INFECTION AND IMMUNITY, Jan. 1989, p. 76-810019-9567/89/010076-06$02.00/0Copyright C) 1989, American Society for Microbiology

Isolation and Characterization of the cx-Galactosyl-1,4-3-Galactosyl-Specific Adhesin (P Adhesin) from Fimbriated Escherichia coli

HEINZ HOSCHUTZKY,1 FRIEDRICH LOTTSPEICH,2 AND KLAUS JANN1*Max-Planck-Institut fur Immunbiologie, D-7800 Freiburg,' and Max-Planck-Institut fur Biochemie, Martinsried,

Federal Republic of Germany

Received 13 July 1988/Accepted 1 October 1988

The ft-galactosyl-1,4-4-galactosyl-specific adhesin (P adhesin) was isolated from the fimbria-adhesin complex(FAC) of recombinant Escherichia coli strains expressing the F71, F8, or F13 fimbrial antigens. Separation intofimbriae and adhesin was achieved by heating the FAC to 80°C in the presence of Zwittergent 3-16. Afterremoval of the fimbriae by precipitation with lithium chloride, the adhesin was purified by anion-exchange fastprotein liquid chromatography in the presence of 4 M urea. The purified adhesins from the three strains hadpls of 4.8 to 5.0 and molecular weights of approximately 35,000. The fimbrillins were smaller, their molecularweights being different with different F antigens. The amino-terminal amino acid sequence of the F71- andF13-derived adhesins were different, that of the F13-derived adhesin being identical to that extrapolated fromthe DNA sequence of the papG gene (B. Lund, G. Lindberg, B.-I. Marklund, and S. Normark, Proc. Natl.Acad. Sci. USA 84:5898-5902). An antiadhesive monoclonal antibody which reacted with the three P adhesinswas prepared. The FAC and the purified adhesins but not the fimbriae from which the adhesins had beenremoved agglutinated erythrocytes and galactose-galactose-coated latex beads. The adhesion of erythrocytes tothe surface-fixed adhesins could be specifically inhibited with ox-galactosyl-1,4-I8-galactosyl-1,4-glucosyl. Theresults indicate that the P adhesin(s) of uropathogenic E. coli represents a group of related proteins withconserved receptor recognition domains. The F13-derived P adhesin is the PapG protein postulated byNormark and his colleagues (Lund et al., Proc. Natl. Acad. Sci. USA 84:5898-5902; B. Lund, F. Lindberg, andS. Normark, J. Bacteriol. 170:1887-1894).

Adherence to host tissue and cell surfaces is an early stepin infections caused by Escherichia coli (5, 25, 28). Thisinteraction, which can easily be monitored in vitro byagglutination of erythrocytes (RBC), is mediated by adhe-sive proteins (adhesins) expressed on the bacterial cellsurface. They recognize complex carbohydrates on theplasma membrane of host cells or in epithelial mucus.

Adherence is frequently associated with the expression offimbriae or pili (4-7). The carbohydrate specificity of thefimbriae has been defined in several instances. Thus, thecommon type 1 fimbriae recognize a-mannose in glycopro-teins (30); the P fimbriae of uropathogenic E. coli interactwith glycolipids containing aGal-(1,4)-PGal, where Gal isgalactosyl (16, 17); the S fimbriae of E. coli causing septice-mia, neonatal meningitis, or urinary tract infections attach toglycoproteins terminating with oaNeuNAc-(2,3)-IGal, whereNeuNAc is N-acetylneuraminidase (15, 27); and the Madhesin of uropathogenic E. coli recognizes on glycoproteins(e.g., blood group M glyophorin AM) a not-yet-definedreceptor containing Gal, N-acetylgalactosamine, NeuNAc,and terminal serine (27, 31).

Fimbriae consist of peptide subunits (fimbrillins) whichdiffer in molecular weight and serological specificity (10, 26).One E. coli strain can exhibit, in addition to type 1 fimbriae,several serologically distinct P fimbriae with different sub-unit molecular weights (1, 10).

