. (i) It allows the identification of a region orass positions (kilo- domain of the polypeptide that may be situatedof each panel. (B) at the surface of the intact pilus, and (ii) it offersrred from a dupli- the possibility of devising synthetic vaccines inr and reacted with cases in which the pilus is involved in bacterial,erum dilution buff- pathogenesis.251I-labeled protein In the case of EDP208 pili, preliminary struc-icate of gel A after tural studies with X-ray fiber diffraction method-

steoinstracnelleuloed ology have indicated that the arrangement ofellulose paper and subunits in EDP208 pili is almost identical tom(1/50) in antiser- that of F pili. Similar layer line spacing and(wt/vol) BSA and overall distribution of density in X-ray fiber

diffraction patterns obtained with both F (8) andEDP208 (W. Folkhard, K. R. Leonard, J. Dubo-chet, D. A. Marvin, and W. Paranchych, Int.

of BSA in the Congr. Biochem. Abstr., 11:181, 1979) pili sug-gest that the two structures are hollow cylinders

it was conclud- 8.0 nm wide with a central hole of about 2.0 nm.ieptide is one of The subunits are arranged in a helical array ofts of the EDP208 3.6 units per turn of 1.28-nm pitch. Moreover,re antigenic sites circular dichroism studies (1) have indicated thatgment ER. both polypeptides have a high a-helical content

(65 to 70%), suggesting that the surface of theintact pilus probably contains at least some a-

:ial role in bacte- helical regions of the protein.structures which Whether the N-terminal portion of theie epithelium of EDP208 pilus is an a-helix remains to be deter-es of pili (conju- mined. However, the ease with which this dode-the process of capeptide is cleaved from the protein with tryp-whereby a con- sin suggests a hinge region at Lys2 and arom one bacteri- possible looping out of the N-terminal dodeca-al plasmids that peptide from a tightly packed subunit which isoften determine highly resistant to denaturation by SDS (1). Thistypes, including tight packing of the pilin polypeptide chain may

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I08OPICLSIESOONEDP298 PILI 961

explain why the pili studied to date (EDP208,K88, Neisseria gonorrhoea) appear to have one,or perhaps two, antigenic sites, whereas otherproteins of a similar size such as sperm wholemyoglobin contain as many as five antigenicallyreactive regions (3).

In future studies it will be of interest todetermine which portions of the ET1 peptide aredirectly involved in the antigen-antibody reac-tion. X-ray diffraction studies (18) have revealedthat the size of a peptide that can be accommo-dated by the antibody-combining site is a pocketor crevice of 1.5 by 0.6 nm, with an approximatedepth of 0.6 nm. This crevice could accommo-date a peptide offive (,-pleated sheet conforma-tion) to ten (a-helix conformation) amino acids.Moreover, Hopp and Woods (14) have suggest-ed that antigenic determinants are usually locat-ed in, or immediately adjacent to, regions ofgreatest local hydrophilicity. On this basis, onewould predict that the C-terminal end (residues 5to 12) of ET1 should contain the antibody-combining site, since this region is much morehydrophilic than the N-terminal region (residues1 to 6). However, it is to be noted that couplingET1 to BSA through the e-amino groups of Lysand Lyst2 did not abolish the antigenic reactivityof the ET1 peptide, suggesting that these resi-dues may not be involved in the antigen reac-tion. It is thus necessary to consider the lesshydrophilic N-terminal portion of ET1 as thereactive site and to ask whether the N-acetylmoiety contributes to the antigenic reactivity.Studies currently in progress in this laboratorywith synthetic analogs of ET1 are designed toprovide an answer to these questions.

ACKNOWLEDGMENTSWe thank Kathy Volpel for excellent technical assistance,

Robert Bradley for photographic assistance, and Pat McDon-ald for typing the manuscript.

This work was supported by the Medical Research Councilof Canada. E.A.W. is the recipient of an Alberta HeritageFoundation for Medical Research Studentship.

LITERATURE CITED

1. Armstrong, G. D., L. S. Frost, P. A. Sastry, and W.Paranchych. 1980. Comparative biochemical studies on Fand EDP208 conjugative pili. J. Bacteriol. 141:333-341.

2. Armstrong, G. D., L. S. Frost, H. J. Vogel, and W.Paranchych. 1981. Nature of the carbohydrate and phos-phate associated with ColB2 and EDP208 pilin. J. Bacter-iol. 145:1167-1176.

