Vol. 42, No. 1INFECTION AND IMMUNITY, OCt. 1983, p. 113-1210019-9567/83/100113-09$02.00/0Copyright X 1983, American Society for Microbiology

Mapping of the Antigenic Determinants of Pseudomonasaeruginosa PAK Polar Pili

TANIA H. WATTS,' PARIMI A. SASTRY,1 ROBERT S. HODGES,2 AND WILLIAM PARANCHYCHl*Department ofBiochemistry' and The Medical Research Council Group on Protein Structure and Function,2

University ofAlberta, Edmonton, Alberta, Canada TG6 2H7

Received 16 May 1983/Accepted 22 July 1983

The polar pili of Pseudomonas aeruginosa are flexible filaments 5.2 nm indiameter and 2.5 ,um in average length. They consist of a single subunit, pilin,which is a 144-residue polypeptide containing a hydrophobic N-terminal region(residues 1 to 30) and eight hydrophilic regions distributed throughout theremainder of the molecule. To delineate the antigenic regions of pilin, we cleavedthe protein at Arg30, Arg53, and Arg120 to produce peptides TCI (residues 1 to 30),TCII (31 to 53), TCIII (54 to 120), and TCIV (121 to 144). TCIII and TCIV werefurther cleaved into several subfragments. The purified peptides were coupled tobovine serum albumin by using the N-hydroxysuccinimide ester of 4-azidobenzoicacid and were then subjected to immunological analysis, using the enzyme-linkedimmunosorbent assay and immunoblot procedures with polyclonal antiserum.Four antigenic regions were identified; one in TCI was found to be common toboth PAK and PAO pilin. The remaining three were found to be specific to PAKpilin. Two of these were subfragments of TCIII, whereas the third was locatedclose to the C-terminus of the molecule, most likely between Cys129 and Cys142.Modification of these cysteines by reduction and carboxymethylation of thedisulfide linkage did not abolish the antigenicity of the C-terminal type-specificantigenic determinant.

Pseudomonas aeruginosa produces polar piliwhich are involved in adherence of the organismto host mucosal surfaces (30) and in a primitiveform of motility known as twitching motility (3).Both of these properties are thought to contrib-ute to the pathogenicity of piliated Pseudomo-nas strains.

Pili isolated from P. aeruginosa PAK (PAKpili) consist of a single type of protein subunit,pilin, of molecular weight 15,000 and consistingof 144 amino acid residues, according to itssequence (22). The protein subunit of pili isolat-ed from P. aeruginosa PAO (PAO pilin) iscomparable in size to PAK pilin according tosodium dodecyl sulfate (SDS)-polyacrylamidegel electrophoresis and sedimentation equilibri-um studies (28). Partial sequencing of PAO pilin(21; P. A. Sastry, J. R. Pearlstone, L. B. Smillie,and W. Paranchych, unpublished data) has shownthat the two pilins are highly homologous fromresidue 1 to 53, but share little homology at theC-terminus of the protein. The sequence of thefirst 22 residues at the N-terminus of PAK andPAO pilin is also strikingly homologous with theN-termini of pilins isolated from Neisseria gon-orrhoeae (16) and Moraxella nonliquefaciens(9), including the unusual amino acid N-methyl-phenylalanine at the N-terminus (11, 16). Thecommon N-terminal sequence is extremely hy-

drophobic, containing only one charged residue,and the region around Tyr24 and Tyr27 has beenshown to be buried at the region of intersub-unit contact in native pili (28a).

Polyclonal antibodies raised against Pseudo-monas pilin in rabbits have been shown to reactwith pilin subunits which have been transferredfrom polyacrylamide gels to nitrocellulose paper(29) in an immunoblot experiment (25). To deter-mine which regions of the pilin subunit wereresponsible for the antigenicity, we have pre-pared proteolytic fragments of PAK pilin andtested them for antigenicity in both an immuno-blot experiment and in an enzyme-linked immuno-sorbant assay (ELISA) (27). Since antigenicdeterminants are generally located on the sur-face of proteins (1, 2, 26), this approach is usefulin identifying surface-exposed regions of thepolypeptide chain. Furthermore, these studiesare potentially useful in identifying antigenicpeptides which could be tested for their ability togenerate antibodies which bind to whole pili toblock the attachment process.

MATERIALS AND METHODSBacteria. The P. aeruginosa strains used were PAK/

2Pfs and PAO/DB2. Both are multipiliated mutantswhich were originally obtained from D. E. Bradley,

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114 WATTS ET AL.

Faculty of Medicine, Memorial University, St. John's,Newfoundland. PAK/2Pfs pili have been shown to beindistinguishable from wild-type PAK pili (10). PAO/DB2 has been described previously (21).

