INFECTION AND IMMUNITY, May 1991, p. 1846-1852
Vol. 59, No. 5
0019-9567/91/051846-07$02.00/0 Copyright X) 1991, American Society for Microbiology
Characterization of Monoclonal Antibodies against Alpha-Hemolysin of Escherichia coli OROPEZA-WEKERLE,l P. KERN,' D. SUN,2 S. MULLER,3 J. P. BRIAND,3 AND W. GOEBEL'* Institute for Microbiology, University of Wurzburg,' and Clinical Research Unit for Multiple Sclerosis, Max Planck Society,2 D-8700 Wurzburg, Federal Republic of Germany, and Biologie Moleculaire et Cellulaire, 67084 Strasbourg Cedex, France3
R. L.
Received 15 November 1990/Accepted 26 February 1991
Monoclonal antibodies (MAbs) were raised against native and denatured alpha-hemolysin (HlyA) of Escherichia coli. Binding of the MAbs to native, denatured, and erythrocyte-complexed active wild-type hemolysin and mutant derivatives was tested. All 15 MAbs analyzed bound to native hemolysin, even when the toxin was complexed with human erythrocytes. While some MAbs were unable to bind to a specific native mutant hemolysin, others could not even bind to mutant hemolysin carrying deletions remote from their actual binding sites. A rough determination of the binding sites of 15 MAbs on HlyA was performed by Western immunoblot analysis using CNBr fragments of HlyA and mutant hemolysin proteins. Interestingly, the binding sites of the MAbs against native hemolysin seem to be more randomly distributed on HlyA than are those of MAbs against denatured hemolysin. Three MAbs inhibited the hemolytic activity significantly. Two of these MAbs bound to the hydrophobic region, and the other one bound to the repeat domain of HlyA. The use of synthetic peptides from these regions allowed determination of the linear epitopes for two of these MAbs.
Escherichia coli alpha-hemolysin (HlyA) is an extracellular protein toxin which is produced predominantly by E. coli strains isolated from patients with pyelonephritis. Hemolysin appears to be an essential virulence factor of such uropathogenic E. coli isolates (11, 12, 15, 33). The cytolytic 110-kDa protein forms pores in erythrocyte membranes of various origins and in artificial lipid bilayer systems (1-3, 26). The diameter of the pores introduced into erythrocyte membranes and lipid bilayers is about 2 nm (1, 26). The average single-channel conductance is 500 pS in 0.15 M KCl, and the mean lifetime of the pore is 2 s at 20 mV (1). The amino acid sequence of HlyA (1,024 amino acids) allows the prediction of several interesting structural domains: (i) an amphiphilic, a-helical N-terminal sequence (amino acids 1 to 40); (ii) a hydrophobic region from amino acids 238 to 410; and (iii) a repeat domain of 11 tandemly arranged repeats of the glycine-rich nine-amino-acid sequence XLXGGXGDD (6, 14). This repeat region is localized in the HlyA protein between amino acids 736 and 849. Recent investigations (7, 19, 21, 22) have indicated that repeat domains with rather conserved basic repeat units but various repeat numbers are common to a collection of different toxins, which is designated the RTX (repeats in toxin) protein family. By using site-specific mutagenesis and monoclonal antibodies (MAbs) against hemolysin (16, 31), part of the functional domains of HlyA was recently unravelled. It was shown (4, 23) that the repeat domain is involved in Ca2+-dependent binding of hemolysin to erythrocytes but not required for pore formation in a lipid bilayer (23). These data suggest that the repeat domain does not directly participate in the pore formation leading to hemolysis but rather may represent the recognition site for an unknown receptor on the erythrocyte membrane. Association of hemolysin with erythrocytes or artificial lipid bilayers is dependent upon modification of HlyA by HlyC (27, 28). Although the *