3. Atass, M. Z. 1975. Antigenic structure of myoglobin: thecomplete immunochemical anatomy of a protein andconclusions relating to the antigenic structures of pro-teins. Immunochemistry 12:423-438.

4. Chong, P. C. S., and R. S. Hodges. 1981. A new heterobi-functional crosslinking reagent for the study of biologicalinteractions between proteins. I. Design, synthesis, andcharacterization. J. Biol. Chem. 256:5064-5074.

5. Erickson, B. W., and R. B. Merrlfield. 1976. Solid phasepeptide synthesis, p. 255-527. In H. Neurath and R. L.Hill, (ed.), The proteins, vol. 2. Publisher, location.

6. Fairbanks, G., T. L. Stick, and D. F. J. Wallach. 1971.Electrophoretic analysis of the major polypeptides of thehuman erythrocyte membrane. Biochemistry 10:2606-2617.

7. Falkow, S., and L. S. Baron. 1962. Episomic element in astrain of Salmonella typhosa. J. Bacteriol. 98:1598-1601.

8. Folkhard, W., K. R. Leonard, S. Malsey, D. A. Marvin, J.Dubochet, A. Angel, M. Acbtman, and R. Helmuth. 1979.X-ray diffraction and electron microscope studies on thestructure of bacterial F pili. J. Mol. Biol. 130:145-160.

9. Frost, L. S., G. D. Armstrong, B. B. Finlay, B. F. P.Edwards, and W. Paranchych. 1983. N-terminal aminoacid sequencing of EDP208 conjugative pili. J. Bacteriol.153:950-954.

10. Gutte, B., and R. B. Merrifield. 1971. The synthesis ofribonuclease A. J. Biol. chem. 246:1922-1941.

11. Hodges, R. S., and R. B. Merrifield. 1975. Monitoring ofsolid phase peptide synthesis by an automated spectro-photometric picrate method. Anal. Biochem. 65:241-272.

12. Hodges, R. S., and R. B. Merrifield. 1975. The role ofserine-123 in activity and specificity of ribonuclease.Reactivation of ribonuclease 1-118 by the syntheticCOOH-terminal tetradecapeptide, ribonuclease 111-124,and its 0-methylserine and alanine analogs. J. Biol.Chem. 250:1231-1241.

13. Hodges, R. S., A. K. Saund, P. C. S. Chong, S. A. St.-Pierre, and R. E. Reid. 1981. Synthetic model for two-stranded a-helical-coiled coils. Design, synthesis, andcharacterization of an 86-residue analog of tropomyosin.J. Biol. Chem. 256:1214-1224.

14. Hopp, T. P., and K. R. Woods. 1981. Prediction of proteinantigenic determinants from amino acid sequences. Proc.Natl. Acad. Sci. U.S.A. 78:3824-3828.

15. Itoh, M., D. Hagiwara, and T. Kamiya. 1975. A new tert-butyloxycarbonylating reagent, 2-tert-butyloxycarbonyl-oxy-imino-2-phenylacetonitrile. Tetrahedron Lett.49:4393-4394.

16. Klemm, P. 1981. The complete amino-acid sequence ofthe K88 antigen, a fimbrial protein from Escherichia coli.Eur. J. Biochem. 117:617-627.

17. Nagy, B., H. W. Moon, R. E. Isaacson, C.-C. To, andC. C. Brinton. 1978. Immunization of suckling pigsagainst enteric enterotoxigenic Escherichia coli infectionby vaccinating dams with purified pili. Infect. Immun.21:269-274.

18. PoUak, R. J., L. M. Amzel, B. L. Chen, Y. Y. Chiu, R. P.Phizackerley, F. Saul, and X. Ysern. 1976. Three-dimen-sional structure and diversity of immunoglobulins. ColdSpring Harbor Symp. Quant. Biol. 41:639-645.

19. Schoolnik, G. K., J. Y. Tai, and E. C. Gotschlich. 1982.Receptor binding and antigenic domains of gonococcalpili, p. 312-316. In D. Schlessinger (ed.), Microbiology-1982. American Society for Microbiology, Washington,D.C.

20. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electro-phoretic transfer of proteins from polyacrylamide gels tonitrocellulose sheets: procedure and some applications.Proc. Natl. Acad. Sci. U.S.A. 76:4350-4354.

21. VoUer, A., D. E. Bidweli, G. Huldt, and E. Engvall. 1974.A microplate method of ELISA and its application tomalaria. Bull. WHO 51:209-221.

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