INFECriON

AND

Vol. 60, No. 11

IMMUNITY, Nov. 1992, p. 4709-4719

0019-9567/92/114709-11$02.00/0 Copyright © 1992, American Society for Microbiology

Functional Heterogeneity of Type 1 Fimbriae of Escherichia coli EVGENI V. SOKURENKO,1 HARRY S. COURTNEY,2'3 SOMAN N. ABRAHAM,4 PER KLEMM,5 AND DAVID L. HASTYl,3* Departments of Anatomy and Neurobiology1 and of Medicine,2 University of Tennessee, and the Memphis VA Medical Center, 3 Memphis, Tennessee 38104; the Departments of Pathology and Molecular Microbiology, Washington University Medical Center, St. Louis, Missouri 631104; and the Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Copenhagen, Denmark5 Received 22 June 1992/Accepted 26 August 1992

Escherichia coli and other members of the family Enterobacteriaceae express surface fibrillar structures, fimbriae, that promote bacterial adhesion to host receptors. Type 1 fimbriae possess a lectinlike component, FimH, that is commonly thought to cause binding to mannose-containing oligosaccharides of host receptors. Since adhesion of type 1 fimbriated organisms are inhibited by mannose, the reactions are described as mannose sensitive (MS). We have studied the adhesion of the type 1 fimbriated CSH-50 strain of E. coli (which expresses onl type 1 fimbriae) to fibronectin (FN). E. coli CSH-50 does not bind detectable amounts of soluble FN but adheres well to immobilized plasma or cellular FN. This adhesion was inhibited by mannose-containing saccharides. By using purified domains of FN, it was found that E. coil CSH-50 adheres primarily to the amino-terminal and gelatin-binding domains, only one of which is glycosylated, in an MS fashion. Binding of the mannose-specific lectin concanavalin A to FN and ovalbumin was eliminated or reduced, respectively, by incubation with periodate or endoglycosidase. Adhesion of E. coli CSH-50 to ovalbumin was reduced by these treatments, but adhesion to FN was unaffected. E. coli CSH-50 also adheres to a synthetic peptide copying a portion of the amino-terminal FN domain (FNspl) in an MS fashion. Purified CSH-50 fimbriae bound to immobilized FN and FNspl in an MS fashion and inhibited adhesion of intact organisms. However, fimbriae purified from HB101(pPKL4), a recombinant strain harboring the entire type lfim gene locus and expressing functional type 1 fimbriae, neither bound to FN or FNspl nor inhibited E. coil adhesion to immobilized FN or FNspl. These novel findings suggest that there are two forms of type 1 MS fimbriae. One form exhibits only the well-known MS lectinlike activity that requires a substratum of mannose-containing glycoproteins. The other form exhibits not only the MS lectinlike activity but also binds to nonglycosylated regions of proteins in an MS manner. hybrid-type carbohydrate chains of glycoproteins (18, 46, 60). From such studies, it has also been proposed that the combining site on the fimbrial filament is in the form of an extended pocket, corresponding to the size of a trisaccharide, with an associated hydrophobic binding region (18, 60). Type 1 fimbriae from different genera within Enterobacteriaceae express MS lectinlike activities that differ in sensitivity to saccharides and in sensitivity to hydrophobic moieties (18). Although there is considerable antigenic crossreactivity among the FimH proteins of Enterobacteriaceae (5), structural differences exist between the FimH proteins from E. coli and Klebsiella pneumoniae (20, 35). It is possible that these primary structural differences could result in conformational effects that result in the different functional sensitivities to sugar. At least some part of the differences in activity may be dictated by the manner in which the lectinlike adhesin, thought to be FimH, associates with other ancillary proteins, FimF or FimG, and/or the primary structural component, FimA. Recombinant organisms expressing hybrid fimbriae composed of K. pneumoniae FimA and E. coli FimH exhibit the sugar sensitivity of the K. pneumoniae strain (30). There are other examples, however, where the lectin sensitivity of hybrid fimbriae reflects that of the strain from which the ancillary protein genes were obtained (47). A number of other classes of fimbrial adhesin have been described for Enterobacteriaceae, such as P (32), S (37), K99 (63), and others. Each of these classes exhibits lectinlike

Adhesion of microorganisms to host receptors is an important factor in the overall pathogenic process (8, 22). One of the classic examples of this phenomenon is the mannosesensitive (MS) adhesion of Escherichia coli to host cells that results from interaction of type 1 fimbriae with receptor molecules. Since the initial description of these fibrillar adhesins (14, 15, 51) and the characterization of the lectinlike sensitivity of their adhesive interactions with epithelial cells to inhibition by mannose (48, 49, 57), an extensive literature has developed regarding the genetics, structure, and function of type 1 fimbriae of E. coli and other members of the

family Enterobacteriaceae (12, 30, 34). Over the years, a generally accepted, albeit incomplete, view of the structure and function of these organelles has emerged. The greatest mass of the fimbrial filament is composed of polymers of FimA, a 17-kDa structural protein arranged in a right-handed helical array surrounding a hollow axillary core (11). Three ancillary proteins, FimF, FimG, and FimH, are also assembled as minor components of the filament. FimH is thought to be primarily responsible for the lectinlike adhesive properties of the fimbriae (3, 35, 36, 38, 43, 44, 54), but association with FimG appears to be necessary for activity (35). Studies with inhibitory saccharides have led investigators to propose that the receptors for the fimbrial adhesins are primarily oligomannoside-type and *

Corresponding author. 4709

4710

SOKURENKO ET AL.

functional activities almost exclusively in that adhesion is inhibited by certain saccharides. In this study, we have investigated the adhesion of a strain of type 1 fimbriated E. coli to human fibronectin (FN), a glycoprotein thought to be involved in the adhesion of a number of different species of microorganisms to host surfaces (26, 27). The data suggest that there are two forms of type 1 MS fimbriae. One form appears to be MS and to exhibit only the well-known lectinlike activity that requires a substratum of mannosecontaining glycoproteins. The other form exhibits the MS lectinlike activity and another activity in which the fimbriae are able to bind to nonglycosylated segments of proteins. Interestingly, the protein-binding activity of this form of type 1 fimbriae is also MS.

