P-Antigen-Recognizing Fimbriae from Human Uropathogenic Escherichia coli Strains

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1 INFECTION AND IMMUNITY, JUlY 1982, p /82/ $02.00/0 Vol. 37, No. 1 P-Antigen-Recognizing Fimbriae from Human Uropathogenic Escherichia coli Strains TIMO K. KORHONEN,l* VUOKKO VAISANEN, HARRI SAXEN,- HANS HULTBERG,3 AND STEFAN B. SVENSON4 Department of General Microbiology, University of Helsinki,1 and Central Public Health LaboratorN,2 SF Helsinki 28, Finland; Department of Organic Chemistry, Arrheniuis Laborcatory, University of Stockholm, S Stockholm,3 and Department of Bacteriology, National Bacteriological Laboratorv, S Stockholm,4 Sweden Received 4 January 1982/Accepted 1 March 1982 P-antigen-recognizing fimbriae (P fimbriae) from four pyelonephritogenic Escherichia coli strains and type 1 fimbriae from an E. coli strain and a Salmonella typhimurium strain were purified. The P fimbriae were morphologically similar to type 1 fimbriae. The purified P fimbriae agglutinated neuraminidase-treated human Pl and p2k erythrocytes but not p erythrocytes, which lack all P-blood group-specific glycosphingolipids. However, coating of neuraminidase-treated p erythrocytes with globoside rendered such erythrocytes agglutinable by the P fimbriae. The hemagglutinations were in all instances fully inhibited by the synthetic oa-d-galp-(1-4)-p-d-galp-1-0-me glycoside. The binding specificity of the P fimbriae could also be demonstrated by using fimbriae coated onto latex particles and nontreated erythrocytes. It was thus concluded that the P fimbriae recognize and bind to the ox-d-galp-(1-4)-r-d-galp carbohydrate sequence occurring in the series of P-blood group antigen-specific glycosphingolipids. In contrast to both type 1 fimbriae, all four P fimbriae preparations showed multiple bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antisera were raised in rabbits against the various E. coli fimbriae. In enzyme-linked immunosorbent assays each one of the antisera to the P fimbriae reacted to titers of log 4 to 7 with both the homologous and the heterologous P fimbriae, but not with the type 1 fimbriae of E. coli. In a reciprocal fashion, the antiserum to the type 1 fimbriae of one E. coli strain reacted only with the homologous type 1 but not with any of the P fimbriae preparations. The ability of bacteria to adhere to epithelial cells of the host has been shown to be a prerequisite for many bacterial infections (1, 11). In human urinary tract infections there is a high correlation between the ability of the infecting bacteria to adhere to human uroepithelium and their virulence (9, 31, 32). The adhesive capacity is likely to endow the bacteria with higher resistance to mechanical elimination by the flow of urine and thus aids their further ascent to the upper urinary tract and kidney. The receptors on human uroepithelial cells and erythrocytes to which pyelonephritogenic Escherichia coli bind have been shown to be glycosphingolipids related to the human P blood group system (12-14, 22, 33). Recently we showed that both trihexosylceramide (correlated to the pk antigen) and globoside (correlated to the P antigen) function as receptors for pyelonephritogenic E. coli by means of the x-d-galp-(l- 4)-4-D-Galp carbohydrate entity of these glycosphingolipids (12, 33). These receptor-active 286 glycosphingolipids occur also on human erythrocytes, and therefore most pyelonephritogenic E. coli strains agglutinate human erythrocytes. Such hemagglutinations are resistant to D-mannose but are readily inhibited by the synthetized cx-d-galp-(1-4)-1-d-galp-1-o-me glycoside (12, 13, 33). We have previously shown that fimbriae isolated from one pyelonephritogenic E. coli strain, causing D-mannose-resistant hemagglutination, bound to human urinary tract epithelial cells in vitro (17) and that adhesion of pyelonephritogenic E. coli to human uroepithelial cells could be inhibited by antifimbrial antibodies (18). This communication demonstrates that fimbriae purified from four different pyelonephritogenic E. coli isolates are able to recognize the cx-d-galp-(1-4)-i- D-Galp carbohydrate portion of P-blood group antigen-specific glycosphingolipids and that these fimbriae are serologically distinct from E. coli type 1 fimbriae. The term P fimbriae therefore appears to be suited to these fimbriae (15).

