by Clinical Isolates of Escherichia coli - Journal of Clinical Microbiology

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1987, p. 26-30

Vol. 25, No. 1

0095-1137/87/010026-05$02.00/0 Copyright C 1987, American Society for Microbiology

Identification with Monoclonal Antibodies of Hemolysin Produced by Clinical Isolates of Escherichia coli FERDINAND HUGO,' MARDJAN ARVAND,' JOHANNES REICHWEIN,' NIGEL MACKMAN,2 IAN BARRY HOLLAND,2 AND SUCHARIT BHAKDIl* Institute of Medical Microbiology, University of Giessen, D-6300 Giessen, Federal Republic of Germany,' and Department of Genetics, University of Leicester, Leicester LEI 7RH, England2 Received 21 July 1986/Accepted 15 September 1986

Murine monoclonal antibodies were generated against the 107,000-dalton hemolysin encoded by the hemolytic determinant from Escherichia coli LE 2001, and colony blotting was used to assay for production of the hemolysin by 35 hemolytic strains of E. coli and other hemolytic members of the family Enterobacteriaceae of clinical origin. Ali hemolytic E. coli strains gave positive reactions with two monoclonal antibodies. In contrast, none of the hemolytic, non-E. coli isolates yielded positive colony blots. In addition, Western blotting showed that the hemolysins produced by all clinical E. coli isolates had a similar molecular weight of about 107,000. Discrete antigenic variation may occur in the molecule, since a third monoclonal antibody did not react with the hemolysin from a number of wild-type E. coli strains. Western blot analysis was used to assess the presence of immunoglobulin G (IgG), IgA, and IgM antibodies to E. coli hemolysin in human sera. All 20 of the tested sera from healthy adults contained antibodies to the toxin, with various constellations among the antibody classes. In contrast, sera from five of eight infants aged 8 to 36 months contained no antihemolysin antibodies. We conclude that the 107,000-dalton hemolysin of E. coli is a widespread immunogen that is produced by most or all hemolytic E. coli strains in the human host.

produced by all tested strains of hemolytic E. coli but not by nonhemolytic E. coli, and we present evidence for discrete structural variations existing among the 107K hemolysins from different E. coli strains. None of the tested hemolytic members of the Enterobacteriaceae other than E. coli produced a similar protein. We report that sera from healthy adults contain specific antibodies to the hemolysin, whereas antibody titers in infants are low or absent. Hence, the hemolysin is an effective immunogen that is produced by E. coli in the human host.

Studies in animal models have indicated that the hemolysin of Escherichia coli is a potentially important pathogenic factor (3, 4, 7, 9, 10, 18-20, 22, 23). Several studies have now led to the identification of the four genes involved in the production and secretion of this extracellular protein (6, 13-15, 21). However, earlier efforts to characterize this toxin at a biochemical, functional, and immunological level were impeded by difficulties in isolating the protein in a functionally intact form (for reviews, see references 3 and 14). Recently, we have been able to identify the hemolysin as a 107,000-dalton protein (107K protein) derived from several wild-type strains of E. coli (12). Other groups have now independently presented evidence that the native and active molecule is secreted as a 107K polypeptide by E. coli strains (5, 8). In a recent study we have shown that the toxin generates functional transmembrane pores of 2- to 3-nm diameter in erythrocytes, possibly through insertion of toxin monomers into the lipid bilayer (1). Before these studies, there have been no systematic studies on the production of the 107K hemolysin by wildtype strains of E. coli. In particular, it is not known whether the hemolysins secreted by all hemolytic strains of E. coli represent immunologically related proteins of the same molecular weight and whether similar toxins are produced by hemolytic members of the family Enterobacteriaceae other than E. coli. Finally, it is not known whether the 107K hemolysin is produced by E. coli in the human host to elicit humoral antibody responses. In the present study, we have used monoclonal antibodies raised against the 107K hemolysin of E. coli LE 2001 to assess the production of this protein by clinical isolates of hemolytic and nonhemolytic E. coli and other members of the Enterobacteriaceae. We show that a 107K polypeptide (or a protein with a very similar molecular mass) is indeed *