Reagents. Octyl glucoside was obtained from SigmaChemical Co. Trypsin (containing tosyl-phenylalaninechloromethylketone) and chymotrypsin were pur-chased from Worthington Diagnostics. Citraconic an-hydride was from Eastman Organic Chemicals. Alka-line phosphatase-conjugated goat anti-rabbitimmunoglobulin G was from Miles Laboratories. p-Nitrophenyl phosphate tablets were from Sigma. 1251_labeled protein A was from New England NuclearCorp.

Pilus purification. PAK and PAO pili were purifiedas described previously (21). Gonococcal pili (from N.gonorrhoeae MSII opaque) were the generous gift ofGary Schoolnik, Department of Infectious Diseases,Stanford University Medical School, Stanford, Calif.CFA/I pili (isolated from Escherichia coli H-10407)and EDP208 pili conjugative pili (encoded by the F0lacplasmid EDP208) were kindly provided by E. A.Worobec, University of Alberta, Edmonton, Alberta,Canada.

Citraconylation, carboxymethylation, and enzymaticdigestion. Citraconylation was carried out by the meth-od of Gibbons and Perham (13) with minor modifica-tions as cited in Sastry et al. (22). Trypsin digestionwas carried out at 37°C for 16 h. The enzyme wasinitially added at a 1:50 molar ratio, with a furtheraddition after 3 h to a final concentration of 0.04 mol oftrypsin per mol of pilin. After trypsin digestion, thesoluble and insoluble fractions (abbreviated TC-solu-ble and TC-insoluble, for trypsin-citraconylated) wereseparated by centrifugation. Deblocking of the lysineresidues was carried out after the initial fractionationof the TC-soluble material on Sephadex G-50 (seebelow). The presence of the citraconyl groups duringthe initial fractionation was important in preventingaggregation. Decitraconylation was accomplished byincubation in 10% formic acid at 25°C for 6 h. Trypticsubfragments of pilin fragment TCIII (residues 54 to120) were prepared by digestion of decitraconylatedTCIII. The subfragment (residues 128 to 144) of pilinfragment TCIV (residues 121 to 144) was obtained bychymotryptic digestion of carboxymethylated wholepilin. Reduction and carboxymethylation of pilin werecarried out in 8 M urea, as described previously byCrestfield et al. (6).

Peptide purification. Pilin fragment TCI (residues 1to 30) was purified from the TC-insoluble material bychromatography in the presence of 30 mM octyl gluco-side. Approximately 2 mg of TC-insoluble materialwas solubilized in 1 ml of 30 mM octyl glucoside-0.05M Na2PO4 (pH 7.0) and centrifuged to remove residualinsoluble material. The supernatant was applied to aSephadex G-75 column (1 by 40 cm) equilibrated in thesame detergent-buffer system. The major peak, TCI,was desalted on a Sephadex G-10 column (1 by 10 cm),which also removes most of the detergent. TCI wasjudged pure by the appearance of a single sharp bandon SDS-polyacrylamide gels, by N-terminal analysis(by dansylation; reference 14), and by amino acidanalysis.The TC-soluble material was initially fractionated

on a Sephadex G-50 column equilibrated with 0.1 MNH4HCO3 (pH 8.1) and eluted at a flow rate of 8 ml/

min. Fractions (5 ml) were collected. Pilin fragmentTCIII obtained from this separation was judged pureby the criteria listed above.

Pilin fragments TCII (residues 31 to 53) and TCIV,which coelute on Sephadex G-50, were further purifiedby high-voltage paper electrophoresis at pH 6.5, fol-lowed by electrophoresis at pH 1.8 at 3,000 V for 50min. The peptides were visualized by cutting away astrip from the electropherogram and staining withninhydrin-cadmium (15). The peptide bands were cutout and eluted with water. The tryptic subfragments ofTCIII and the chymotryptic fragment of TCIV (resi-dues 128 to 144) were also purified by high-voltagepaper electrophoresis. Details of these separations willappear elsewhere. (P. A. Sastry, J. R. Pearlstone, L. B.Smillie, and W. Paranchych, manuscript in prepara-tion).Amino acid analysis. Amino acid analyses were

carried out on a Durram D-500 amino acid analyzer.Samples were hydrolyzed in 6 N HCI containing 0.1%phenol in evacuated sealed tubes for 24 h at 110°C.

Coupling of peptides to BSA. The photosensitivecross-linking reagent, the N-hydroxysuccinimide esterof 4-azidobenzoic acid (AB-OSu), was synthesized asdescribed previously by Chong and Hodges (5). Thestarting material for the 14C reagent was 4-amino[1-14C]benzoic acid. The specific activity of AB-OSu was6,100 cpm/nmol. Cross-linking of the peptide to bovineserum albumin (BSA) was carried out as describedpreviously by Worobec et al. (31) with the followingmodifications. Only 50 to 75 nmol of each peptide wasused. The peptide in 30 ,ul of 0.16% NaHCO3 wasadded to 1 ,umol (0.3 mg) of AB-OSu in 30 p.l ofdioxane. The Sephadex G-25 column used was ofdimensions 1 by 25 cm and was eluted at a flow rate of3 ml/h. Fractions (0.25 ml) were collected. A sample ofthe peptide, derivatized at its amino group {the N-(4-azido[1-14C]benzoyl)-peptide} was hydrolyzed foramino acid analysis before the addition of BSA. Fromthis analysis, we could determine the degree of incor-poration of radiolabel in the peptide. From the specificactivity of "4C-labeled AB-OSu, the peptide concen-tration in the peptide-BSA conjugate after photolysiscould be determined by radioactivity measurements.