MATERIALS AND METHODS Reagents. Concanavalin A (ConA)-peroxidase was purchased from Sigma Chemical Co., St. Louis, Mo. All saccharides used in the inhibition assays were purchased from Sigma, except for D-galactose, D-mannose, and N-acetyl-Dglucosamine, which were purchased from Fluka AG, Buchs, Switzerland. FN. Human plasma FN was purified by affinity chromatography as described previously (6, 17). FN was stored at -80°C in 0.1 M cycloaminopropanesulfonic acid buffer-0.15 M NaCI-1 mM CaCl2 (pH 11.0). Prior to use, the samples were thawed and dialyzed against the appropriate buffer as indicated in the text. Labeling of FN with 25I was performed by using an iodination kit according to the manufacturer's recommendations (New England Nuclear, Boston, Mass.). The specific activity of the radiolabeled FN was 146,000 cpm/,ug. Heat treatment of FN was performed for 5 min at 96°C at a concentration of 10 ,ug/ml in 0.02 M NaHCO3 buffer. Cellular FN (human fibroblast FN) was purchased from Fibrogenex, Evanston, Ill. Plasma FN fragments were generated by digestion with thermolysin and purified over a hydroxylapatite column by the methods outlined by Zardi et al. (76). The synthetic peptide FNspl copies the first 30 amino acid residues of the FN molecule and was synthesized in the Protein Chemistry Laboratory of the VA Medical Center, Memphis, Tenn. To degrade or remove oligosaccharides, the following procedures were used. FN- or ovalbumin-coated microtiter wells were treated with 20 mM NaIO4 in phosphate-buffered saline (PBS) for 15 h at 4°C in the dark, washed three times with 0.1 M Tris-maleate buffer (pH 7.4), treated with 5 mM NaBH4 in the same buffer for 30 min at 37°C, washed three times with PBS, and quenched with PBS-bovine serum albumin (BSA). Alternatively, the substrates were treated with an endoglycosidase. A 100-,ul aliquot of FN or ovalbumin (0.5 mg/ml in PBS) was heated to 100°C for 5 min, and 100 ,ul of PBS containing 20 mM EDTA, 20 mM ,-mercaptoethanol, and 0.2% NaN3 was added. One hundred microliters of this mixture was used as a control and incubated with no additions, while the other 100 ,ul was incubated overnight at 37°C with 2.5 RI of endoglycosidase F (0.6 U). The treated proteins were diluted to 10 ,ug/ml with 0.1 M sodium bicarbonate buffer (pH 8.5) and used to coat microtiter wells. Bacterial strains. E. coli CSH-50, which was used in most of the experiments, is a Cold Spring Harbor K-12-derived strain that expresses only type 1 fimbriae and has been utilized in several previous studies from this laboratory (1, 4). A nonfimbriated strain of E. coli, ORN 103 (43) (originally obtained from Paul Orndorff), and its recombinant deriva-

INFECT. IMMUN.

tives which express intact type 1 fimbriae [i.e., E. coli ORN 103(pSH2) (29) (originally obtained from Sheila Hull)] or Fim A+H- type 1 fimbriae [i.e., E. coli ORN 103(pUT2002)] have been described previously (3, 5). HB101(pPKL4), harboring the fim gene cluster from E. coli K-12 strain PC-31, expresses intact type 1 fimbriae as described previously (35). It should be noted that HB101 strains from different laboratories are not identical. There is also evidence that some, possibly all, of these contain in their chromosome many if not all of the genes required to make type 1 fimbriae (10, 16). The HB101 strain we utilized under the conditions of growth employed during these studies did not express fimbriae. A wild-type K-12 strain, MG1655, was kindly provided by I. C. Blomfield. Clinical isolates of E. coli were obtained from the clinical microbiology laboratory of the Memphis VA Medical Center. The Staphylococcus aureus Cowan 1 strain used was obtained from the collection of the State Tarasevich Institute of Standardization and Control of Biomedical Products (Moscow, Russia). All strains were stored in small aliquots in brain heart infusion (BHI) broth at -80°C and grown in BHI broth for 18 h at 37°C before being used in experiments. Mutant strains of E. coli were grown in the presence of chloramphenicol (25 ug/rml) in BHI broth. Bacteria were harvested by centrifugation at 5,000 x g for 5 min, washed in 0.1 M sodium phosphate buffer-0.15 M NaCl (pH 7.0) (PBS), and adjusted to an appropriate optical density at 530 nm (OD530) before use. Antisera. Anti-E. coli antiserum was obtained by immunization of New Zealand White rabbits with E. coli CSH-50. Heat-killed bacteria were injected intravenously every 2 weeks, and sera were collected on a weekly basis. These antisera did not cross-react with any of the substrates used in these experiments and gave a strong reaction with all of the strains of E. coli used here. Production of rabbit anti-human FN and anti-type 1 fimbriae monoclonal antibodies have been reported previously (4, 62). Peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (and other conjugated anti-IgGs cited below) was purchased from Cappel (Organon Teknika Corp., Durham, N.C.) Goat anti-human FN and anti-rabbit IgG were purchased from Sigma. Binding of radiolabeled FN to bacteria. E. coli CSH-50 and S. aureus Cowan 1 were immobilized on radioimmunoassay microtiter plates (Nunc, Inc., Roskilde, Denmark) by incubating 100 ,ul of bacteria in PBS (OD530, 2.0) for 30 min at 37°C. Nonattached organisms were removed by four washes with PBS. Any remaining sites on the plastic that might bind protein were blocked by incubation with 0.1% BSA in PBS for 1 h at 37°C. The wells were then washed with PBS, and twofold dilutions of 125I-labeled FN (100 pA in 1% BSA-PBS) from a maximum concentration of 6 p,g/ml were added to wells coated with bacteria or with BSA only. After incubation at 37°C for 45 min and four washes with PBS, individual wells were removed and radioactivity was measured by using a Packard gamma spectrometer. Detection of bacterial adhesion and fimbriae binding to immobilized FN by ELISA. Flat-bottom polystyrene microtiter plates (Nunc) were coated by incubating wells with different concentrations of plasma or cellular FN in 0.02 M NaHCO3 buffer (100 pul per well) for 1 h at 37°C. The FN-coated wells were washed two times with the same buffer, and unreacted protein-binding sites were quenched with 0.1% BSA in NaHCO3 (125 ,ul per well). Alternatively, microtiter plates were coated with FN bound to gelatin by serially incubating wells first with gelatin (50 p,g/ml in bicarbonate buffer), then with BSA (1% in PBS), and, finally, with plasma FN (20 ,ug/ml in 0.1% BSA-PBS), each for 1 h at