Genetic studies of type 1, P, and S fimbriae (8, 9, 13, 19,21, 23, 32, 33) have revealed that the bacterial properties ofadherence and fimbriation are determined at distinct chro-mosomal sites. Of special interest was the finding thatmutants which lacked fimbriae but were still adhesive or

which exhibited fimbriae but were not adhesive could be

* Corresponding author.

constructed (9, 19, 21, 33). These results indicated thatseveral proteins are involved in the expression of adhesivefimbriae. The protein present in fimbrial preparations in thelargest amounts is the fimbrial subunit. Several minor pro-teins are also determined by the fimbria-adhesin gene com-plex and some of these can be separately expressed inminicells (18, 19).

Recently, we isolated and characterized the aNeuNAc-(2,3)-PGal-specific adhesin (S adhesin) from fimbriated py-elonephritogenic E. coli and showed it to be associated with(presumably the tips of) the fimbriae (24). The isolation ofthe a-mannose-specific adhesin from E. coli exhibiting type1 fimbriae was reported (2). In this communication we

describe the isolation and characterization of the otGal-(1,4)-3Gal-specific adhesin (P adhesin) from uropathogenic E.

coli.

MATERIALS AND METHODS

Bacteria and cultivation. The strains used are listed inTable 1. They were grown at 37°C for 18 h on Loeb agarcontaining 0.1% glucose and the appropriate antibiotics (100,ug of ampicillin or 25 jig of tetracycline per ml). Bacteriawere selected by passage of colonies which agglutinatedhuman P adhesin-specific RBC and Gal-Gal-coated beadsbut did not agglutinate Saccharomyces cerevisiae cells (11,25).

Isolation of the FAC. A suspension of agar-grown bacteriain 75 mM NaCl was heated to 65°C for 30 min. After beingcooled to room temperature, the suspension was centrifuged(15,000 x g, 30 min) to remove the defimbriated bacteria. Tothe supernatant glycine and EDTA were added to finalconcentrations of 20 and 5 mM, respectively. The crudefimbria-adhesin complex (FAC) was precipitated with am-monium sulfate (10% saturation) and collected by centrifu-

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FIMBRIATED E. COLI P ADHESIN 77

TABLE 1. E. coli strains used

Strain Serotype Plasmida F antigen Reference or source

21086 02:K5:H4 9 520025 04:K12:H- 12rel, 13, 14, 1C 1121614 K-12 (HB101) pDAL201B (Apr) 71 D. Low (unpublished data)21391 K-12 (HB101) pANN921 (Tcr) 8 821464 K-12 (HB101) pRHU845 (Tcr) 13 23

a Apr, Resistant to ampicillin; Tcr, resistant to tetracycline.

gation (15,000 x g, 30 min). To remove contaminating lipids,we dissolved the pellet in 50% ethanol and precipitated theFAC with LiCl (250 mM). After centrifugation, the pelletwas suspended in 10 mM Tris chloride (pH 7.8). To removeresidual lipopolysaccharide, we added deoxycholate to 0.5%and heated the mixture to 60°C for 30 min. The pure FACwas precipitated with LiCl (250 mM), collected by centrifu-gation, dissolved in a small amount of water, and stored at-700C.Separation and purification of fimbriae and adhesin. To a

solution of FAC (10 mg/ml) in 10 mM Tris chloride (pH 8)Zwittergent 3-16 was added to a final concentration of 0.5%(corresponding to an approximate ratio of protein to deter-gent of 10:1, by weight). The mixture was heated to 80°C for30 min, causing dissociation of the FAC into fimbriae andadhesin. The fimbriae were precipitated with LiCl (250 mM)and collected by centrifugation. The supernatant was ad-justed to 4 M urea and subjected to high-resolution anion-exchange chromatography with a Pharmacia MonoQ HR5/5column under the following conditions: buffer A (20 mM Trischloride, 20 mM glycine, 4 M urea, 0.05% Zwittergent 3-16[pH 8.5]); buffer B (buffer A containing 1 M LiCl); flow rate,5 ml/min; gradient slope, 20 mM/ml. Fractions were col-lected and assayed for protein content, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pat-tern, and hemagglutination activity. Pooled fractions weredialyzed against 50 mM ammonium bicarbonate and lyoph-ilized.Monoclonal antibodies. The fusion of spleen cells (108)

from immunized female BALB/c mice (8 weeks old) withPAI nonproducer myeloma cells (2 x 107), the cloning ofhybridoma cells by limiting dilution, and the production andcharacterization of the monoclonal antibodies have beendescribed previously (1, 14, 24).