MATERIALS AND METHODS Production and isolation of monoclonal antibodies. Prewarmed Todd-Hewitt broth (200 ml) containing 10 mM CaCl2 was inoculated with 1 ml of an overnight culture of E. coli LE 2001. The bacteria were cultured at 30°C in a water bath with vigorous shaking for 4 to 5 h. Cell-free culture supernatants were then obtained by centrifugation (25,000 x g, 10 min, Sorvall centrifuge RC 2B, 4°C), and the hemolysin was obtained in concentrated form by precipitation with 20% polyethylene glycol as described previously (1). BALB/c mice were immunized with the hemolysin by published protocols (11). Cells of the myeloma fine X 63-Ag 8.6.5.3 cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U of penicillin per ml, and 100 ,ug of streptomycin per ml were fused with the spleen cells derived from immunized mice in the conventional manner by using 50% polyethylene glycol as the fusing agent. Fused cells were diluted in RPMI 1640 containing 1 ,uM hypoxanthine, 0.4 FM aminopterin, and 16 ,uM thymidine and distributed into four Nunc trays (96 wells per tray). Hybrid cultures producing specific antibodies were cloned three times by limiting dilution. To isolate monoclonal antibodies, approximately 600 ml of culture supernatant was concentrated 10-fold in a pressurized Amicon concentration chamber (PM 10 membrane

Corresponding author. 26

MONOCLONAL ANTIBODIES TO E. COLI HEMOLYSIN

VOL. 25, 1987

..

a

, b

c

O

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FIG. 1. SDS-PAGE immunoblots of E. coli hemolysin probed with five monoclonal antibodies: a, clone h2A; b, clone 13H; c, clone h9G; d, clone IIC; e, clone hllE.

filter; exclusion limit, 10,000 daltons), and the immunoglobulins were precipitated by the addition of sodium sulfate (18% [wt/vol], final concentration). After 60 min of incubation at room temperature, the precipitated immunoglobulin G (IgG) was pelleted at 5,000 x g, and the pellet was suspended, washed with 18% Na2SO4 twice, and dialyzed against 0.1 M sodium phosphate buffer (pH 8.5) overnight. Final purification of IgG antibodies was achieved with the use of Affi-Gel protein A (MAPS-kit; Bio-Rad Laboratories, Munich, Federal Republic of Germany). The purity of the immunoglobulin preparations was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoglobulin samples were concentrated to yield preparations containing 0.8 to 2.0 mg of protein per ml, assuming an absorbance at 280 nm of 1.4 for a 1-mg/ml IgG solution. Routine screenings for the presence of antihemolysin antibodies were performed with micro-enzyme-linked immunosorbent assays. Plates (micro-ELISA plates; Nunc, Wiesbaden, Federal Republic of Germany) were coated with the purified 107K hemolysin (2 to 5 ,ug/ml) in bicarbonate buffer (pH 9.6) overnight at room temperature or for 2 h at 43°C. Hybrid culture supernatants were applied to the antigen-coated plates and incubated for 6 to 8 h at 37°C. The mouse antibodies bound to the antigen were detected by using biotinylated anti-mouse IgG from sheep (Amersham, Braunschweig, Federal Republic of Germany) diluted 1:1,000. Streptavidin-biotinylated horseradish peroxidase (Amersham) diluted 1:1,000 was used as enzyme, and the substrate reaction was carried out in citrate buffer (38 mM, pH 5.0, 0.002% H202) with ortho-phenylendiamine (0.43 mg/ml; Sigma, Munich, Federal Republic of Germany) as the chromogen. The diluting and washing buffer was 20 mM Tris hydrochloride (pH 7.5)-150 mM NaCl containing 0.05% Tween 20 (E. Merck AG, Darmstadt, Federal Republic of Germany). Neutralization tests. Samples (50 ,ul) of purified IgG were doubly diluted in microtiter plates and mixed with 1 volume of freshly prepared hemolysin (8 hemolytic units per ml). One hemolytic unit per milliliter was defined as that toxin concentration yielding .90% hemolysis of an equal volume of 2.5% rabbit erythrocytes (2.5 x 108 cells per ml) after 60 min at 37°C. After incubation at 4°C for 30 min, 50 ltl of a