HETEROGENEITY OF TYPE 1 FIMBRIAE

VOL. 60, 1992

37°C followed by three washes in PBS. In all experiments where FN was used as a substratum, the relative amounts of immobilized FN were determined by rabbit anti-FN antibody in a direct enzyme-linked immunosorbent assay (ELISA) to ensure accuracy of assays comparing binding of E. coli, S. aureus, or ConA-peroxidase. After the wells were washed two times with PBS, 100-,ul bacterial suspensions were added in 0.1% BSA-PBS at the concentrations indicated in the text. After incubation at 37°C for the indicated times, wells were washed three times with PBS and adherent bacteria were detected by using anti-E. coli serum (diluted 1:1,000 in 0.5% BSA-PBS). The use of anti-E. coli antibody to detect the presence of S. aureus Cowan 1 was based on the ability of staphylococcal protein A to bind rabbit IgG with high affinity. After incubation for 30 min at 37°C, wells were washed three times with PBS, peroxidase-conjugated goat anti-rabbit IgG (diluted 1:1,000 in 0.5% BSA-PBS) was added, and the wells were incubated for an additional 30 min at 37°C. ConA-peroxidase was incubated in wells similarly, except that the intermediate incubations in second antibody were, of course, obviated. The plates were then washed four times with PBS, the peroxidase substrate 5-aminosalicylic acid was added, and reaction product was measured at 405 nm after 30 min by using an automatic microplate reader (Molecular Devices, Inc., Menlo Park, Calif.). The background reaction product obtained by incubating substrate in BSA-coated wells was subtracted, and values in some experiments are reported as a percentage of maximal binding. Binding of fimbriae to FN was detected by a similar assay using (i) rabbit anti-E. coli serum, (ii) rabbit anti-type 1 fimbria serum, or (iii) mouse anti-type 1 fimbria monoclonal antibodies (detected with peroxidase-conjugated goat antimouse IgG), each giving comparable results. Purification of fimbriae. Fimbriae were prepared from CSH-50 and from HB101(pPKL4) by the method of Dodd and Eisenstein (13) as described in a previous publication from this laboratory (4). The purity of these preparations was determined by electron microscopic analysis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of fimbriae dissociated by heating the fimbriae in 0.1 N HCl for 5 min and neutralizing the samples with NaOH. Contaminants were not detected nor were differences between the two fimbrial preparations found by using either of these techniques. Inhibition experiments. For testing the effects of various compounds on adhesion of bacteria to FN, each of the inhibitors was prepared in 0.1% BSA-PBS at twofold more than the final concentration and mixed with a suspension of bacteria in the same buffer at an OD530 of 1.0 just prior to being added to wells of the microtiter plate. RESULTS Reaction of the CSH-50 strain of E. coli with intact FN. Soluble "NI-labeled FN bound in a dose-related manner to the Cowan 1 strain of S. aureus used as a control for some of the experiments reported below (Fig. 1). In contrast, the E. coli CSH-50 strain did not bind soluble FN in amounts detectably greater than background amounts (Fig. 1). In contrast to its lack of reactivity with soluble FN, the CSH-50 strain of E. coli adheres well to immobilized FN. Increasing numbers of E. coli (Fig. 2) are bound when increasing amounts of FN are adsorbed to the polystyrene wells. Adhesion reached a plateau at a coating concentration of 7.5 ,g of unlabeled FN per ml, approximately where concentrations of FN saturating the plastic are reached.

4711

12000

,

10000

1

0

2

3 4 Fn added

5

6

7

(jig/ml) FIG. 1. Binding of soluble "2I-labeled FN to immobilized E. coli CSH-50 (A) or S. aureus Cowan 1 (0). Bacteria were bound to microtiter wells as described in Materials and Methods. Indicated amounts of FN were incubated with bacteria, and then wells were washed and bound radioactivity was counted. Means ± standard deviations are indicated.