Agglutination and adhesion tests. The hemagglutinatingactivities of bacteria, FAC, and adhesins were determinedwith human RBC at room temperature or on ice as describedpreviously (17, 24).Adhesion of FAC and adhesins to erythrocytes (hemad-

hesion) was determined in a microassay. Microdilutionplates (polystyrene round-bottom wells) were coated withthe fractions (0.5 ,ug of protein in 50 Fl of coating buffer perwell) at 4°C. The wells were washed once with phosphate-buffered saline (PBS), and free binding sites were blockedwith bovine serum albumin (1% in PBS) at 4°C for 4 h. Afterbeing washed twice with PBS, the wells were incubated witha suspension of human RBC (1% in PBS) at 20°C for 30 min.The wells were carefully washed five times with PBS, theadherent RBC were lysed (50 ,ul of water per well), and theA405 of hemoglobin was taken as a measure of the number ofadherent RBC. Putative inhibitors of adhesion were tested.

Analytical methods. SDS-PAGE (13% gels) was performedin the presence or absence of mercaptoethanol. Molecularmass markers were bovine serum albumin (67 kilodaltons[kDa]), ovalbumin (46 kDa), bovine carbonic anhydrase (28

kDa), soybean trypsin inhibitor (23 kDa), sperm whalemyoglobin (17.8 kDa), lysozyme (14.3 kDa), and cytochromec (12.3 kDa). Isoelectric focusing gels (0.5% agarose gels, pH3 to pH 10; Pharmalyte; Pharmacia) were standardized withthe Pharmacia isoelectric focusing calibration kit. Proteinconcentrations were determined as described by Bradford(3). Amino acid analyses were performed as describedpreviously (12). For N-terminal amino acid sequence analy-sis the purified adhesin was analyzed by SDS-PAGE (13%gels) and electroblotted onto siliconized glass fiber (Glassy-bond; Biometra) (6a). The P adhesin-containing band wasexcised and sequenced in an Applied Biosystems 470Agas-phase sequencer.

All buffers were sterilely filtered. Tubes and dialysis bagswere coated with 1% polyethylene glycol 6000 prior to use.

RESULTS

Bacteria. For the isolation of the FAC, wild-type uropath-ogenic E. coli strains expressing one or several F antigens aswell as recombinant strains each expressing one F antigenwere used (Table 1). We observed that the recombinantstrains were profusely fimbriated and agglutinated humanRBC well, while both properties were less pronounced andless constant in the wild-type strains. The recombinantstrains expressing F71 and F13 fimbriae were used to elabo-rate the isolation procedure described here. The procedure isequally applicable to the other strains shown in Table 1.

Isolation of the FAC. The FAC was isolated from thebacteria by heating bacterial suspensions at low salt concen-trations. After removal of the defimbrillated bacteria bycentrifugation, the FAC was precipitated from the superna-tant with ammonium sulfate. A low precipitating concentra-tion of ammonium sulfate as well as the presence of glycineand EDTA minimized an association of outer membranecomponents with the FAC. However, FAC prepared in thisway was contaminated with phospholipids and lipopolysac-charide. These were removed by extraction with ethanol anddeoxycholate, and the pure FAC was subsequently precip-itated with lithium chloride.The purified FAC agglutinated RBC and latex beads

coated with aGal-(1,4)-,Gal at pH 5 but not at pH 7.Agglutination as well as adherence was specifically inhibitedwith aGal-(1,4)-,BGal-(1,4)-PGlc, where Glc is glucosyl.These results show that the purified FAC had retained its Padhesin specificity. The agglutinating capacity of the FAC atpH 5 but not at pH 7 was most likely due to aggregation ofthe FAC at the lower pH. This was indicated by an increasein the turbidity of a suspension of FAC (1 mg/ml) in 150 mMNaCl-20 mM phosphate buffer when the suspension wasadjusted from pH 7 (optical density at 405 nm, 0.15) to pH 5(optical density at 405 nm, 0.65).

