Escherichia coli fimbriae recognizing sialyl galactosides

JOURNAL OF BACTERIOIOGY. Aug. 1984. p. 762-766 0021-9193/84/080762-05$02.00/0 Copyright © 1984, American Society for Microbiology

Vol. 159, No. 2

Escherichia coli Fimbriae Recognizing Sialyl Galactosides TIMO K. KORHONEN,l* VUOKKO VAISANEN-RHEN,' MIKAEL RHEN,' AULI PERE,' JAAKKO PARKKINEN,- AND JUKKA FINNEDepartments of General Microbiologv' and Medical Chemistry,2 Universitv of Helsinki, SF-00280 He/sinki 28, Fin/and Received 12 March 1984/Accepted

1

May 1984

Fimbriae recognizing sialyl galactosides (S fimbriae) were purified from an Escherichia coli strain. The S fimbriae were morphologically identical to type 1 and P fimbriae of E. coli and showed a hemagglutination that was abolished when erythrocytes were treated with neuraminidase. Hemagglutination by the purified fimbriae was inhibited by orosomucoid but not by its desialylated derivative. Of the oligosaccharides tested, sialyl-(cL2-3)lactose and sialyl-(o2-3)-N-acetyllactosamine had the strongest inhibitory activities. It was concluded that S fimbriae have the strongest affinity for (ot2-3)-linked sialyl galactosides. In the enzyme-linked immunosorbent assay, the hyperimmune serum to the S fimbriae reacted strongly with the homologous antigen but not with type 1, P, or nonhemagglutinating KS71C fimbriae of E. coli. Analogously, the hyperimmune sera to the other E. coli fimbriae did not react with the purified S fimbriae. The immunoprecipitation assay showed that S fimbriae on different E. coli serotypes shared immunological cross-reactivity.

Agglutination tests. Yeast cell agglutination and hemagglutination assay on glass slides were performed over crushed ice as described previously (12). Hemagglutination titrations and inhibition tests with purified IH3084 fimbriae were performed in microtiter plates as described previously (12). The microtiter plates were kept at 4°C for 2 h before the results were recorded. Human OP, and Op erythrocytes were kindly donated by Anna Pirkola, Finnish Red Cross Blood Transfusion Service, Helsinki, Finland. The erythrocytes were treated with Vibrio cholerae neuraminidase (Koch-Light Laboratories Ltd., Coinbrook Berks, England) as described by Gahmberg et al. (9). Hemagglutinations with whole bacterial cells were performed in the presence of 5% (wt/vol) o-methyl-D-mannoside (Sigma Chemical Co., St. Louis, Mo.) to measure only MR hemagglutination. Inhibitors. Colomic acid, N-acetyl-neuraminic acid, and lactose were purchased from Sigma. Orosomucoid was kindly provided by G. Myllyla (Finnish Red Cross Blood Transfusion Service, Helsinki). Desialylated orosomucoid was prepared as described earlier (24). N-Acetylneuraminyl-(co2-3)-galactose-(l1-4)-glucose, N-acetyl-neuraminyl-(o.2-6)-galactose-( p1-4)-glucose, N-acetyl-neuraminyl(o2 -3)- galactose - (f31 -4)- N- acetyl - glucosamine, N-acetylneuraminyl -(c2-6)- galactose -(r,1-4)- N-acetyl- glucosamine,