Adhesion of E. coli reached saturation by 25 min of incubation. Incubating increasing numbers of bacteria with a single concentration of adsorbed FN also resulted in increased adhesion. Adhesion was maximal at pH values between 6.0 and 8.0. The effect of ionic strength was tested by varying NaCl concentrations. Adhesion was maximal at an NaCl concentration of 0.15 M and fell by more than 50% at concentrations below 0.07 M or above 0.3 M. Adhesion to FN almost doubled when bacteria were passed several times in static broth culture, which is known to increase expression of type 1 fimbriae (14, 52).

0.8

0.6-

§,0.4-

0.00

0

5

10

,,, 15 20

,., , 25 30 35 40

FN (jIg/ml) FIG. 2. Attachment of E. coli CSH-50 to increasing concentrations of immobilized unlabeled plasma FN. Indicated amounts of FN were added to microtiter wells. Saturation of binding of radiolabeled FN to assay wells occurred at approximately 5 to 10 FLg/ml. Bacteria were diluted in buffer to an OD530 of 0.5 for incubation in wells. Means ± standard deviations are indicated.

4712

INFECT. IMMUN.

SOKURENKO ET AL.

TABLE 1. Comparison of E. coli CSH-50, S. aureus Cowan 1, and ConA-peroxidase binding to different substrata Substrate

% of control binding" E. col CSH-50 S. aureus Cowan 1

Native plasma FN Cellular FN Gelatin Gelatin + plasma FN

100 126 ± 2 11 ± 2 41 ± 10

100 76 ± 6 60 ± 4 150 ± 4

Treated FN Glycosidase Periodate Heated

125 ± 30 88 ± 3 85 ± 21

ND ND 7±2

Ovalbumin Native Glycosidase Periodate

TABLE 2. Effects of various substances on attachment of E. coli or S. aureus to FN"

100 56 ± 4 21 ± 2

ND ND ND

ConA

100 106 ± 7 9±2 41 ± 2

16 ± 13 6±2 96 ± 23 100 50 ± 10 18 ± 5

a Mean percent is given plus or minus variation in percent binding to provide an indication of agreement in assays. One hundred percent binding equals the OD450 of binding of bacteria to native plasma FN or to native ovalbumin.

When the effectiveness of immobilized plasma FN was compared with that of immobilized cellular FN, adhesion of E. coli to cell surface FN was slightly greater than that to plasma FN (Table 1). By contrast, adhesion of S. aureus to cell surface FN was slightly reduced in comparison with that to plasma FN. Interestingly, E. coli did not attach very well to gelatin inimobilized on plastic (Table 1). While attachment increased when FN was added to the gelatin substrate (Table 1), attachment of E. coli to this substrate never reached levels of bacterial adhesion to FN immobilized directly on plastic. Heat treatment of FN reduced its ability to support the attachment of S. aureus dramatically but had little effect on adhesion of E. coli (Table 1). A variety of compounds were tested for their ability to inhibit the adhesion of 'E. coli or S. aureus to FN-coated micrdtiter wells. Soluble EN, up to a concentration of 150 ,ug/ml (10-fold excess over the ~oating concentration), did not inhibit the adhesion of either E. coli CSH-50 or S. aureus Cowan 1 to imnmobilized FN (Table 2). While anti-FN antiserum inhibited E. coli adhesion almost completely, a control antibody had essentially no effect. Of a large number of saccharides, amino acids, and other substances tested, only mannose and fructose inhibited adhesion, both of which are specific inhibitors of type 1 fimbriae (14, 18, 49, 75). The relative abilities of several a-glycosides of mannose to inhibit agglutination of yeast cells by type 1 fimbriated E. coli have been reported previously by Firon et al. (18). We determined the relative molar ratios of the concentration of D-mannose and three ax-mannosides necessary to achieve 50% inhibition of adhesion of CSH-50 to FN and yeast mannan (Table 3). The relative activities of the saccharides in inhibiting adhesion of CSH-50 to mannan are similar to those reported by Firon et al. for inhibition of aggregation of yeast cells by another type 1 fimbriated strain of E. coli. While the inhibition of adhesion to FN by these saccharides showed a similar trend, aromatic a-mannosides were considerably more potent inhibitors than D-mannose. Organisms incubated in the presence of mannosides and washed behaved similarly to control, untreated organisms, indicating that mannose inhibition was not due to elution of a fimbriabound component by the saccharide.

Binding to FN (% of control) E. coli CSH-50 S. aureus Cowan 1

Substance

Soluble FN at concn (,ug/ml) of: 2 5 10 20 40 75 150

80 ± 12 77 ± 17 76 ± 11 88 ± 10 90 ± 11 84 ± 21 107 ± 8

ND 110 ± 3 99 ± 2 93 ± 10 105 ± 12 ND

Anti-FN serum Anti-IgG serum

1±4 105 ± 4

ND ND

NDb

Sugars (1% concn) Galactose Glucose Mannose Fructose

90 ± 26 83 ± 7 9±2 16 ± 4 a The following substances had essentially no effect on E.