Separation and purification of fimbriae and adhesin. Thedissociation of the FAC into fimbriae and adhesin wasachieved by heating the FAC in the presence of Zwittergent

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78 HOSCHUTZKY ET AL.

OD275A

200

Fim

Adh~ ~ ~ -100

FIG 1.Sprto fPfmradhscaedesn(d)fo

o ~~~~~~~~~~~E

0D275 Bddh

200

3-16.Th fmrieFimC

03 ~~~~~~~~~~~10015 ~~~~~~~~~~~E

FIG. 1. Separation of P fimbria-associated adhesin (Adh) fromfimbrial subunits of the F71 fimbriae (Fim) by anion-exchangechromatography on a MonoQ HR5/5 column in the presence of 4 Murea. (A) F71 FAC. (B) Zwittergent 3-16 extract of the F71 FAC.0D275, Optical density at 275 nm.

3-16. The fimbriae from which the adhesin was removedwere precipitated (like FAC, see above) with lithium chlo-ride. They could also be sedimented by centrifugation(180,000 x g, 2 h). On electron microscopic examinationthey had the characteristic appearance of fimbriae (data notshown). The adhesin could be purified to apparent homoge-neity by anion-exchange fast protein liquid chromatography(FPLC) on a MonoQ HR5/5 column with a lithium chloride

TABLE 2. Molecular weights, ionic strength for elution inanion-exchange FPLC (EIS), and isoelectric points of the adhesin

and the fimbrillins from cloned F antigens

Fimbrillin AdhesinF

antigen Mol wt EIS p1b Mol wt EIS b(103) (MM)' p (103) (MM)' p

71 22 68 4.8-5.0 35 115 4.8-5.08 18.5 65 4.8-5.0 35 115 4.8-5.013 18 75 4.8-5.0 35 115 4.8-5.0

a Elution was done with LiCI.b The preparations had three closely spaced bands in the pH gradient gel.

gradient. FPLC profiles obtained with the F71 preparationfrom E. coli 21614 are shown in Fig. 1, and the SDS-PAGEanalysis of the fimbrillins and adhesin preparations from thedifferent strains is shown in Fig. 2.

Characterization of fimbriae and adhesin. SDS-PAGE in-dicated that the purified adhesin is a 35-kDa peptide, largerthan those of the subunits of P fimbriae (Fig. 2). SDS-PAGEof purified FAC revealed in addition to the 35-kDa adhesintwo minor proteins of 16.5 and 15 kDa (Fig. 2). Afterremoval of the adhesin from the FAC with Zwittergent 3-16,only the 15-kDa protein copurified with the adhesin. It couldbe separated from the adhesin with anion-exchange FPLC.Only the adhesin was studied further. The subunit weights offimbriae and adhesin from F71, F8, and F13 FACs as well astheir isoelectric points and the ionic strength for elution froma MonoQ column in FPLC are compared in Table 2.The amino-terminal amino acid sequences of the adhesins

from F71 and F13, from which most material was available,were found to be XNNIVFY(S)LGNVN(S)YQGG andGWHNVMFYAFNDYLTTNA, respectively. The amino-terminal amino acid of the adhesin from F71 could not bedetermined, owing to impurities in the buffer system. TheN-terminal sequence of the first amino acids of the adhesinfrom F13 was identical to that extrapolated by Lund et al.(22) from the DNA sequence of the papG gene. For compar-ison, it is shown here at the same length as the F71N-terminal sequence.The FAC and purified adhesins caused agglutination at pH

5 but not at pH 7 to pH 8, in concentration ranges ofnanograms per milliliter and micrograms per milliliter, re-

kO

67 - -~

46 - -"

28 - -

17.8 - -m

12.4 -m

1 2 3 4 5 6 7 8FIG. 2. SDS-PAGE analysis of the P fimbriae and adhesin. The Coomassie blue stained-lanes contained the following: 1, crude F71 FAC;

2, purified F71 FAC; 3, purified F71 fimbriae; 4, purified F71 adhesin; 5, purified F8 fimbriae; 6, purified F8 adhesin; 7, purified F13 fimbriae;8, purified F13 adhesin. kD, Kilodaltons.