27

2.5% suspension of rabbit erythrocytes suspended in isotonic saline was added, and the extent of lysis was determined after 60 min at 37°C by measurement of hemoglobin (absorbance at 412 nm) in the supernatants. As a positive control, rabbit IgG prepared by adsorption and desorption of hyperimmune serum on protein A-Sepharose was also used in these experiments. Colony blots. Clinical isolates of hemolytic E. coli and other members of the Enterobacteriaceae were obtained from the diagnostic laboratories of the Institute of Medical Microbiology, Giessen. Species identification was performed with the conventional API-20E identification system (API System S.A., Montalieu Vercieu, France). A total of 28 hemolytic E. coli isolates, 3 hemolytic Serratia strains, 2 hemolytic Morganella strains, and 2 unidentifiable hemolytic species of Enterobacteriaceae were examined. We also analyzed 8 hemolytic strains of Pseudomonas aeruginosa and 20 nonhemolytic E. coli and other Enterobacteriaceae species. The bacteria were cultured on Diagnostic Sensitivity Test (DST) agar containing 5% defibrinated sheep blood, and colony immunoblotting was performed essentially as previously described (17) with polymyxin B (20 ,ug/ml) dissolved in phosphate-buffered saline (pH 7.4) to permeabilize the bacteria. The nitrocellulose filters were blocked by incubation in 0.1% Tween 20 and then incubation with 10% culture supernatant containing a monoclonal antibody overnight at room temperature. Further processing and development of the blots followed the procedure that was used for Western blot analyses of SDS-polyacrylamide gels. SDS-PAGE Western blot analyses. The analyses were performed as previously described (1, 16). To determine the molecular size of the hemolysins secreted by clinical E. coli isolates, the bacteria were cultured on blood agar overnight. The cells were then removed from the surface of the agar, samples of the underlying agar containing the hemolyzed erythrocytes were mixed with 4% SDS and boiled briefly (3 to 5 s), and 100-,ul samples were applied to SDSpolyacrylamide gels as described previously (16). Western blots were probed with the monoclonal antibodies. To test for the presence of antibodies to E. coli hemolysin in human sera, blots were incubated with 2% serum solutions overnight and developed with the respective, peroxidase-labeled second antibodies to IgG, IgM, and IgA (all from Dakopatts Immunoglobulins, Copenhagen, Denmark).

A*

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FIG. 2. Colony blots of hemolytic and nonhemolytic members of the family Enterobacteriaceae probed with monoclonal antibody h2A. Blots: a, b. and c, E. coli; x, P. aeruginosa; y, Morganella sp.; z, unidentified isolate. A, Agar culture; B, colony blot. Only the hemolytic E. coli isolates yielded positive colony blots.

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HUGO ET AL.

J. CLIN. MICROBIOL.

A

B

FIG. 3. A, Six hemolytic E. coli isolates were inoculated onto blood agar, and the lysed erythrocytes contained in the agar were subsequently analyzed by SDS-PAGE immunoblotting. The 107K hemolysin was detected by monoclonal antibody h2A in all cases. B, Similar agar samples were probed with monoclonal antibody clone l1C; this antibody recognized only three of the six hemolysins. The corresponding isolates also yielded positive colony blots, whereas the isolates producing nonreactive hemolysin yielded respective negative colony blots (Fig. 4). Arrow indicates E. coli hemolysin.