95 ± 26 88 ± 6 89 ± 14 70 ± 17

coli adhesion: Tween 20 (0.1%),p-nitrophenol (5 mM); heparin (1.5 mg/ml); amino acids at 0.1 M (alanine, arginine, aspartic acid, glycine, glutamine, isoleucine, leucine, methionine, histidine, proline, serine, and threonine); other saccharides at 1% (D-arabinose, L-arabinose, fucose, glucuronic acid, N-acetylglucosamine, lactose, maltose, ribose, xylose, and a-methyl glucoside). Concentrations given for substances are final. b ND, not done.

Reaction of the CSH-50 strain ofE. coli with FN fragments, treated FN, and synthetic peptides. All of the results above suggested that the adhesion of E. coli CSH-50 to immobilized FN was probably due to a typical type 1 fimbriamediated interaction of the bacteria with the mannosecontaining oligosaccharide moieties of FN. Therefore, it was expected that strain CSH-50 would be found to adhere to the gelatin-binding domain of FN or another of the glycosylated domains of FN (Fig. 3) (for a review of structures, see reference 55). We studied CSH-50 adhesion to purified, therinolysin-generated fragments, which together span essentially the entire length of the FN molecule (Fig. 4). The bacteria adhered as well to the glycosylated 40-kDa gelatinbinding fragment as they did to intact FN but did not adhere very well to the glycosylated 139- to 148-kDa 4+5 domains. TABLE 3. Relative inhibitory activity of mannosylated derivatives on E. coli CSH-50 adhesion to immobilized FN and mannan and on E. coli aggregation of yeast cells Inhibitor

Relative activity" againstto:adhesion FN

D-Mannose

Methyl ca-mannoside Phenyl a-mannoside

4-Methylumbelliferyl

1 30

1,000 5,500

Relative activity against

yeast cell

aggregationb

Mannan

1 2.3 77

1,400

2.3 92

1,380

a-mannoside I Serial dilutions of saccharides were prepared in BSA-PBS, and the molar

concentrations required to cause 50% inhibition were determined. Relative activities were then calculated, setting the concentration of D-mannose at 1. Thus, as an example, methyl a-mannoside is 30fold more active than D-mannose because the molar concentration required for 50% inhibition is 30-fold less. b Adapted from Firon et al. (18).

HETEROGENEITY OF TYPE 1 FIMBRIAE

VOL. 60, 1992

4713

A.

(1)

1 HIep-l/Fib-1

NTjb

2

3

Gelatin/collagen

Heparin /DNA

5

Cell

Hep-2

Fib-2 a

COOl.I

EEC

E

6

4 + 5A/ SB

2 + 3

6

20 kDa }l_2928

1 10 k)a

m~a4

N

(3)

4

B. 13 k 1 lOkD'-_

54kD -_ 4imm

AOkDD--

28 kDm-

a-

2OkD'-

_s

1

2

3

4

5

6

7

FIG. 3. FN fragments tested for ability to support adhesion of E. coli CSH-50. (A) Model of FN showing domains and common interactions (part 1), regions of type 1, 2, and 3 homology and sites of N-linked glycosylation (part 2), and fragments used in this study (part 3). (B) SDS-PAGE of purified fragments. Lanes: 1, complete thermolysin digest; 2, domain 2; 3, domain 6; 4, domain 2+3; 5, domain 4+5a; 6, 4+5p; 7, domain 1. Coomassie blue-stained gel.

Interestingly, the bacteria also bound to the 28-kDa aminoterminal fragment, which is known not to be glycosylated (55), in somewhat greater numbers than they did to intact FN. Strain CSH-50 also bound to the 20-kDa carboxylterminal fragment, which is also known not to be glycosy-

lated (55). All of the bacterium-FN peptide interactions were inhibited 80% or more by D-mannose. The adhesion of E. coli CSH-50 to FN was then compared with adhesion to another glycoprotein, ovalbumin, which is known to possess oligomannose-type oligosaccharide moi-

4714

SOKURENKO ET AL.

INFECT. IMMUN.

.8 I,q)

100

*1- 00

.6

80-

E

.4

'~60

.2

40

0

20 EN

EN 1

EN 2+3

EN 4+5

FN 6

FIG. 4. Adhesion of E. coli CSH-50 to intact FN and to FN fragments immobilized on plastic. Microtiter wells were coated with proteins at a concentration of 50 p,g/ml to ensure saturation. E. coli cells were added to wells at an OD530 of 0.5. Symbols: _, controls; X, in the presence of D-mannose. Other conditions were as specified in Materials and Methods. Means ± standard deviations are indicated.

eties capable of inhibiting the activity of type 1 fimbriated organisms (46). Adhesion to untreated substrates as well as to substrates treated with periodate or with endoglycosidase F to degrade or remove the oligosaccharide chains was determined. Mannose removal was monitored by using ConA. Strain CSH-50 adhered to periodate- or glycosidasetreated FN as well as or better than to untreated FN, even though ConA reactivity was virtually eliminated (Table 1). Adhesion of E. coli CSH-50 to ovalbumin, however, was diminished by periodate or glycosidase treatment to essentially the same degree as ConA reactivity was diminished. E. coli CSH-50 fimbriae, therefore, exhibit both lectinlike and protein-adhesive activities, depending upon the substrate used. To ensure the absence of oligosaccharide moieties from the protein substratum, we studied adhesion of CSH-50 to synthetic peptides copying portions of the amino-terminal region of FN or other unrelated molecules. We found that the bacterium also adhered to certain of these synthetic peptides (Fig. 5) and that even this adhesion was inhibited by mannose. Comparison of CSH-50 and HB101(pPKL4) adhesion. To determine if MS adhesion to protein or peptide devoid of sugar moieties is shared by other type 1 fimbriated E. coli isolates, the adhesion of CSH-50 was compared with that of other clinical isolates of E. coli that express MS hemagglutination and yeast cell agglutination and with recombinant strains which contain the entire 9-kb fim gene cluster and express fully functional type 1 fimbriae. While CSH-50 adhesion to FN is unaffected by periodate, adhesion of HB101(pPKLA) (35) is dramatically inhibited (Fig. 6). Fimbriae purified from CSH-50, but not those from HB101(pPKILA), bind to intact FN and to FNspl in an MS manner (Fig. 7). Purified CSH-50 fimbriae, but not HB101(pPKILA) fimbriae, inhibit the adhesion of CSH-50 bacteria to FN and to FNspl (Fig. 8). Another recombinant organism expressing intact fimbriae [ORN103(pSH2)] as well as a reference wild-type K-12 strain (MG1655) also exhibited only lectinlike activity (i.e., did not bind to periodate-treated FN or to FNspl). When 12 clinical isolates of MS E. coli