Photolysis was carried out as described previouslyby Worobec et al. (31), and the reaction products wereseparated on a Sephadex G-50 column (1 by 25 cm)equilibrated with 1 mM HCl and eluted at a flow rate of4 ml/h. Fractions (0.25 ml) were collected. The pep-tide-BSA conjugate, which eluted at the void volume,was pooled and lyophilized.

Preparation of antisera. The antipilus antisera usedin the immunoelectrophoresis experiments were pre-pared by intraveneous injection of New Zealand whiterabbits as described previously by Watts et al. (29).For the ELISA and immunoblot experiments, antiseraagainst PAO and PAK pili were obtained from NewZealand white rabbits as follows: 100 p.g of pure pilidissolved in an equal volume of Freund completeadjuvant was injected subscapularly and intramuscu-larly in the gluteal area. Booster injections were ad-ministered at two 1-month intervals with 100 p.g of pilidissolved in an equal volume of saline and Freundincomplete adjuvant. The rabbits were bled 2 weeksafter the second booster injection.

Immunoelectrophoresis. Counterimmunoelectro-phoresis and rocket immunoelectrophoresis were car-

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ANTIGENIC DETERMINANTS OF PSEUDOMONAS PILIN

ried out as described previously by Tsang and Maru-syk (24).

Polyacrylamide gel electrophoresis and transfer tonitrocellulose paper. SDS-polyacrylamide gel electro-phoresis was carried out by the method of Lugtenberget al. (18) with the following modifications. For experi-ments with the peptides TCI and TCIII, the runninggel contained 20% acrylamide-0.36% methylene bisa-crylamide and the stacking gel contained 7% acryla-mide-0.2% bisacrylamide. For the BSA-conjugatedpeptides, the running gel contained 10% acrylamide-0.18% bisacrylamide and the stacking gel contained4% acrylamide-0.1% bisacrylamide. Samples wereboiled in SDS for 10 to 20 s before application to thepolyacrylamide gel. The gels were 0.8 mm thick.

Transfer to nitrocellulose paper was carried out withan Electro-blot apparatus (E-C apparatus) accordingto the procedure of Towbin et al. (25). Transfer wascarried out at 4 mA/cm2 for 3 h. Duplicate sampleswere stained directly by the method of Fairbanks et al.(7). Staining of the gels after transfer of the proteins tonitrocellulose showed that transfer was complete with-in 3 h.

Immunological assays. Immunological detection ofpeptides bound to nitrocellulose was carried out asdescribed previously by Towbin et al. (25) with theexception that 1251I-labeled protein A was used ratherthan 1251I-labeled sheep immunoglobulin G.The ELISA was carried out exactly as described

previously by Worobec et al. (31) according to theprinciples described by Voller et al. (27). The ELISAswere repeated two to five times. Replicate experi-ments gave a standard deviation of 0.02 to 0.21 units ofabsorbance at 405 nm (A405) (0.11 on average).

RESULTS AND DISCUSSIONTo determine whether one or several antigenic

sites were responsible for the antigenicity ofpilin, we subjected purified PAK pili to counter-immunoelectrophoresis and rocket immunoelec-trophoresis as described above. At least threePAK pili-antipilus antibody precipitin lines wereevident by both methods (Fig. 1). The occur-rence of multiple precipitation zones can beattributed to several causes (reviewed in refer-ence 19). The presence of several antigenicspecies of the protein, the presence of more thanone class of immunoglobulin against the antigen(immunoglobulins G and M, for example), andthe presence of multiple antigenic determinantswithin a single antigenic protein each could giverise to multiple precipitation zones. This occursbecause each antibody-antigen interaction has aspecific dissociation constant (Kd) associatedwith it. Therefore, the concentration of antigenand antibody required to form a precipitationzone and thus the position in the gel of thatprecipitation zone are dependent on this Kd.