2 VOL. 37, 1982 MATERIALS AND METHODS Bacterial strains. The following E. coli strains were all urinary isolates from patients with acute pyelonephritis: ER2 (serotype 04:K12), KS71 (04:K12), IH11002 (01:K1), and 3048 (04:K3). The strains ER2 and KS71 were kindly provided by G. Kallenius, National Bacteriological Laboratory, Stockholm, Sweden, and the strains IHE11002 and 3048 were provided by J. Elo, Aurora Hospital, Helsinki, Finland, and C. Svanborg Eddn, Department of Clinical Immunology, University of Gothenburg, Sweden. The type 1-fimbriated E. coli strain 2131 was kindly donated by K. Jann, Max-Planck-Institut fur Immunbiologie, Freiburg, West Germany. S. typhimurium SH6749 was available from previous work (19). Purification of fimbriae. Fimbriae from E. coli strains ER2, KS71, IH11002, and 3048 were prepared from cultures grown at 37 C on colonization factor antigen agar (CFA-agar) (8) by using deoxycholate and concentrated urea (20). Type 1 fimbriae of E. coli 2131 and S. typhimurium SH6749 were prepared by the same technique but from stationary cultures in Luria broth (19, 20). Agglutination tests. Human erythrocytes of the phenotypes Pl, P2k, and p were kindly provided by Anna Pirkola, Finnish Red Cross Blood Transfusion Service, Helsinki, Finland, and Bertil Cedergren, Blood Bank, Umea, Sweden. The erythrocytes were washed with phosphate-buffered saline (ph 7.1), and samples (100,ul of a 20% [vol/vol] suspension) of the various erythrocytes were treated with 1 U of neuraminidase (Sigma Chemical Co., St. Louis, Mo.) for 3 h at 37 C. The erythrocytes were then washed three times with phosphate-buffered saline and resuspended to 2%. Neuraminidase-treated p erythrocytes and washed horse erythrocytes were coated with globoside (Supelco, Bellefonte, Pa.) as described by Leffer and Svanborg Eddn (22). Briefly, 50 p.l of a 20% suspension of washed erythrocytes was added to a 1-ml suspension of globoside (200,ug/ml), and the mixture was incubated for 3 h at 37 C with occasional shakings. After washings, the erythrocytes were suspended to 2% before agglutination tests. The coating of latex particles (Difco Laboratories, Detroit, Mich.) with purified fimbriae was carried out as described by Evans et al. (7). Hemagglutinations and yeast cell agglutinations were performed on glass slides as described previously (16). The erythrocyte suspensions were kept at 4 C, and the agglutinations were performed on crushed ice. The inhibitory effect of a-methyl-d-mannopyranoside (Sigma) was tested at the concentration of 2.5% (wtl vol), whereas the inhibitory effect of the synthetic methyl-cx-d-galactopyranosyl-(1-4)-,-d-galactopyranoside (Hultberg and Garegg, manuscript in preparation) was tested at the concentration of 0.5% (wt/vol). SDS-PAGE. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was performed in 1-mm-thick slab gels (gel concentration, 15%) by the system of Laemmli (21). In one experiment, a 15 to 25% gradient gel was employed. Peptide bands were visualized by staining with Coomassie brilliant blue R250. A low-molecular-weight electrophoresis calibration kit (Pharmacia, Uppsala, Sweden) was used as a standard. The type 1 fimbriae preparations were denaturated at low ph before the SDS treatment (25). P FIMBRIAE OF E. COLI 287 a b c d e f '... &-m-o %-o i FIG. 1. SDS-PAGE analysis of the purified P fimbriae. Each lane in lanes A to D contains 25 p.g of the fimbriae preparation; lanes E and F have 15 p.g of protein. (A) ER2 fimbriae, (B) KS71 fimbriae, (C) IH11002 fimbriae, and (D) 3048 fimbriae. (E) The type 1 fimbriae of E. coli 2131 and (F) S. typhimurium SH6749 (19) were run on the same gel and are shown for comparison. The positions of the standard proteins are indicated on the right. Protein determinations. Protein was estimated by the modified Lowry procedure as described in reference 24, using bovine serum albumin as a standard. Electron microscopy. Purified fimbriae were negatively stained with 2% (wt/vol) phosphotungstic acid in 0.1 M sodium phosphate buffer (ph 6.5). All micrographs were taken at the Department of Electron Microscopy, University of Helsinki, with a JEM-1OOB electron microscope operating at 80 kv. Immunological methods. Antisera against the various purified fimbrial preparations were produced in rabbits as described (20), and the