FN spl

sM5(117-146)

FIG. 5. Adhesion of E. coli CSH-50 to immobilized synthetic peptides. Microtiter wells were coated with synthetic peptides at a concentration of 100 p.g/ml. E. coli cells were added to wells at an OD530 of 0.5. Symbols: _, controls; EN, in the presence of D-mannose. Other conditions were as specified in Materials and Methods. Means + standard deviations are indicated.

were tested for lectinlike versus protein-adhesive activities, 3 exhibited the protein-adhesive phenotype and adhered to FN, periodate-treated FN, and FNspl in an MS fashion, suggesting that the two adhesive phenotypes are not restricted to laboratory strains.

DISCUSSION The fraction of gram-negative bacteria that reacts with FN is small relative to that of gram-positive bacteria (26). The gram-negative strains used in our own previous studies were among those that appeared not to interact with FN. Soluble FN failed to bind to the strains of K. pneumoniae and

100 80 0

60 40

20

ovalbumin FN periodate-FN FN spl FIG. 6. Effect of periodate treatment on adhesion of HB101(pPKLA) to immobilized FN. Microtiter wells were coated with ovalbumin (10 ,g/ml), FN, and periodate-treated FN (50 pg/ml) or FNspl (100 p,g/ml). E. coli cells were added to wells at an OD530 of 0.5. Symbols: _, controls; 03, in the presence of D-mannose. Other conditions were as specified in Materials and Methods. Mean percentages of maximal adhesion ± variations in percentage of maximal adhesion are indicated to show agreement between wells.

HETEROGENEITY OF TYPE 1 FIMBRIAE

VOL. 60, 1992

.6 Q

.4

.2

FN

FN

FN

FN

spl

spi

FIG. 7. Binding of purified fimbriae to immobilized FN and FNspl. Microtiter wells were coated with FN at a concentration of 50 ,ug/ml or synthetic peptides at a concentration of 100 pg/ml. Fimbrial binding was detected by using antibodies to type 1 fimbriae as described in Materials and Methods. Symbols: _, controls; in the presence of 13 ,ug of purified fimbriae per ml. Other conditions were as specified in Materials and Methods. Means + standard deviations are indicated. -,

Pseudomonas aeruginosa that were tested (62). While Streptococcus pyogenes bound preferentially to buccal epithelial cells exhibiting surface EN, E. coli bound preferentially to cells devoid of surface FN (2). We also found that exogenous FN could bind to buccal epithelial cells (61) and to tissue culture cells (66) and inhibit the adhesion of several strains of E. coli. It was logical to assume, then, that these adhesive organelles of E. coli would not interact with FN. This apparent lack of interaction with FN seemed logical. Studies with the most active oligosaccharide inhibitors of type 1 fimbriated E. coli adhesion suggest that nonsubstituted oa-(13)-linked terminal mannose residues are critical components

FN substrate

FNspl subgtrte

100

~so

~60 ~40 20JJ

+

+

+

+

FIG. 8. Inhibition of adhesion of E. coli CSH-50 to immobilized FN by purified fimbriae. Microtiter wells were coated with FN (50 pg/ml) or FNspl (100 zg/ml) and incubated with E. coli CSH-50 (OD530, 0.5) in the presence or absence of 130 p,g of CSH-50 fimbriae or HB101(pPKL4) fimbriae per ml. Other conditions were as specified in Materials and Methods. Means standard deviations are indicated.