Since pilin has been shown to be immunologi-cally pure by the technique of immunoblotting(29), the multiple precipitin lines observed inFig. 1 are most likely due to the presence ofthree kinds of antibodies against pilin in the

A+

B

FIG. 1. Immunoelectrophoresis of PAK pili. (A)Counterimmunoelectrophoresis carried out for 16 h at3 V/cm. The lower well contained 10 ,ug of pili, theupper well contained 10 1±l of a 1:8 dilution ofantiserum. (B) Rocket immunoelectrophoresis carriedout for 20 h at 3 V/cm. The upper gel contained 5%(vol/vol) anti-PAK pilus antiserum; the well contained10 ,ug of PAK pili.

rabbit antipilus antiserum. As mentioned above,these could be due to the presence of immuno-globulins of more than one class reacting with asingle type of antigenic site or due to the pres-ence of antibodies against multiple antigenicsites in the protein. In light of the results pre-sented below, we favor the latter explanation.Since these techniques are fairly limited in reso-lution, this represents the minimum number ofantigenic sites that one expects to find in pilin.

Preparation of proteolytic fragments of PAKpilin. A schematic representation of the PAKpilin sequence (22) is shown in Fig. 2. There arethree arginine residues at positions 30, 53, and120, which conveniently divide the protein intofour regions. Therefore, arginine-specific cleav-age was used to generate the four fragments TCI(residues 1 to 30), TCII (residues 31 to 53), TCIII(residues 54 to 120), and TCIV (residues 121 to144). Arginine-specific cleavage was accom-plished by modification of the 12 lysines in pilin

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I I I I I I --l4

a~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

20 40 60 80 100 120 1.40

Position in Sequence

M , is----I

II I a

III LOOH

2-1 IS-s -i

53TCIII+

59 68 70 8182a a a

_ + +

2 3l

I 1

120 144TCIV~

110 128 144

n + e,

4.1

FIG. 2. Schematic representation of locations of antigenic peptides in P. aeruginosa PAK pilin. (a) Thehydrophilicity index calculated for pilin by the method of Hopp and Woods (17), reproduced from Sastry et al.(22). (b) Schematic representation of the sequence of pilin. The buried tyrosines (residues 24 and 27) andtryptophans (residues 55 and 127) are indicated. The disulfide linkage between residues 129 and 142 is alsoshown. (c) Map of the TC-peptides of PAK pilin. (+) indicates a positive reaction with anti-PAK pilus antiserum,and (-) indicates a negative reaction. (d) Map of the smaller fragments of pilin that have been tested forantigenicity. cmc indicates that the disulfide linkages have been reduced and modified to form carboxymethyl-cysteine. (e) Proposed antigenic determinants in pilin based on studies with peptide fragments, cross-reactivitystudies, and chemical modification. 1, residues 1 to 9; 2 and 3 are the same as the peptides in (d); 4, residues 130to 141.

with citraconic anhydride (13), followed by tryp-sin digestion as described above.TCI, which encompasses the extremely hy-

drophobic N-terminus of pilin, was largely foundin the insoluble pellet obtained after centrifuga-tion of the tryptic digest. This material could besolubilized in the detergent octyl glucoside andfurther purified on Sephadex G-75 in the pres-ence of octyl glucoside (Fig. 3A). Octyl gluco-side has been used in the past to dissociate pilininto dimers (28, 28a). The major peak obtainedon Sephadex G-75 elutes at an apparent molecu-lar weight of 33,000 and consists of aggregatedTCI plus bound detergent. After removal ofdetergent, the preparation was found to consistof pure TCI (see above).The trypsin-soluble citraconylated material

was initially fractionated on a Sephadex G-50column (Fig. 3B). Some TCI is present in the

TC-soluble material and elutes as an aggregate atthe void volume (peak a). Peak b was found tobe pure TCIII (Mr = 6,800), whereas peak cconsisted of an approximately equimolar mix-ture of TCII and TCIV. TCII and TCIV wereseparated by high-voltage paper electrophoresisas described above.TCIII was further subdivided by digestion of

the decitraconylated peptide with trypsin. Inaddition, a subfragment of TCIV was preparedby chymotryptic digestion of carboxymethylat-ed whole pili. The positions in the pilin sequenceof the peptides which were used for furtherstudy are summarized in Fig. 2c and d. Thepurity of each of the peptides was assessed asdescribed above.

Antigenicity ofPAK pilin peptides. The proteo-lytic fragments of PAK pilin, described above,were tested for their ability to react with rabbit

x +20

*: 0

0

0 O., 4

.,C

=s -2

b

C

d

I I1- I:r' 1r

1 30TCI+ TCII-

1e f

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ANTIGENIC DETERMINANTS OF PSEUDOMONAS PILIN 117

antipilus antiserum in an ELISA and in animmunoblot experiment. Both TCI (which existsas an aggregate in aqueous media) and TCIII aresufficiently large to be used directly in theseassays. The remaining peptides were chemicallycross-linked through their primary amino groupsto BSA, using AB-OSu as described previouslyby Worobec et al. (31) and above. The resultingmolar ratios of peptide to BSA were 2 ± 1:1 forall six peptides.The results of the ELISA assay for PAK pilin

and its proteolytic fragments versus anti-PAKpilus antiserum are shown in Fig. 4. The wells ofthe microtiter plates are saturated with respectto PAK pili at a coating concentration of be-tween 4 and 5 jig/ml (data not shown); therefore,PAK pili were used at a concentration of 5 pug/ml. The peptides and BSA-peptide conjugateswere coated at a concentration of 1 to 1.5 nmollml. Increasing these concentrations twofold didnot result in an increase in A405 for any of thepeptides. BSA controls (not shown) gave nodetectable A405.