Fimbriae are filamentous surface proteins that serve as bacterial binding factors (2, 13, 29). Escherichia coli strains carry many types of fimbriae, which are characterized by their hemagglutination and serological properties, by the serotypes of the strains carrying them, and by the associated clinical condition (2, 8, 32). Although the genetic and structural properties of fimbrial types on E. coli are relatively well known (3, 8, 25, 26, 29), knowledge of fimbrial binding specificities is still rather limited. The binding specificity of type 1 fimbriae for mannosides was first demonstrated by Duguid and Old (2), and since then, it has become customary to divide fimbrial hemagglutinins into mannose-sensitive and mannose-resistant (MR) ones. However, it is evident that the MR fimbriae include those with many binding specificities, only some of which have been characterized on a molecular level (11, 19, 24, 30, 31, 33). We have previously described E. coli strains that recognize sialyl galactosides (24). In this communication, we show that this binding specificity is mediated by fimbriae and establish thus a novel fimbrial type on E. coli. On the basis of receptor specificity, we termed the fimbriae S fimbriae. MATERIALS AND METHODS Bacteria. The strain IH3084 (serotype RK1H47) of E. coli used in fimbrial purification was isolated from a neonatal meningitis patient. In addition, 16 E. coli strains were tested for precipitation with anti-IH3084-fimbriae serum; they were isolated from neonatal patients with sepsis or meningitis and were of serotypes 018acKlH7 (10 strains), RK1H33 (two strains), RK1H- (2 strains), 06K+ and 02K2 (1 strain of each). The bacteria were grown on colonization factor antigen (CFA) agar (6) as described previously (15). For yeast cell agglutination, IH3084 was grown in static Luria broth (12). Fimbriae. Fimbriae of the strain IH3084 were purified by using deoxycholate and concentrated urea (14). Type 1 fimbriae of E. coli 2131 and nonhemagglutinating KS71C fimbriae, as well as their antisera, were available from previous work (15, 25-27). P fimbriae of E. coli IH11024 and antiserum to it will be described in a separate report (A. Pere et al., manuscript in preparation). *

N-acetyl-neuraminyl-(ot2-8)-N-acetyl-neuraminyl-(o(23)-galactose-(31-4)-glucose were isolated from human urine

and

and bovine colostrum as described earlier (23, 24). All inhibitors were in 10 mM phosphate-buffered saline (sodium phosphate-0.15 M NaCl) adjusted to pH 7.1 SDS-PAGE. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was performed in slab gels (gel concentration, 15%; 1 mm thick) by the system of Laemmli (17). Peptide bands were visualized by staining them with Coomassie brilliant blue R250. A low-molecular-weight electrophoresis calibration kit (Pharmacia Fine Chemicals, Uppsala, Sweden) was used as a standard. Protein determination. Protein was estimated by a modified Lowry procedure (21), with bovine serum albumin as a standard. Electron microscopy. Bacterial cells and 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 Microsco-

Corresponding author. 762

VOL. 159, 1984

S FIMBRIAE OF E. COLI

py, University of Helsinki, with a Jem-lOOB electron microscope operating at 80 kV.

9467-

Immunological methods. Antiserum against the purified was produced in rabbits as described previously (14). Enzyme-linked im.munosorbent assay (4) was performed as described previously (15). Immunoprecipitation of fimbrial extracts (27) was performed as follows. Bacterial growth from 10 petri dishes was suspended in 5 ml of phosphate-buffered saline and treated for 2 min in an ice bath with an Ultra-Turrax homogenizator (Janke & Kunkel KG, Breisgau, Federal Republic of Germany). The cells were then removed by centrifugation (15 min, 7,780 x g, 4°C) in a Sorvall RC-5B centrifuge (DuPont Instruments, Newtown, Conn.), and sodium deoxycholate (Merck Co., Darmstadt, Federal Republic of Germany) was added to the supernatant to a concentration of 0.1% (wt/vol). The suspension was kept at 4°C overnight and centrifuged for 10 min (10,000 x g, 40C). The supernatants were diluted with sodium deoxycholate-phosphate-buffered saline to give a protein concentration of 200 ,ug/ml, and 25 ,ul of the antiIH3084 fimbriae serum was added to 1 ml of the diluted extracts in centrifuge tubes (Eppendorf, Hamburg, Federal Republic of Germany). The suspensions were kept at 37°C for 3 h and then overnight at 4°C. Immunoprecipitates were pelleted (7 min, 4C) in a Hemifuge (Heraeus Christ, Oesterboede, Federal Republic of Germany), washed three times with phosphate-buffered saline, and suspended in 50 1L of the SDS-PAGE solubilization mixture. In each case, 25 ,ul of the solution was analyzed by SDS-PAGE. fimbriae