4715

of receptors (18, 46). However, most analyses of FN oligosaccharides show N-acetylglucosamine, galactose, and sometimes N-acetyl neuraminic acid linked distally to such a mannosylated core (55). Nevertheless, the growing examples of E. coli interacting with FN (19, 53, 69-73) required reexamination of the interaction of a well-defined type 1 fimbriated strain of MS E. coli with FN. It was found that E. coli CSH-50 does not bind soluble FN but does adhere avidly to immobilized FN. This interaction is mediated by fimbriae and is MS but appears not to be due to adhesion to oligosaccharide moieties of FN but to adhesion to protein. Perhaps the last of these findings is the most intriguing in the present study because it extends earlier observations (25) suggesting a nonlectin adhesive phenotype for type 1 fimbriae. The observation that certain type 1 fimbriated organisms (CSH-50 and 3 of 13 clinical isolates) adhere in an MS manner to protein structures that have few, if any, oligosaccharide moieties was, nevertheless, surprising. It has become common practice to ascribe all MS adhesive interactions of E. coli to the interaction of type 1 fimbriae with mannosylated host receptors. While we believe that this is true much of the time, it appears not to be true all of the time and the data reported here suggest that studies of type 1 fimbria-mediated events should not rely solely on mannose sensitivity and should take into consideration the possibility of this additional functional activity. That fimbrial components have binding sites for protein should not be too surprising, however, since a fimbrial filament is a polymeric structure formed through proteinprotein interactions. Even if one does not take into account the likely interaction of fimbrial components with chaperone molecules (30), FimA molecules must have binding sites for other FimA molecules and for FimH molecules. FimH must, likewise, have binding sites for other FimH molecules and/or FimA molecules. It is perhaps one of these sites on FimA or FimH that is involved in adhesion of E. coli CSH-50 and other clinical isolates to protein structures. The concept that lectins have binding sites that interact with noncarbohydrate ligands is not new (8). Several years ago, 10 or more lectins were known or presumed to be bifunctional in that they exhibited both specific carbohydrate- and protein-binding sites. In one case, the binding of a lectin to a nonglycosylated protein was inhibited by a sugar specific for the carbohydrate-binding site of the lectin. Further, the possible role of protein-adhesive properties of FimH (PilE) was suggested previously in a study of lesions of the fimH (pilE) gene that affected pellicle formation (25). The results presented by these authors suggested that pilE gene products in fimbriae on adjacent cells interact in the formation of the pellicle. Evidently, the phenomenon described by Harris et al. (25) is different from that described here, since the property of pellicle formation is shared by most type 1 fimbriated strains and the ability to adhere to FN, periodatetreated FN, and synthetic peptide is shared by a limited fraction of the isolates we have surveyed thus far. The adhesion of E. coli CSH-50 to FN, as compared with that to other glycoproteins such as ovalbumin, appears to be predominantly due to interaction with protein portions of the molecule. As mentioned above, a priori one would have predicted little, if any, interaction of type 1 fimbriated organisms with FN because the oligosaccharide moieties are all of the complex type (55). The possibility that truncated forms of oligosaccharide chains with mannose in the terminal positions may be present because of incomplete glycosylation or plasma glycosidases cannot be excluded. However, while there may be some interaction of CSH-50 with

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the oligosaccharides of FN, it appears to be negligible. Strain CSH-50 adheres to domain 2, the glycosylated, gelatinbinding domain, but adhesion to intact FN is virtually unaffected by periodate or endoglycosidase treatment. Further, adhesion to unglycosylated domains of FN is equal to or greater than adhesion to the intact molecule. Adhesion of these organisms to synthetic peptides underscores the apparent lack of a requirement for oligosaccharide components of the substrate, but, as yet, we have tested too few synthetic peptides to allow a detailed analysis of reactivity. We believe that the adhesion of strain CSH-50 to FN is mediated by fimbriae for several reasons. First, adhesion is sensitive to inhibition by D-mannose and other a-mannosides but is not sensitive to numerous other compounds. The relative activities of these saccharides in inhibiting adhesion to FN is similar to their inhibition of E. coli aggregation of yeast cells. Also, growth conditions known to increase expression of type 1 fimbriae (passage in broth) (14, 52) increases adhesion to FN. Purified E. coli CSH-50 fimbriae bind to FN and inhibit adhesion of the organisms. The inability of fimbriae purified from E. coli HB101(pPKILA) to bind to FN or synthetic peptide and their inability to inhibit adhesion of E. coli CSH-50 to FN or synthetic peptide underscore that there are at least two different functional forms of type 1 fimbriae. Differences in the two functional forms of type 1 fimbriae may be due to structural differences in one or more of the known proteins making up the fimbrial filament (FimA, FimF, FimG, FimH) or to quantitative or qualitative differences in the way they are polymerized into a fimbrial filament. It is possible that the type 1 fimbrial filament can influence the conformation of the adhesin protein and, thereby, its binding properties; recombinant strains expressing hybrid fimbriae composed of K. pneumoniae FimA and E. coli FimH have been reported to express binding activity typical of the K.pneumoniae strain (30). In another instance, however, the functional activity of hybrid Serratia marcescens-K. pneumoniae fimbriae correlated with the K. pneumoniae strain from which the fimH gene was obtained (47). Because two of the strains used in this study exhibiting lectinlike activity [HB101(pPKIA) and ORN103(pSH2)] expressed genes encoded on multicopy plasmids, one might suggest that the differences observed are due to copy number of one or more of thefim genes rather than to differences in gene structure. However, preliminary genetic experiments in our laboratory suggest that structural differences in fim genes confer the different adhesive phenotypes (65). We cannot, as yet, explain the mechanism of inhibition of adhesion to protein structures by mannose. It has been postulated that the combining site of the type 1 fimbrial lectin is a cleft or pocket on the surface of the lectin (presumably FimH) corresponding in size to a trisaccharide (18, 60). The greatly increased activity of certain aromatic a-mannosides suggests the presence of an adjacent hydrophobic binding region that acts in concert. The avidity of mannosides for the components of this interaction are not sufficient to allow pretreatment of substrate or microorganism, but it has been assumed that the inhibitors interfere with adhesion by filling the combining site on the bacterial lectin, i.e., by competitive inhibition. There is, however, very little direct evidence for the binding of mannose by fimbriae or for the precise mechanism by which mannose exerts its inhibitory effect. While competitive inhibition may be logically assumed, it is not necessary that the mannoside inhibitors bear structural identity with the receptor or that they even belong to the same class of molecule for classical competitive inhibition to