A vov

GA - I v

1411kG2 - \° / I/-!! '.,,; _ _

15

Ve (ml)

FIG. 3. Chromatography of trypsin-treated citra-conylated pili. (A) Purification of TCI from the TC-insoluble pellet on a Sephadex G-75 column (1 by 40cm) equilibrated with 30 mM octyl glucoside. A280,Absorbance at 280 nm. (B) Separation of TC-solublepeptides on Sephadex G-50 (2.5 by 120 cm) in 0.1 MNH4HCO3 (pH 8.1). The bars indicate the pooledregions. a, TCI; b, TCIII; c, mixture of TCII andTCIV. A230, Absorbance at 230 nm.

FIG. 4. ELISA of PAK pilin and its peptide frag-ments with anti-PAK pilus antiserum. The results areshown for a 1:500 dilution of antiserum. The locationof the peptides in the sequence is diagrammed in Fig.2. PAK indicates native PAK pili. Details are providedin the text.

It can be seen that three of the four TCpeptides show a positive reaction with antipilusantiserum. The reaction with TCII-BSA wasconsidered negative. The small A405 obtainedwith this peptide (5% of the reading for nativepili) may be due to slight contamination of TCIIwith TCIV since the two peptides are verysimilar with respect to size and charge. Of theTCIII subfragments tested, residue 59 to 68 wasnegative, whereas residue 70 to 81 and residue82 to 110 were positive.The two cysteines in pilin form an intrachain

disulfide bridge between residues 129 and 142(Fig. 2b) (Sastry et al., manuscript in prepara-tion). The chymotryptic fragment (residues 128to 144) was derived from carboxymethylatedpilin, and therefore the disulfide linkage hasbeen reduced and the cysteines modified in thispeptide. Since residue 128 to 144-BSA gives acomparable reaction to TCIV-BSA in theELISA assay, it appears that neither the disul-fide linkage nor the cysteine residues are impor-tant in the antigen-antibody interaction. Thisfinding is contrary to those of Schoolnik et al.(23) with pili isolated from N. gonorrhoeae. Inthis case, the antigenicity of a C-terminal cyano-gen bromide fragment is lost when the disulfidelinkage is reduced.We obtained similar results when the peptides

and the peptide-BSA conjugates were subjectedto SDS-polyacrylamide gel electrophoresis, fol-lowed by electrophoretic transfer to nitrocellu-lose, reaction with antipilus antibodies, and de-tection with 125I-labeled protein A. The resultsare shown for the TC-peptides of PAK pilin inFig. 5. It can be seen that PAK pilin, TCI-,

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118 WATTS ET AL.

TCIII-, and TCIV-BSA, but not TCII-BSA, re-act with antipilus antiserum in this assay.We have therefore identified four antigenic

regions in the PAK pilin subunit. These areresidues 1 to 30, 70 to 81, 82 to 110, and 128 to 144(Fig. 2). Figure 2a shows the hydrophilicityindex calculated for pilin by the method of Hoppand Woods (17). On the basis of an analysis ofthe hydrophilicity index of 12 proteins for whichextensive immunochemical data were available,Hopp and Woods (17) suggested that antigenicdeterminants are invariably found at or immedi-ately adjacent to points of highest local hydro-philicity. In the case of PAK pilin, however,only two of the four antigenic peptides encom-pass peaks of hydrophilicity (residues 82 to 110and residues 128 to 144). The antigenic fragmentof residues 70 to 81 falls between two such localmaxima, and TCI is in the most hydrophobicregion of the molecule.As mentioned above, modification of the two

cysteines in the C-terminal antigenic peptidedoes not appear to effect its antigenicity. There-fore, we propose that the C-terminal antigenicsite is contained within the region of residues130 to 141 as indicated in Fig. 2e, antigenic site4. We further propose that the N-terminal anti-genic determinant (located in TCI) is at the N-terminal end of TCI and includes N-methylphe-nylalanine, on the basis of the following ratio-nale. First, residues 24 and 27 have been shownto be buried in native pilin (28a). Since antigenicdeterminants are generally found on the surfaceof proteins, one would not expect to find anantigenic site at the C-terminal end of TCI.Second, the amino acid N-methylphenylalanine

is a good candidate for an antigenic determinantowing to its rare occurrence. Finally, as will beshown below, antibodies raised against PAK pilicross-react with both PAO pili and with gono-coccal pili. Since the region of highest sequencehomology between these strains is found atresidues 1 to 9, where the sequences are identi-cal, this region is the most likely candidate forthe common antigenic determinant. Also consis-tent with the data is the fact that Trp55 andTrp127, which are buried in native pilin (28a), donot fall into any of the antigenic regions pro-posed above.