estimation of antibody titers and serological cross-reactivity was done by enzyme-linked immunosorbent assays (ELISA) (6) in microtiter plates essentially as described by Buchanan (2). The antigen concentration used for coating was 5 jig/ml, and the fimbriae suspensions were briefly sonicated before coating. The antibody titers are given as the logarithm of the reciprocal of the highest serum dilution yielding an absorbance of 0.5 at 400 nm. RESULTS Hemagglutinating properties of pyelonephritogenic E. coli. When grown on CFA-agar plates, E. coli strains ER2, KS71, IH11002, and 3048 were abundantly fimbriated but lacked type 1 fimbriae as tested by yeast cell agglutination. The strains were considered to carry P fimbriae by the following criteria: (i) they agglutinated human Pl and p2k erythrocytes but not p erythrocytes, (ii) the hemagglutinations were not inhibited by a-methyl-d-mannopyranoside but were sensitive to the a-d-galp-(1-4)-13-d-galp-1-0-me glycoside, and (iii) the strains agglutinated horse erythrocytes after passive coating with globoside. Characterization of purified P fimbriae. All four P fimbriae preparations consisted of long filaments (up to 1 to 2 pum in length) with a

3 288 KORHONEN ET AL. diameter of 5 to 7 nm, i.e., their morphology was similar to that of type 1 fimbriae. No contaminating membrane vesicles or other nonfimbrial material was observed in the electron microscopic examinations (data not shown). The SDS-PAGE analysis of the various P fimbriae preparations is shown in Fig. 1. In contrast to the type 1 fimbriae preparations from E. coli 2131 and S. typhimuriium SH6749 (lanes e and f in Fig. 1), all four P fimbriae showed multiple bands in SDS-PAGE (lanes a to d in Fig. 1). The KS71 fimbriae preparation (lane b) exhibited three bands with apparent molecular weights of 17,300 (17.3K), 19.0K, and 22.0K. The ER2 fimbriae (lane a) had two bands with apparent molecular weights similar to the lower bands in the KS71 fimbriae preparation. The fimbriae from E. coli IH11002 (lane c) showed two bands with apparent molecular weights of 18.0K and 20.5K. The apparent molecular weights of the two bands of the strain 3048 fimbriae (lane d) were 17.0K and 18.4K. The SDS-PAGE shown in Fig. 1 was overloaded to visualize the purity of the preparations. An identical pattern of bands was observed also in a gradient SDS-PAGE (15 to 25%) and in all three to five different batches of each fimbriae preparation tested (data not shown). Hemagglutinating properties of purified P fimbriae. The different purified P fimbriae did not readily agglutinate human erythrocytes. To improve hemagglutination, the low-ionic-strength buffer of Sedlock et al. (30) was applied, but without success. However, after treatment of the erythrocytes with neuraminidase, the Pl and p2k erythrocytes became agglutinable by all four P fimbriae preparations. Figure 2 shows a typical hemagglutination of neuraminidase-treated erythrocytes by the IHE11002 fimbriae. All four P fimbriae preparations induced agglutination of Pl (Fig. 2A) and p2k erythrocytes at the concentration of 125 p.g/ml, and these hemagglutinations were in all instances fully inhibited by the synthetic ct-d-galp-(1-4)-4-d-galp-1-0-me glycoside (Fig. 2B), but not affected by co-methyl-dmannopyranoside. None of the fimbrial preparations agglutinated p erythrocytes before or after neuraminidase treatment (Fig. 2C). To ascertain the ability of the fimbriae to recognize the P-glycosphingolipid receptors, neuraminidase-treated p erythrocytes were passively coated with globoside. All four fimbriae preparations agglutinated the globoside-coated p erythrocytes (Fig. 2D), and again these hemagglutinations were totally inhibited by the Cx-D- Galp-(1-4)-p-D-Galp-1-0-Me glycoside (Fig. 2E). Hence we conclude that the purified P fimbriae indeed recognized the Ot-D-Galp-(1-4)- P-D-Galp carbohydrate entity of the P-blood group antigen-specific glycosphingolipids. INFECT. IMMUN. An alternative method to demonstrate the hemagglutinating activity of the purified P fimbriae was to coat them onto latex particles. Latex particles coated with each of the P fimbriae agglutinated human Pl but not p erythrocytes, and again these hemagglutinations were inhibited by the synthetic cx-d-galp-(1-4)-1-d- Galp-1-0-Me glycoside. Serological