INFECT. IMMUN. occur (for an example, see reference 64). In addition to the possibility that there is actual competitive inhibition due to mannose binding to the combining site of the adhesin, it is also possible that there is an allosteric effect of mannose on

the conformation of the FimH lectin or on another aspect of the fimbrial structure. The fact that the E. coli isolates used in this study interact with soluble and immobilized FN differently is not surprising. In fact, there have been a number of reports of such differences in reaction with FN. Streptococcus sanguis adheres avidly to immobilized FN, but the adhesion is not inhibited by a high concentration of soluble intact FN (42). Soluble FN does not inhibit the adhesion of S. aureus to immobilized FN (39; this study). A P-fimbriated strain of E. coli used in another study (73) adheres to immobilized FN but does not bind soluble FN. Plasminogen (59), lipoprotein A (58), and hyaluronate (40) interact with immobilized, but not soluble, FN. A similar phenomenon has also been seen in the interaction of Actinomyces viscosus LY7 with prolinerich proteins (PRPs) from human saliva, where the bacteria bind to immobilized PRPs in the presence of soluble PRPs (23). In most of these studies, authors suggest that cryptic receptors are exposed when the molecules are immobilized. The hypothesis that a conformational change takes place when FN adsorbs to a surface, whether it is plastic or gelatin, thereby exposing a cryptic receptor, could also explain the results obtained in the current studies. It is thought that FN does change conformation and does undergo an unfolding into a more-linear configuration accompanied by enhanced activity ("surface activation") when it becomes bound to certain other molecules or substrates such as gelatin, collagen, or plastic (7, 45, 74). It could also be that the failure of E. coli to bind soluble FN is because the interaction of type 1 fimbriae with protein is of relatively low affinity, similar to their affinity for oligosaccharide receptors (21, 56). FN molecules in solution might exhibit a monovalent, low-affinity interaction with individual fimbriae, but when FN becomes adsorbed to a substrate, the interaction between the bacteria and the substrate would be multivalent, increasing the effective strength of the interaction. While both of these hypotheses are plausible, the data presented here do not allow us to determine which provides the correct explanation. Although work remains to be done before we will understand precisely how FN can act as a receptor for type 1 fimbriated E. coli under certain conditions and as an inhibitor of adhesion under other conditions, it is clear that nonglycosylated domains of substrate-adsorbed FN can serve as receptors for type 1 fimbriated E. coli and perhaps other type 1 fimbriated species of the family Enterobacteriaceae. Members of the family Enterobacteriaceae appear to have several different ways in which to bind to this important extracellular matrix molecule. A lectin-independent interaction of P fimbriae of E. coli with immobilized (but not soluble) FN has also been reported, but this was not sensitive to the receptor analog a-D-Gal-a-(1-4)-13-D-Gal-1-0-Me and was not dependent upon the presence of the FsoG lectin but on the FsoE and FsoF proteins (72, 73). The results in the present study and those in the reports of Westerlund et al. (72, 73) differ from those of Froman et al. (19). These investigators described the adhesion of a strain of E. coli to immobilized FN but also found that soluble FN bound to the bacteria as well. This is clearly yet another mechanism whereby E. coli can interact with FN, even though the amino-terminal domain of FN appears to be one of the primary binding sites in each case. Olsen et al. (53) recently

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reported what is probably another E. coli surface component capable of binding FN. This fibrillar appendage, called curli, is only assembled on the bacterial surface at reduced temperatures (26°C) and is a polymer of a protein component termed curlin. Although the crl gene is apparently present in most E. coli strains and curlin is transcribed in many, its assembly into surface structures is limited. We have never seen any indication of curli on the strains we have used in this study nor have we grown any of the organisms at

temperatures other than 37°C. It is commonly believed that specific interactions between adhesins on the surfaces of bacteria and receptors associated with host mucosal membranes or other tissues lead to attachment of bacteria to host surfaces and thus serve as an important factor in the pathogenic process (9, 22). For this reason, it becomes important to determine the nature of the host and bacterial molecules involved, the mechanisms of interaction, and the factors that might affect their ability to interact. There is a distinct association between certain fimbrial types and disease, the best example of which may be the association of P fimbriae and pyelonephritis (33, 50, 67, 68). Although there is still considerable controversy as to the relationship of the expression of type 1 fimbriae to virulence (24, 31, 41), these organelles are expressed by a large fraction of clinical isolates of Enterobacteriaceae (28). Since type 1 fimbriae are the most commonly expressed fimbriae, it is perhaps this ubiquity that makes determination of their role in virulence more difficult. Nevertheless, the study of their adhesive interactions with host receptor molecules remains of considerable interest. The data reported here suggest that there are two distinct functional forms of type 1 fimbriae, one exhibiting only the classical lectinlike activity and the other exhibiting both lectinlike and protein-adhesive activities. Expression of this activity appears to be dependent not only upon the bacterial adhesin but also on certain specificities of the substratum. Future studies should give consideration to the possible role of this protein-adhesive phenotype of type 1 fimbriae. ACKNOWLEDGMENTS We thank Jane Hurley and Loretta Hatmaker for excellent technical assistance. We also thank I. Ofek, N. Sharon, D. Ohman, R. Doyle, and W. A. Simpson for insightful discussions. These studies were supported by research funds from the National Institutes of Health (grants DE-07218 [D.L.H.] and AI-13550 [S.N.A.]) and by VA Medical Research Funds. REFERENCES 1. Abraham, S. N., J. P. Babu, C. S. Giampapa, D. L. Hasty, W. A. Simpson, and E. H. Beachey. 1985. Protection against Esche-

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