Cross-reactivity between pilus strains. In thepast (4), the polar pili associated with P. aeru-ginosa PAK and PAO were considered to beserologically unrelated. However, these conclu-sions were based upon agglutination tests andupon observation of antibodies bound to pili inthe electron microscope. Using the more sensi-tive ELISA assay, we find that anti-PAK pilusantiserum does recognize PAO pili and viceversa. Figure 6 shows the results of an ELISA inwhich five types of pili were tested for theirability to react with anti-PAK and anti-PAOpilus antisera. It can be seen that PAK and PAOpili show a significant cross-reactivity in bothdirections. On the other hand, little or no reac-tion was observed between anti-Pseudomonasantisera and pili isolated from N. gonorrhoeae,E. coli H-10407, or E. coli(EDP208).

This cross-reactivity could also be demon-strated in an immunoblot experiment (Fig. 7).Surprisingly, in this assay, the anti-PAK pilusantiserum recognized not only PAK and PAOpilin but also gonococcal pilin. Again, no reac-

I A

_

A B C.N DFIG. 5. Polyacrylamide gel electrophoresis and transfer to nitrocellulose, followed by immunological

detection of PAK TC-peptides as described in the text. (A) 20% polyacrylamide gel stained with Coomassie blue.1, PAK pilin; 2, TCI; 3, TCIII; 4, protein standards, with molecular weights as indicated in thousands. (B)Peptides from a duplicate gel to that in (A) were transferred to nitrocellulose and reacted with antipilus antiserumand 1251I-labeled protein A. The lanes are the same as in (A). (C) 10% polyacrylamide gel stained with Coomassieblue. 1, TCII-BSA; 2, TCIV-BSA. (D) Duplicate of the gel shown in (C), after transfer of the peptide-BSAconjugates to nitrocellulose and immunological detection.

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ANTIGENIC DETERMINANTS OF PSEUDOMONAS PILIN 119

A B

0X op 0(0iu 0R0.antiseawwithtu1.0-

1.0.Al piiwr otda onetaino u/l

0.51II

PA,PA il;PO, PO pil;G,gncca ii

FIG. 6. ELISA assay showing cross-reactivity be-tween pili isolated from P. aeruginosa PAK and PAO.(A) Results obtained with a 1:500 diution of anti-PAKpilus antiserum. (B) Results obtained with a 1:500dilution of anti-PAO pilus antiserum. The results of(A) and (B) were normalized so that the reaction ofantisera with the homologous pilus gave an A d5of1.50. All pili were coated at a concentration of 5 ~xg/ml.PAK, PAK pii; PAO, PAO pili; GC, gonococcal piil;CFAII, pili isolated from E. coli H-10407; EDP208, piliencoded by the Folac plasmid EDP208.

tion was observed with CFAII or EDP208 pili. Asimilar result was obtained with anti-PAG pilusantiserum (data not shown). Since the onlyregion of homology among PAK, PAO, andgonococcal pilins is found at the N-terminus(residues 1 to 22) of the proteins, it is likely thatthis region is responsible for the cross-reactivity

1 2 3 4 5

A B

in the immunoblot experiment. We attribute thefailure of anti-Pseudomonas pilus antisera torecognize gonococcal pili in the ELISA to thepossibility that the N-terminus of gonococcalpilin is buried in the native state and becomesexposed in SDS-polyacrylamide gels.Of the four PAK TC-peptides, only TCI reacts

with anti-PAO pilus antiserum (Fig. 8). Thecross-reactivity observed with this peptide doesnot appear to account for the total cross-reactiv-ity between the intact pilins. This could beattributed to several possible causes. It is likelythat TCI in intact pili exists in a fairly limitednumber of conformations. In solution, however,TCI may be more flexible, and therefore theproportion of TCI that is in a suitable conforma-tion for binding with antibodies may be dimin-ished. It is also possible that proteolytic cleav-age of the polypeptide has interrupted a secondcommon sequential antigenic determinant. Athird possibility is that an antigenic determinantthat is present in intact pili and consists ofresidues contributed by more than one subunitmight have been lost upon proteolytic cleavage.This type of antigenic determinant (called aneotope) has been observed with tobacco mosa-ic virus (26). A further possibility is that aconformational antigenic determinant, which isdependent on the native tertiary structure ofpilin, has been lost upon proteolytic digestion ofpili. The latter possibility is less likely sincecarboxymethylated pili, which retain only 50%

2 3 4 5

i_.a....