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TABLE 3. Hemagglutination and adhesive properties of FACs,fimbriae from which adhesins were removed,

and purified adhesinsa

Agglutination ofHemagglutination Gal-Gal-coated Adherence of RBC

Prepn latex beads

Optimal Optimal OptimalActivity pH Activity pH Activity pH

FAC + 5 + 5 + 7-8Fimbriae - -Adhesins + 5 + 5 + 7-8

a Agglutination and adherence of FAC and adhesins were both inhibited bycaGal-(1,4)-IGal-(1,4)-Glc.

spectively, as opposed to agglutination by fimbriated bacte-ria at pH 7 to pH 8. Both, like fimbriated bacteria, adheredto cells at a pH range of 5 to 9. Adherence and agglutinationdata obtained with FACs, purified fimbriae, and adhesinsfrom F71, F8, and F13 are compared in Table 3.For adherence studies, immobilized FAC and purified

adhesin were used in a microassay. As shown in Fig. 3 withthe adhesin from F71, the adhesion of RBC to the purifiedadhesin was inhibited by aGal-(1,4)-IGal-(1,4)-IGlc, indi-cating P adhesin specificity.Monoclonal antibodies. With crude adhesin from the F71-

specific FAC of E. coli 21614 as the immunogen, monoclonalantibodies were produced by the hybridoma technique (14)as previously described (24). Antibodies specific for thefimbrillin, the adhesin, and the rough K-12 lipopolysaccha-ride were detected by the enzyme-linked immunosorbentassay. The properties of some antiadhesin and antifimbrillinantibodies are summarized in Table 4. Several antifimbrillinantibodies were specific for F71 fimbrillin, and others cross-reacted with F8-, F12-, F13-, or F14-specific fimbrillin.Antiadhesin antibody 6B10 reacted with the 35-kDa adhesinsfrom all the bacteria used. It was strongly antiadhesive,inhibiting hemagglutination as well as hemadhesion of hu-man RBC.

DISCUSSION

Despite the long-standing knowledge on the phenomenonof cell interaction, receptor specificity, and involvement ofadhesion in E. coli infections, the molecular basis for thisinteraction and the chemical nature of the adhesins remainedunknown until recently. Molecular analysis of the genecomplexes responsible for the adhesion of E. coli to type 1-,P-, and S-specific fimbriae showed that the properties offimbriation and adherence are directed from different chro-mosomal sites (8, 9, 19, 21, 23, 32, 33). These findings led tothe concept of a distinct adhesin molecule associated withthe fimbriae. The first fimbria-associated adhesin isolatedand characterized was the S-specific adhesin (24). We de-scribe here the P-specific adhesin of uropathogenic E. coli,isolated by a modified procedure.

In a first step the FAC was isolated and purified. The pureFAC could be dissociated efficiently by heating in thepresence of Zwittergent 3-16. Subsequent purification of theseparated adhesin and fimbriae was achieved by anion-exchange FPLC in the presence of Zwittergent 3-16 andurea.SDS-PAGE revealed that the adhesins from the F71-, F8-,

and F13-specific FACs had molecular masses of approxi-mately 35 kDa, whereas the molecular masses of the respec-tive fimbrillins were lower and differed with the various Fantigens (see also reference 10). The amino-terminal aminoacid sequences of the F71- and F13-derived adhesins weredifferent, that of the F13-derived adhesin being identical tothat translated by Lund and co-workers from the DNAsequence of the papG gene (22).

All three adhesins studied here exhibited Gal-Gal speci-ficity, as demonstrated by agglutination ofRBC and Gal-Gal-modified latex beads as well as inhibition of hemadhesion bythe trisaccharide otGal-(1,4)-,BGal-(1,4)-Glc. Whereas adher-ence occurred at a pH range of 5 to 9, agglutination withFAC or purified adhesin occurred at pH 5 and not at pH 7 topH 9. We assume that, like S adhesin-specific FAC (24), Padhesin-specific FAC also does not aggregate and appears to

V ~g8 ~g-7- ~8

0

0

0n- 0 -

. 0.0

0.3 0.08 0.02 0.005

Inhibitor (rmM)

FIG. 3. Inhibition of the adhesion of human RBC to immobilized F71-derived P adhesin by aGal-(1,4)-PGal-(1,4)-Glc (0). Raffinose (A)was used as a control. Details of this inhibition of hemadhesion are given in the text. OD405, Optical density at 405 nm.