cross-reactivity of P fimbriae. The serological cross-reactivity of the four P fimbriae preparations with each other and type 1 fimbriae of E. coli 2131 was tested by ELISA (Table 1). The ELISA titrations revealed a low titer of antibodies directed to the E. coli 2131 type 1 fimbriae in the rabbit preimmune sera. However, the level of these antibodies was not enhanced in the antisera obtained after hyperimmunization with any of the four P fimbriae preparations, whereas their titer to the homologous P fimbriae preparation had risen from <2 to 4-7. In the hyperimmune serum to the type 1 fimbriae, the level of antibodies against any and each of the P fimbriae was just above the detection limit. Thus, we conclude that the P fimbriae are not serologically cross-reactive with the type 1 fimbriae of E. coli In contrast, a considerable, but variable, degree of cross-reactivity was observed between the four P fimbriae, suggestive of a large structural homology (Table 1). DISCUSSION Several reports have shown that the adherence of E. coli cells to human urinary tract epithelial cells is correlated with their virulence, and thus the ability to adhere can be considered as a virulence factor (9, 31, 32). The minimal structure recognized by the bacteria on uroepithelial cells and on human erythrocytes has recently been shown to be the ot-d-galp-(1-4)-3- D-Galp disaccharide entity (13-15), which comprises part of the carbohydrate moieties of the P blood group antigen determinants (23, 27). Both globoside [P-D-GalNAcp-(1-3)-cx-D-Galp-(1-4)-p- D-Galp-(1-4)-P-D-Glcp-Ce] and trihexosyl ceramide [ax-d-galp-(1-4)-13-d-galp-(1-4)-r-d-glcp- Ce] are recognized by pyelonephritogenic E. coli strains (12, 22, 33). On the basis of structural similarities it has also been suggested that the P1 glycosphingolipid [0t-D-Ga1p-(1-4)-P-D-Galp-(1-4)-p-D-GIcNAcp-(1-3)-p-D-Galp-(1-4)-p-D-Glcp- Ce] can function as receptor for these bacteria (13, 14). We have previously shown that the adhesion of E. coli to human uroepithelial cells is mediated by fimbriae (17, 18). This communication demonstrates that fimbriae purified from four pyelonephritogenic E. coli strains recognize P-blood group antigen-specific glycosphingolipids. These fimbriae are serologically distinct from the type 1 fimbriae of E. coli 2131 causing

4 VOL. 37, 1982 P FIMBRIAE OF E. COLI 289 b~~~~~~~~~ v L s ta~,"' (d 4l" O a, 0 _J_. W ;y~~~~~~~~~"i d-- *SW i}.., --+S I '- O I9 v- *.I I_ I, G -Lero C, -td, r r;;i 'E_;. t,,. e r%: 4.- FIG. 2. Agglutination of neuraminidase-treated human erythrocytes by the E. coli IH11002 fimbriae. (A) The fimbriae agglutinate human P5 erythrocytes, and (B) this agglutination is inhibited by cs-d-galp-(1-4)-,3-d-galp-1-0-me glycoside (0.5%, wt/vol). (C) Erythrocytes with the phenotype p are not agglutinated by the IH11002 fimbriae. (D) After coating of the p erythrocytes with globoside they become agglutinable by the fimbriae, and (E) again this agglutination is inhibited by the disaccharide derivative. The fimbriae concentration was 125,ug/ml. D-mannose-sensitive agglutinations, and we have termed them P fimbriae according to their binding properties. The purified P fimbriae were morphologically indistinguishable from E. coli (20) or S. typhimurium (19) type 1 fimbriae, i.e., they were filaments of 0.5 to 2 p.m in length and 5 to 7 nm in diameter. A striking difference from the type 1 fimbriae was the multiplicity of protein bands seen when the P fimbriae preparations were run in SDS-PAGE (Fig. 1). All P fimbriae yielded two bands in the SDS-PAGE; one of the preparations (KS71) even yielded three bands. A similar multiple SDS-PAGE pattern of purified fimbrial preparations has recently been reported from an E. coli strain causing diarrhea (3), as well as from other strains (10). We do not consider the various bands to be nonfimbrial contaminants since different batches of purified fimbriae gave identical SDS-PAGE patterns. TABLE 1. Cross-reactions between the purified E. coli fimbriae' Antiserum to fimbriae Antibody titer to fimbriae from E. coli strain: from E. coli strain: IH11002 ER2 KS IH ER KS Preimmune serum 2.9 <2 <2 <2 <2 a Enzyme-linked immunosorbent assay; the antibody titer is given as the logarithm of the reciprocal of the highest dilution of the antiserum giving an absorbance of 0.5 at 400 nm.