chemical nature of the component which is covalently linked to a defined amino acid of HlyA (7a, 32) is not known, this component appears to represent an anchor which fixes the modified but not the unmodified HlyA protein to the membrane (29). The function of the a-helical, amphiphilic N-terminal sequence is unclear. Removal of this region, which possesses membrane-seeking properties (13), leads to a mutant hemolysin with increased specific hemolytic activity (24, 25). We have recently proposed a model for the structure of the hemolysin pore (24) which anticipates the possibility that four amphipathic and four hydrophobic a-helical segments of the hydrophobic region of HlyA are inserted into the membrane. On the basis of this model, we have suggested that the amphiphilic N-terminal sequence can compete with the amphipathic transmembrane segments of the pore-forming structure, thereby restricting the lifetime of the hemolysin pore to a short period. The short lifetime may be an important factor for the in vivo function(s) of E. coli hemolysin during infection, since this hemolysin not only exhibits cytolytic activity but also triggers induction of synthesis and release of inflammatory substances, such as leukotrienes, from granulocytes, mast cells, and macrophages (8, 18). The latter activity, caused in vitro by sublytic doses of hemolysin, may require a short lifetime of the pore. Here we report on the isolation and characterization of MAbs raised against native and denatured hemolysin and the binding of these MAbs to wild-type and mutant hemolysins which carry deletions at the N terminus, the hydrophobic region, or the repeat domain of hemolysin. It is shown that some MAbs fail to bind to a specific mutant hemolysin, whereas other MAbs are unable to bind to several mutant hemolysins with extended deletions in various regions of the HlyA protein. By using synthetic peptides, we localized the epitopes of two MAbs which inhibit hemolytic activity. Our data confirm and extend those of previous studies done with HlyA-specific MAbs (16, 31).
Corresponding author. 1846
MAbs AGAINST E. COLI ALPHA-HEMOLYSIN
VOL. 59, 1991
MATERIALS AND METHODS Bacterial strains and plasmids. The E. coli strains and the plasmids that encode the mutant hemolysins used in this study have already been described (23, 24, 30). Isolation of extracellular HlyA protein. An overnight culture of the respective E. coli strains was inoculated into 20 ml of 2x YT medium and incubated with shaking at 37°C. Cells in the logarithmic growth phase were pelleted by centrifugation and discarded. The cell-free supernatant was mixed with 75% ammonium sulfate, and complete precipitation was achieved by incubation of the mixture on ice for 1 h. The pellet was suspended in 10 ml of TCU buffer (20 mM Tris, 150 mM NaCl, 6 M urea, pH 7.0) and dialyzed overnight against TCU buffer. After 24 h, the hemolytic activity was tested and the preparation was stored at -70°C. Equal volumes of hemolysin and sample buffer containing 50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% mercaptoethanol, and 0.5% bromophenol blue were used for SDS-polyacrylamide gel electrophoresis as described by Laemmli (20). Immunoblotting. Hemolysin separated on SDS-10% polyacrylamide gels was transferred to 0.2-pum-pore-size nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, N.H.). Electrophoretic transfer was accomplished overnight at 4°C at 70 mA in 0.025 M Tris buffer (pH 8.3, containing 0.19 M glycine) by using a TE 52 Transphor unit (Hoefer Scientific Instruments, San Francisco, Calif.). Residual protein-binding sites on the membrane were blocked by incubation in 50 mM Tris-buffered saline (pH 7.5) containing 5% bovine serum albumin (BSA) for 60 min at room temperature. This nitrocellulose was transferred to an antibody test solution containing either mouse polyclonal antiserum or mouse monoclonal ascitic fluid, each diluted 1:1,000 in Tris-buffered saline-BSA. The membrane was incubated with the antibody probe for 90 min and then washed four times (10 min each time) in Tris-buffered saline. Reaction of the antibody probes with hemolysin was detected by incubation of the nitrocellulose in horseradish peroxidase-conjugated rabbit anti-mouse antibodies (DAKO PATTS, Roskilde, Denmark) with specificity for either immunoglobulin G or M. After incubation and washing, the nitrocellulose