0.2 -

0.1 -

5 1.25

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80 HOSCHUTZKY ET AL.

TABLE 4. Properties of monoclonal antibodies obtained by immunization with crude F71-specific adhesin

Monoclonal Immunoglobulin Inhibition of ELISAI reactivity with:antibody subclass hemagglutination F71 adhesin F71 fimbrillin F8 adhesin F8 fimbrillin F13 adhesin F13 fimbrillin

Antiadhesin6B10 Gl + + - + - + -5G10 M - + - - -

Antifimbrillin2A12 G3 - - + - - - -8B1 M - - + - + - -

10A9 Gl - - + - + - +

a ELISA, Enzyme-linked immunosorbent assay.

be functionally monovalent at a neutral pH. At a lower pH(and also in the presence of bivalent cations) FAC aggre-gates, thus becoming multivalent and mediating agglutina-tion. This effect does not influence adhesion, which occurs ata pH at which FAC is apparently monovalent.The fact that an antiadhesive monoclonal antibody which

acted with the adhesins despite differences in their primarystructures was obtained indicated that their receptor bindingsite is conserved. Recently, Lund et al. (20) reported that theF71- and F13-specific adhesins seem to be serologicallydistinct, as shown by the reactivities of respective mutants inabsorbed antisera. This result does not disagree with ourfindings but rather shows that the concentration of antibod-ies reacting with the adhesin receptor-binding domain mustbe very low in the absorbed sera used.

Genetic studies by Normark and co-workers (19-22) indi-cated the association of several minor proteins with thefimbrial subunit. The minor 16.5- and 15-kDa proteins whichwe observed in SDS-PAGE of purified FAC may be identicalto two of these proteins. They may serve to maintain thefunctional FAC. The 35-kDa protein(s) described in thispaper is the Gal-Gal (P)-specific adhesin of uropathogenic E.coli. Our data indicate that in such strains, the P adhesin isin fact a group of closely related but not identical recognitionproteins with a conserved receptor recognition site. TheF13-derived adhesin is the PapG protein postulated byNormark and co-workers (20, 22).

ACKNOWLEDGMENTS

We thank J. Hacker, Wurzburg, Federal Republic of Germany,for the recombinant F8 strain, D. Low for the recombinant F71strain, and S. Normark for the recombinant F13 (Pap) strain. Thetechnical assistance of Monika Held and Matthias Berg is gratefullyacknowledged.

This work was supported by the Deutsche Forschungsgemein-schaft and the Bundesministerium fur Forschung and Technologie.

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12. Klemm, P. 1982. Primary structure of the CFA1 fimbrial proteinfrom human enterotoxigenic Escherichia coli strains. Eur. J.Biochem. 124:339-348.

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20. Lund, B., F. Lindberg, and S. Normark. 1988. Structure andantigenic properties of the tip-located P pilus proteins of uro-pathogenic Escherichia coli. J. Bacteriol. 170:1887-1894.

21. Lund, B., F. P. Lindberg, M. Baga, and S. Normark. 1985.Globoside-specific adhesins of uropathogenic Escherichia coliare encoded by similar trans-complementable gene clusters. J.Bacteriol. 162:1293-1301.

22. Lund, B., G. Lindberg, B.-I. Marklund, and S. Normark. 1987.The papG protein is the a-D-galactopyranosyl-(1-4)-3-D-galac-topyranose-binding adhesin of uropathogenic Escherichia coli.Proc. Natl. Acad. Sci. USA 84:5898-5902.

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24. Moch, T., H. Hoschutzky, J. Hacker, K. D. Kroncke, and K.Jann. 1987. Isolation and characterization of the a-sialyl-3-2,3-galactosyl-specific adhesin from fimbriated Escherichia coli.Proc. Natl. Acad. Sci. USA 84:3462-3466.

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