RESULTS

Monoclonal antibodies to E. coli hemolysin. Figure 1 depicts the immunoblots of polyethylene glycol-purified E. coli hemolysin developed with five murine monoclonal antibodies; 49 of 54 clones thus tested recognized the SDSdenatured antigen in the Western blots. The remaining five antibody clones gave positive micro-enzyme-linked immunosorbent assay reactions with nondenatured toxin but did not stain the Western blots. IgG antibodies from four clones which recognized the SDS-denatured antigen were isolated by protein A affinity chromatography from cell culture supernatants and tested for neutralizing activity. Polyclonal rabbit antibodies were tested similarly. The IgG fraction of the polyclonal antibodies was found to neutralize toxin activity, but we were unable to demonstrate toxin neutralization by any of the tested monoclonal antibodies. Hence, none of the tested monoclonal antibodies was directed against epitopes of the molecule that were essential for its hemolytic function. Production of 107K hemolysin by clinical isolates of E. coli and other enterobacteria. A total of 35 clinical isolates of

hemolytic species of Enterobacteriaceae, 8 isolates of P. aeruginosa, and 20 nonhemolytic species of Enterobacteriaceae were examined for the production of hemolysin. Figure 2 shows the results of colony blots probed with monoclonal antibody clone h2A. The hemolytic isolates analyzed in this experiment produced morphologically similar zones of P-hemolysis surrounding the bacterial colonies. The three hemolytic E. coli examined in this fashion (blots a, b, and c) yielded positive colony blots, whereas no single non-E. coli isolate (blots x, y, and z) reacted with the antibodies. The same results were obtained for other E. coli and non-E. coli isolates, i.e., nonhemolytic E. coli and other species of Enterobacteriaceae, as well as the hemolytic P. aeruginosa isolates yielded negative colony blots. The E. coli hemolysin thus appears to be species specific. Molecular size of E. coli hemolysin produced by clinical isolates. It is often not possible to recover nondegraded hemolysin in the culture supernatants of hemolytic E. coli isolates, and the agar-blot method was therefore also used as an additional method to analyze the secreted hemolysin from these strains. The blot method revealed that all hemolytic E. coli isolates indeed secreted a polypeptide similar in molecular weight to the 107K hemolysin when tested with the monoclonal antibodies (clone h2A) (Fig. 3A). The 107K band was never observed with non-E. coli or nonhemolytic E. coli strains. It is noteworthy that when culture supernatant samples from hemolytic E. coli clinical isolates are analyzed by SDS-PAGE followed by staining with Coomassie brilliant blue, small variations in the molecular weight of the secreted hemolysins are occasionally detected (12). Immunological microheterogeneity of E. coli hemolysin. An unexpected observation was made with one monoclonal antibody (clone 11C). In this case, colony blots revealed that the antibody recognized the hemolysin from some, but not from other, E. coli isolates (Fig. 4). Similarly, Western blots of agar samples showed that this antibody stained the 107K band derived from the homologous antigen and the 107K protein from some wild E. coli strains but did not react with the protein secreted by those strains that had yielded negative colony blots (Fig. 3B). These results suggested that this monoclonal antibody reacted with a variable epitope in the toxin molecule. In support of this contention, another antibody clone (fl2E) was found to stain some hemolysinpositive colonies more weakly than others (Fig. 4C). The possibility that denaturation and the extent of renaturation

EL e

*

A

e

e

e

*S B

c

D

FIG. 4. A, Agar culture inoculated with 12 isolates including three nonhemolytic E. coli and one hemolytic Morganella sp. (x) All other strains were clinical isolates of E. coli. B, colony blot probed with monoclonal antibody h2A; C, a similar colony blot probed with antibody clone fl2E; D, colony blot probed with antibody clone l1C. Note the differences in staining intensities observed with clone fl2E compared with clone h2A and the total absence of staining of many E. coli isolates with clone l1C, indicating immunological microheterogeneity among the 107K hemolysins produced by these E. coli isolates.