5 290 KORHONEN ET AL. Also in our hands several different type 1 fimbriae, purified by the same procedure, always give only one band in SDS-PAGE (19, 20; unpublished data) (Fig. 1). Neither the pyelonephritogenic bacteria nor the purified P fimbriae used in the present study expressed any detectable D- mannopyranosyl binding as tested by yeast cell agglutination, which is a very sensitive method for screening of the presence of type 1 fimbriae (16). Hence, we conclude that none of the bands found in SDS-PAGE of the P fimbriae preparations is due to contaminating type 1 fimbriae. This conclusion is further supported by the observation that the various P fimbriae preparations did not serologically cross-react with E. coli type 1 fimbriae and that immunization with P fimbriae did not enhance anti-type 1 fimbrial antibody levels (Table 1). On the other hand, type 1 fimbriae of different E. coli strains are thought to be cross-reactive (4, 18). One explanation for the multiple bands observed in SDS-PAGE is that these strains possess multiple types of fimbriae, of which some have P antigen-binding specificity, some may bind to receptor structures unknown at present, and some may even be nonbinding. It has been demonstrated that one E. coli strain and even one cell may produce several types of fimbriae (26, 33). At present we cannot say which of the various bands seen in the P fimbriae preparations are responsible for the recognition of and binding to the P receptors. Experiments designed to elucidate this question are under way. The purified P fimbriae differed from the type 1 fimbriae of both E. coli and S. typhimlurium (16, 19, 28) in that neuraminidase treatment of target erythrocytes or coating of the fimbriae onto latex particles was necessary to demonstrate their binding specificity (Fig. 2). Similar findings have been reported for the CFA/I fimbrial antigen, which is a poor agglutinator unless coated onto latex particles (7). It is likely that both the neuraminidase treatment of erythrocytes and the coating of fimbriae onto latex particles enhance agglutination by overcoming the repulsive forces caused by charge-charge interactions between target erythrocytes. Neuraminidase treatment has been used in an analogous fashion to enhance agglutination by lectins (29) and type 1 fimbriae (28). The four P fimbriae were serologically crossreactive with each other as measured by ELISA (Table 1). However, none of them cross-reacted with the E. coli 2131 type 1 fimbriae. The possible use of P fimbriae as vaccines to prevent urinary tract infections is currently being explored; the extent of cross-reactivity may be important in this context. Duguid et al. (5) have recently emphasized the heterogeneity of the D-mannose-resistant class INFECT. IMMUN. of fimbriae from E. coli; however, the binding properties of their strains were not described in detail. We too have found that a considerable number (about 6%) of pyelonephritogenic E. coli strains carry another non-p but D-mannose-resistant class of fimbriae which were provisionally termed X fimbriae (33; unpublished data). Hence, the finding of D-mannose-resistant binding properties of any E. coli strain should be specified in detail before any comparisons are made. ACKNOWLEDGMENTS We thank P. Helena Makela, Per Garegg, Gunilla Kallenius, and Ronald Mollby for helpful discussions. S.B.S. was supported by the Swedish Medical Research Council (grant no. 19X765). LITERATURE CITED 1. Beachey, E. H Bacterial adherence: adhesin-receptor interactions mediating the attachment of bacteria to mucosal surfaces. J. Infect. Dis. 143: Buchanan, T. M Antigen-specific serotyping of Neisseria gonorrhoeae. I. 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