-15

-15

FIG. 7. Cross-reactivity between pilins demonstrated by the immunoblot technique, using anti-PAK pilusantiserum. (A) Coomassie blue-stained 20o polyacrylamide gel. Lanes 1 through 5 contain, respectively,gonococcal pilin (4 Fig), PAK pilin (4 ,ug), standard proteins with molecular weights as indicated in thousands,CFA/I pilin (4 pLg), and EDP208 pilin (4 ,ug). (B) Results obtained after electrophoresis of pilins on a 20%polyacrylamide gel, transfer to nitrocellulose, and reaction with anti-PAK pilus antibodies and 125"-labeledprotein A as described in the text. 1, PAO pilin, 1.5 ,ug; 2, PAK pilin, 0.5 ,ug; 3, GC pilin, 4 ,ug; 4, CFA/I pilin, 4,ug; 6, EDP208 pilin, 4 ,ug. The arrows indicate the expected migration positions of the four pilins.

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1.5-

Lr1.0-

0.51

n nrIV ,.u _

FIG. 8. ELISA assay showing reaction of PAKpilin and its TC-peptides with anti-PAO antiserum.The results shown are for a 1:500 dilution of anti-PAOantiserum and are normalized relative to an A405 of 1.5for PAO pilin with its homologous antiserum.

of the secondary structure of native pili, accord-ing to circular dichroism studies, are equally asreactive in the ELISA as native pili (data notshown).

In conclusion, we have identified four proteo-lytic fragments of PAK pilin which contain se-quential antigenic determinants. Of the four anti-genic regions, three appear to be specific toPAK pilin, whereas the N-terminal antigenicpeptide, TCI, contains an antigenic site which iscommon to PAK, PAO, and gonococcal pilins.This is in keeping with the findings of Sastry etal. (unpublished data) that the N-terminal one-third of PAK and PAO pilins represents a con-stant sequence, whereas the C-termini are vari-able. Similarly, Schoolnik et al. (23) have shownthat the C-termini of gonococcal pilins showantigenic variability, whereas the N-termini ofthese proteins are conserved. On the other hand,comparative sequencing of antigenically distinctK88 pilins (12) has shown that most changesoccur at the center of the protein primary se-quence. Yet another motif is observed with thedistribution of antigenic sites in EDP208 pilin(31). The major antigenic site in this protein isfound at the N-terminus, whereas the C-termi-nus probably contains one or more weaker anti-genic sites.These findings are consistent with the obser-

vation that diffraction patterns of PAK andgonococcal pilins indicate some structural simi-

larity between the two proteins (8; T. H. Watts,G. K. Schoolnik, and W. Paranchych, unpub-lished data), whereas the diffraction pattern ofEDP208 pilin does not indicate any such struc-tural homology (W. Folkhard, K. R. Leonard, J.Dubochet, D. A. Marvin, and W. Paranchych,Abstr. IXth Int. Congr. Biochem., 03-3-S116,1979).Although the four peptides shown in Fig. 2c

and d are antigenic, it has not been establishedthat they are immunogenic. To establish thisfact, it will be necessary to raise antibodiesagainst the BSA-conjugated peptides. Work iscurrently under way to chemically synthesizeanalogs of the four antigenic sites proposed inFig. 2e, to further delineate the antigenic sitesand to prepare sufficient material for raisingantibodies against the peptides.

Similarly, one must be cautious in interpretingthese results as indicating that the antigenicpeptides are on the surface in native pili, be-cause of the danger of dissociation or break-down of pili in the immunized animals. Toestablish this fact, it will be necessary to demon-strate that antibodies raised against the frag-ments are capable of binding to native pili.

ACKNOWLEDGMENTS

We would like to thank P. C. S. Chong for preparing ["4C]-AB-OSu, K. Volpel for technical assistance, Gary Schoolnikfor donating gonococcal pili, Mike Nattriss for carrying outthe amino acid analyses, and L. B. Smillie for the use of hisfacilities.

This research was supported by the Medical ResearchCouncil of Canada and the Alberta Heritage Foundation forMedical Research (AHFMR). T.H.W. was supported by anAHFMR studentship.

LITERATURE CITED'1. Attassi, M. Z. 1975. Antigenic structure of myoglobin: the

complete immunochemical anatomy of a protein andconclusions relating to the antigenic structures of pro-teins. Immunochemistry 12:422-438.

2. Attassi, M. Z., and C.-S. Lee. 1978. The precise and entireantigenic structure of native lysozyme. Biochem. J.171:429-431.

3. Bradley, D. E. 1980. A function of Pseudomonas aerugin-osa PAO polar pili: twitching motility. Can. J. Microbiol.26:146-154.

4. Bradley, D. E., and T. L. Pitt. 1975. An immunologicalstudy of the pili of Pseudomonas aeruginosa. J. Hyg.74:419-431.

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

6. Crestfield, A. M., S. Moore, and W. H. Stein. 1963. Thepreparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J. Biol. Chem. 238:622-627.

7. 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.