RESULTS

Agglutination properties of IH3084. When grown on CFA agar plates, strain IH3084 was fimbriated but lacked type 1 fimbriae as tested by-yeast cell agglutination. Type 1 fimbriae were not formed in static broth either. Instead, the bacteria showed an MR hemagglutination of human erythrocytes. The strain was considered to carry S fimbriae because (i) hemagglutination was observed with both PI and p erythrocytes, indicating noninvolvement of the P fimbriae, and (ii) treating erythrocytes with neuraminidase abolished hemagglutination. Characterization of IH3084 fimbriae. Purified IH3084 fimbriae had morphology typical of E. coli fimbriae, i.e., they were 1- to 2-pm-long filaments with a diameter of 5 to 7 nm. No contaminating membrane vesicles or other nonfimbrial material was observed in electron microscopic examinations (data not shown). SDS-PAGE analysis of the fimbrial preparation revealed only one peptide band, with an apparent molecular weight of 17,000 (Fig. 1). Hemagglutination properties of purified IH3984 fimbriae. The agglutination titer of purified IH3984 fimbriae for untreated human 0 erythrocytes was 7.3 ,ug/ml. No hemagglutination was observed with neuraminidase-treated erythrocytes; the highest concentration tested was 117.0 ,ug of purified fimbriae per ml. These results indicate that fimbriae recognized a neuraminic acid-containing structure. For a more detailed characterization of the binding specificity, we tested different sialyl oligosaccharides for hemagglutination inhibition (Table 1). Sialyl-(a2-3)-lactose and sialyl-(a2-3)-N-acetyllactosamine inhibited the hemagglutination at a concentration of 0.3 mM, whereas corresponding (a2-6)-linked isomers were four times less effective. Disialyl lactose, which has an (a2-8)-linked residue bound to an (ax23)-linked sialic acid, was less inhibitory. Colomic acid, a polymer of a28-linked sialic acid, sialic acid, or lactose

763

43-

30-

20.114.4FIG. 1. SDS-PAGE analysis of purified IH3084 fimbriae (15 p.g). The positions of standard proteins (in kilodaltons) are indicated on the left.

had no effect on hemagglutination. Hemagglutination was also inhibited by orosomucoid but not by its desialylated derivative (Table 1). Serological properties of IH3084 fimbriae. Serological cross-reactions of IH3084 fimbriae with type 1 fimbriae of E. coli 2131, P fimbriae of E. coli IH11024, and nonhemagglutinating (pseudo-type 1) KS71C fimbriae were analyzed in an enzyme-linked immunosorbent assay (Table 2). Anti-IH3084 fimbriae hyperimmune serum reacted strongly with the homologous antigen but showed only weak or no reaction with the other fimbriae; the reaction with the 2131 fimbriae was 1.3% of the homologous reaction. Each of the heterologous cross-reactions of the purified IH3084 fimbriae was less than 0.4% of the corresponding homologous reaction. We therefore concluded that the IH3084 fimbriae were serologically distinct from the other fimabrial antigens used in this study. In other respects, the results in Table 2 confirmed our previous findings and showed no significant serological cross-reactivity between type 1, P, and KS71C fimbriae (15, 25). Immunoprecipitation of fimbrial extracts with anti-IH3084 fimbriae serum was used to determine whether S fimbriae on other E. coli strains cross-react with IH3084 fimbriae. Fimbrial extracts were obtained from bacterial cells by homogenization, sodium deoxycholate was added to TABLE 1. Inhibition of hemagglutination by fimbriae Inhibitor'

purified IH3084

NeuAc(a2-3)Gal1(1-4)Glc ........................... NeuAc(a2-3)Gal1(1-4)GlcNAc .......................

NeuAc(a2-6)Ga1(P1-4)G1c ........................... NeuAc(a2-6)GaI(P1-4)G1cNAc ....................... NeuAc(a2-8)NeuAc(a2-3)Gal(q1-4)Glc ......

.........

MIC (mM) 0.3 0.3 1.3 1.3 5.0

NeuAc ........................................... >10.0h Gal(p1-4)Glc ....................................... >10.0 Colomic acid ............ .......................... >10.0' Orosomucoid ...................................... 0.001' Desialylated orosomucoid ......... .................. None" aNeu, Neuraminic acid; Ac, N-acetyl; Gal, galactose; GIc, glucose; GIcN, glucosamine. b MIC required to prevent hemagglutination. 'No inhibition at 10 mM. d Calculated as sialic acid. Highest concentration tested corresponded to 128 times the concentration of native orosomucoid.

764

J. BACTERIOL.

KORHONEN ET AL.

TABLE 2. Cross-reactions between E. c(oli fimbriae Antiserum to the Antibody titer to E. co/i fimbriae' following E. coli fimbriae:

IH3084 IH11024 2131 KS71C Preimmune serum

IH11024

IH3084 4.9 2.9