was transferred to a peroxidase substrate solution containing 6 ml of 0.3% 4-chloro-1-naphthol (Sigma), 42 ,ul of 30% H202 (Fischer Scientific Co.), and 94 ml of Tris-buffered saline for detection of horseradish peroxidase on the membrane. Determination of hemolytic activity. Hemolytic activity was determined essentially as described before (29). Antibody production. Male BALB/c mice (8 weeks old) obtained from the Max-Planck-Institut fur Immunbiologie, Freiburg, Federal Republic of Germany, or from the Zentralinstitut fur Versuchstierzucht, Hannover, Federal Republic of Germany, were used as a source of immune lymphocytes for hybridoma production. BALB/c mice were injected with three doses of hemolysin at weekly intervals. The first dose, administered intraperitoneally, contained 10 ,ug of hemolysin per ml in Freund incomplete adjuvant. The second 10-,ug/ml dose of hemolysin in 6 M urea was administered intraperitoneally and subcutaneously. The final injection (5 R.g of hemolysin per ml) was given by tail vein. The mice were killed 2 days postimmunization by cervical dislocation. Lymphocytes were obtained from their axillary, inguinal, brachial, and popliteal lymph nodes and fused with X63-Ag8.653 mouse myeloma cells as previously described (17). For ascites production, mice were sensitized with an intraperitoneal injection of pristane (Aldrich Chemical Co.,
1847
Inc., Milwaukee, Wis.) and 24 h later they were injected with 107 hybridoma cells. After 2 to 3 weeks, ascitic fluid was harvested. Polyclonal sera were prepared by an immunization protocol similar to that described above. ELISA. Mouse sera and ascitic fluid were tested for antibody reactivity by a direct enzyme-linked immunosorbent assay (ELISA). Wells of polyvinyl assay plates (Costar, Cambridge, Mass.) were coated with hemolysin and BSA as previously described (10). Activity of mouse polyclonal or monoclonal antibodies with hemolysin was detected with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins (DAKO PATTS). Binding of the conjugated antibody was detected with ABTS (Serva). Monoclonal antibody subclass determination was done by a similar assay, using a panel of peroxidase-conjugated rabbit immunoglobulins specific for individual mouse antibody subclasses (Miles). When ELISA was used in inhibition experiments with synthetic peptides, the peptides were first incubated overnight at 4°C with 10 ,ug of MAb per ml and the mixture was then added to wells precoated with 3 jig of hemolysin. Binding of MAbs to hemolysin was measured by a previously described ELISA (30). Binding of wild-type and mutant hemolysins to human erythrocytes. Wild-type hemolysin and mutant hemolysins AL2, AL9, AL13, AL10-1, AL10-4, and AL10-9 (23, 24, 29) were isolated from the supernatant of E. coli 5K strains carrying the corresponding plasmids, which all carry hlyC+ hlyB+ hlyD+. Binding of hemolysin was determined in a fluorescence-activated cell sorter (FACS) using mouse polyclonal antibodies or MAbs as the first reagent and fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin antibodies as the second reagent (29). CNBr cleavage. Chemical cleavage of hemolysin with CNBr was performed by the method of Gross and Witkop (9). The protein was dissolved in 1 ml of 70% formic acid containing 100 mg of CNBr (Pierce Chemical Co., Rockford, Ill.). The reaction mixture was dried under vacuum in a Speed Vac centrifuge. The pellet was suspended in water and dried again to remove traces of CNBr and formic acid. Peptide synthesis. Synthetic peptides corresponding to residues 300 to 309 (peptide 1), 310 to 317 (peptide 2), and 758 to 774 (peptide 3) of the hemolysin of E. coli were synthesized by the solid-phase method using an NPS 4000 peptide synthesizer. Peptides 1, 2, 3A, and 3B were coupled to BSA by using glutaraldehyde, and the peptide/carrier ratio was determined by amino acid analysis. To facilitate coupling to the carrier protein in a defined orientation, an additional cysteine was added to peptide 1. Synthesis of peptides was performed at Neosystem (Strasbourg, France).