8. Folkhard, W. F., D. A. Marvin, T. H. Watts, and W.Paranchych. 1981. Structure of polar pili from Pseudomo-

) C.)m m

II

~e->

CL A F-I I-

nrm

INFECT. IMMUN.

on February 14, 2021 by guest

http://iai.asm.org/

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ANTIGENIC DETERMINANTS OF PSEUDOMONAS PILIN 121

nas aeruginosa strains K and 0. J. Mol. Biol. 149:79-93.9. Frjholm, L. O., and K. Sletten. 1977. Purification and N-

terminal sequence of a fimbrial protein from Moraxellanonliquefaciens. FEBS Lett. 73:29-32.

10. Frost, L., and W. Paranchych. 1977. Composition andmolecular weight of pili purified from Pseudomonas aeru-ginosa K. J. Bacteriol. 131:259-269.

11. Frost, L. S., M. Carpenter, and W. Paranchych. 1978. N-methyl-phenylalanine at the N-terminus of Pseudomonasaeruginosa K pili. Nature (London) 271:87-89.

12. Gaastra, W., P. Klemm, J. M. Walker, and F. K. deGraaf. 1979. K88 fimbrial protein: amino- and carboxy-terminal sequences of intact proteins and cyanogen bro-mide fragments. FEMS Microbiol. Lett. 6:15-18.

13. Gibbons, I., and R. N. Perham. 1970. The reaction ofaldolase with 2-methyl-maleic anhydride. Biochem J.116:843-849.

14. Hartley, B. S. 1970. Strategy and tactics in protein chemis-try. The first BDH lecture. Biochem. J. 119:805-822.

15. Heathcoat, J. G., and C. Haworth. 1969. An improvedtechnique for the analysis of amino acids on thin layers ofcellulose. II. The quantitative determination of aminoacids in protein hydrolysates. J. Chromatogr. 43:84-92.

16. Hermodsen, M. A., K. C. S. Chen, and T. M. Buchanan.1978. Neisseria pili proteins: amino-terminal sequencesand identification of an unusual amino acid. Biochemistry17:422-445.

17. 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.

18. Lugtenberg, B., J. Meyers, N. Peters, P. van der Hoek, andL. van Alphen. 1975. Electrophoretic resolution of themajor outer membrane proteins of E. coli K12 into 4bands. FEBS Lett. 58:254-258.

19. Oudin, J. 1980. Immunochemical analysis by antigen-antibody precipitation in gels. Methods Enzymol. 70:166-198.

20. Paranchych, W., L. S. Frost, and M. Carpenter. 1978. N-terminal amino acid sequence of pilin isolated from Pseu-domonas aeruginosa. J. Bacteriol. 134:1179-1180.

21. Paranchych, W., P. A. Sastry, L. S. Frost, M. Carpenter,

G. D. Armstrong, and T. H. Watts. 1979. Biochemicalstudies on pili isolated from Pseudomonas aeruginosastrain PAO. Can. J. Microbiol. 25:1175-1181.

22. Sastry, P. A., J. R. Pearlstone, L. B. Smillie, and W.Paranchych. 1983. Amino acid sequence of pilin isolatedfrom Pseudomonas aeruginosa PAK. FEBS Lett.151:253-256.

23. Schoolnlk, G. K., [. Y. Tai, and E. C. Gotschllch. 1982. Apilus peptide vaccine for the prevention of gonorrhoea.Prog. Allergy 33:314-331.

24. Tsang, L. W-L., and R. G. Marusyk. 1980. Purificationand partial characterization of a 33,000 molecular weightendonuclease associated with human adenovirus type 5.Can. J. Microbiol. 26:1224-1231.

25. Towbin, H., T. Staehelm, 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.

26. Van Regenmortel, M. H. V. 1982. Serology and immuno-chemistry of plant viruses. Academic Press, Inc., NewYork.

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

28. Watts, T. H., C. M. Kay, and W. Paranchych. 1982.Dissociation and characterization of pilin isolated fromPseudomonas aeruginosa strains PAK and PAO. Can. J.Biochem. 60:867-872.

28a.Watts, T. H., C. M. Kay, and W. Paranchych. 1983.Spectral properties of three quaternary arrangements ofPseudomonas pilin. Biochemistry 22:3640-3646.

29. Watts, T. H., E. A. Worobec, and W. Paranchych. 1982.Identification of pilin pools in the membranes of Pseudo-monas aeruginosa. J. Bacteriol. 152:687-691.

30. Woods, D. E., D. C. Strauss, W. G. Johanson, V. K. Berry,and J. A. Bass. 1980. Role of pili in adherence of Pseudo-monas aeruginosa to mammalian buccal epithelial cells.Infect. Immun. 29:1146-1151.

31. Worobec, E. A., A. K. Taneja, R. S. Hodges, and W.Paranchych. 1983. Localization of the major antigenicdeterminant of EDP208 pilin. J. Bacteriol. 153:955-961.

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