VOL. 25, 1987

MONOCLONAL ANTIBODIES TO E. COLI HEMOLYSIN

B

A

E

D

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29

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FIG. 5. SDS-PAGE immunoblotting of 107K E. coli hemolysin probed with sera from healthy human adults (A through C). The blots depict the various constellations of antibody classes found. Blots were developed with (a) anti-IgG, (b) anti-IgA, or (c) anti-IgM second antibodies. All adults had IgG antibodies, some with both IgA and IgM (A) or with IgA only (B) or IgM only (C). In contrast, 6- to 24-month-old infants often had no detectable antibodies (D). In one out of eight cases, a significant level of IgG with only a little IgA was observed (E).

might influence antibody recognition has not been excluded but seems improbable to us at present. Presence of antibodies to E. coli hemolysin in human sera. Antibodies to the 107K E. coli hemolysin were detected in the sera of all 20 healthy human adults examined, although there were considerable differences in the distribution of antibody classes (Fig. 5A through C). All sera contained detectable IgG antibodies, whereas IgM and IgA were present in 6 and 13 of the tested sera, respectively. Preliminary analyses with micro-enzyme-linked immunosorbent tests indicated that quantitation of the respective antibodies was feasible (data not shown). In contrast to the results derived from adult sera, five out of eight sera from infants aged 8 to 36 months contained no detectable antibodies (Fig. 5D). Two infants with hemolysin antibodies had high IgG titers with only very weak accompanying IgM or IgA antibodies (Fig. SE). DISCUSSION Recent studies have indicated that the hemolysin produced by three different strains of E. coli is a polypeptide of Mr 107,000 (5, 8, 12). In this study, we sought to determine whether hemolytic E. coli as well as other hemolytic non-E. coli clinical isolates produced a similar toxin. It was also of interest to assess whether antihemolysin antibodies could be detcted in human sera, since this would directly indicate whether the toxin is produced by bacteria and represents an immunogen in the human host. At present no published data are available on these issues. To address the question of production of toxin among clinical isolates, monoclonal antibodies raised against the 107 K toxin from E. coli LE 2001 were used to develop colony and Western blots. These studies revealed that all hemolytic E. coli strains produced immunologically related toxins of approximately Mr 107,000, although small variations in size were detected. It therefore appears very likely that all hemolytic E. coli strains produce hemolysins that are biochemically and immunologically similar. In contrast to

hemolytic E. coli isolates, other hemolytic members of the Enterobacteriaceae that we tested did not produce an immunologically related hemolysin. This may indicate the presence of distinct hemolysins in the family Enterobacteriaceae. However, in view of the small number of strains tested, the presence of a protein produced by other species, which is immunologically related to the E. coli hemolysin, cannot be ruled out. Interestingly, one monoclonal antibody detected antigenic microheterogeneity among the hemolysins from different E. coli isolates. This antibody recognized the hemolysin from some strains but not from others. Such antigenic variation has not been noted previously among other pore-forming bacterial cytolysins (2). If similar antigenic variations were found in epitopes that are important for hemolysin function, antibodies to the hemolysin of a given E. coli strain might fail to neutralize the hemolysin of variant strains. Since none of the presently available, purified monoclonal antibodies possessed neutralizing activity, further work will be required to test whether such functionally significant antigenic variation exists. Western blot analyses were used to assess the presence of antibodies to E. coli hemolysin in sera of healthy human adults and infants. Significantly, sera of all of the adults contained antibodies to the toxin, whereas most infants aged 8 to 36 months had no or only low levels of antibodies. These results indicate that the hemolysin of E. coli is an effective immunogen that is produced by these bacteria in the human host. Future studies should reveal whether determination of antibody class and titer could become a useful diagnostic tool in cases of chronic infections with members of the family Enterobacteriaceae. ACKNOWLEDGMENTS We thank Sylvia Kramer and Marion Muhly for excellent technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft (Bh 2/2).

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