RESULTS Isolation and characterization of MAbs against native and denatured hemolysin. Eight hemolysin-specific hybridoma clones producing MAbs A4D3, A4D11, A4F9, A4F10, A4G2, A4G4, A4G5, and D7G2 were isolated by fusing mouse myeloma cell line X63-Ag8-653 with lymphocytes from BALB/c mice which were immunized against native hemolysin obtained from E. coli 5K carrying plasmid pANN202-812 (29). In a second fusion, denatured hemolysin, isolated as a 110-kDa protein by SDS-polyacrylamide gel electrophoresis, was used as the antigen. The hemolysinspecific MAbs obtained in this experiment are designated C7D1A8, C7D1C1O, C7D3D5, D7D8E11, C7D3F8, D7B12, and DllH1. A total of about 2,000 hybrids from both fusions were screened. The isolated MAbs belonged to five isotypes
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OROPEZA-WEKERLE ET AL.
INFECT. IMMUN.
TABLE 1. MAbs raised against native and denatured E. coli hemolysin Antigen and
A. B.
Class or subclass
MAb
Native hemolysin A4D3 .............. A4D11 ............... A4F9 .............. A4F1O .............. A4G2 ............... A4G4 .............. A4G5 .............. D7G2 ..............
Denatured hemolysin C7D1A8 .............. C7D1C1O .............. C7D3D5 ............... C7D8E11 .............. C7D3F8 .............. D7B12 ............... DllH1 ..............
110-
110-.-
--
.
IgGl IgGl
IgG2b IgM IgM IgGl IgM IgM
1
2
3
4
5
6
S
7
1
2
3
45
6
9
7
FIG. 1. Immunoblots of MAbs raised against native hemolysin (A) and denatured HlyA protein (B) with the 110-kDa HlyA protein. Lane 1 (A and B), binding of polyclonal anti-HlyA antibodies. Lanes in panel A: 2, A4G2; 3, A4G5; 4, D7G2; 5, A4F1O; 6, A4D11; 7, A4D3; 8, A4G4. Lanes in panel B: 2 and 3, D7B12; 3, D7B1O; 4, C7D3D5; 5, C7D1C1O; 6, C7D1A8; 7, C7D3F8; 8, C7D8E11; 9, DllHl.
IgGl IgM IgA IgG2a IgG2a IgGl IgGl
80%, compared with polyclonal anti-HlyA serum at the same immunoglobulin G [IgG] concentration), indicating that the epitopes recognized by most of the MAbs are not directly involved in hemolytic activity, e.g., in binding to erythrocytes and/or pore formation. This assumption is in line with the observation that all MAbs still were able to bind to hemolysin which was bound to human erythrocytes. The assay for measuring antibody binding to the erythrocytehemolysin complex was performed as previously described (29), by using a FACS after coupling the erythrocytehemolysin-MAb complex with rabbit anti-mouse immunoglobulin antibodies conjugated with fluorescein isothiocyanate (Table 2; Fig. 2).
(Table 1). The MAbs were selected by binding to activated hemolysin (HlyA modified by HlyC) in an ELISA. All selected MAbs also reacted in Western blots with the denatured 110-kDa HlyA protein (Table 2; Fig. 1), albeit with different binding efficiencies, indicating that most MAbs recognize linear epitopes on the HlyA protein. Only three of the isolated MAbs (A4G2, A4G4, and A4F10) were able to inhibit hemolytic activity to an appreciable extent (50 to
TABLE 2. MAb binding to native or denatured hemolysin and preformed hemolysin-erythrocyte complexesa Binding to: Wild-type
Antibody
NH
Polyclonal antibody MAbs A4D3 A4D11 A4F9 A4F1O A4G2 A4G4 A4G5 D7G2 C7D1A8 C7D1C1O C7D3D5 C7D8E11 C7D3F8 D7B12 DllHl
Mutant AL13
hemolysin N NHEDH E
-
Mutant AL2
EDH E
Mutant AL9
Mutant
Mutant AL10-4
AL10-1
CNBr
Mutant 10-9
fragment:
NH NH DH DH~NH ~~~ DH~NHH NH DH NH + E DH -E DH NH ~~~~~~ -E ED DH AA B ~~~~~~~~~~~~~~+E
+
+
+
+
+
+
+
-
+
+
-
+
+
+
+
+
-
+
+
-
+
+ + +
+ + + + + + + +
+ + +
+ + +
+ + +
+ + +
+ + +
-
+ + +
+ + +
-
+ + +
+
+
+
+ .
+ + + + + + + + + + +
+ + + + + + + + + + +
+ + + + + +
+ + + + +
+ + + + + +.+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + +
+ + + + +
-
+ + + + -
-
-
-
+ + + + + + + + + + + + + + +
-
-
+ + -
-
+ + + + + + + + + + + + + +
+
+ + + + + + + + + + +
+ + + + + + +
.
.
.
.
.
+ + + + -
-
+ +
C
D
E
+
+
+
ND + + +
+ +
+ +
+ + + +
ND +
ND
a Binding of MAbs to native, HlyC-modified HlyA proteins (NH) was measured in the ELISA described in Materials and Methods. Constant concentrations (50 ng/ml) of the mutant hemolysins were used in the binding assay, and the concentration of the MAb was adjusted to 20 ,ug of protein per ml of ascitic fluid. Binding of hemolysin to a MAb was considered positive at an optical density higher than 0.03. The background optical density was 0.01 or lower. NH-E indicates binding of the MAb to a preformed hemolysin-erythrocyte complex. Formation of hemolysin-erythrocyte-antibody complexes was determined by FACS analysis (29). DH indicates binding of the MAbs to denatured HlyA protein determined by Western blot analysis. Binding of MAbs to CNBr fragments A (16.9 kDa), B (26.0 kDa), C (0.9 kDa), D (51 kDa), and E (14.4 kDa) was also determined by immunoblotting of the separated CNBr fragments with the MAbs. The mutants carried the following deletions: AL13, amino acids 9 to 37; AL2, amino acids 300 to 319; AL9, amino acids 200 to 599; AL10-1, amino acids 775 to 783 and repeat 5; AL10-4, amino acids 775 to 783 and 814 to 822 and repeats 5 and 8; 10-9, amino acids 757 to 822 and repeats 3 to 8. ND, not done.
MAbs AGAINST E. COLI ALPHA-HEMOLYSIN
VOL. 59, 1991 100F
C,D,As
a
C7D,As
b
f LISA
0tUSA
C7D,A
C
LISI
|
A,G4
A,Goe
+
AG
ELISO-
"45LISA
1849
(L1504A
-t .
400
.
A00 0
400
400
09
go
.400
Fluorescenc
FIG. 2. FACS analysis of complex formation between wild-type hemolysin and human erythrocytes after binding of polyclonal anti-HlyA antibodies (a), MAb A4G4 (b), MAb A4F10 (c), MAb A4G5 (d), MAb C7D3D5 (e), and MAb D7B12 (f).
Binding of MAbs to native mutant hemolysins. We have recently constructed and characterized several mutant hemolysins which are altered in their pore-forming properties and/or binding to erythrocytes. Some of these mutant hemolysins were tested for binding to the MAbs described. Mutant hemolysin AL13, encoded by pANN202-312AL13* (23), lacks amino acids 9 to 37 from the N-terminal end of HlyA. This hemolysin has threefold higher specific hemolytic activity and forms pores with a much longer lifetime than those formed by wild-type hemolysin (23, 24). As shown in Table 2, all MAbs bound to this mutant hemolysin (in the native or denatured form), but interestingly, five MAbs (A4G2, A4G4, C7D1C1O, C7D8E11, and D7B12) no longer bound to this protein when it was complexed with erythrocytes (Table 2). This suggests that binding of mutant hemolysin AL13 to the erythrocyte membrane protects or alters the epitopes recognized by these five MAbs. Mutant hemolysin AL10-1, produced by E. coli carrying plasmid pANN202-312AL10-1*, lacks repeat 5 within the repeat region of hemolysin (23). This hemolysin has reduced hemolytic activity which can be restored to the wild-type level by higher Ca2+ concentrations (23). As shown in Table 2 and Fig. 3, all MAbs bound to this mutant hemolysin, even when it was complexed with erythrocytes. While the former two mutant hemolysins still possess hemolytic activity, which implies that they are able to bind to the erythrocyte membrane and form pores, a second group of hemolysins, described below, have a loss of hemolytic activity in common. The reason for this lies either in the lack of Ca2+-dependent binding of the mutant hemolysins to the erythrocyte membrane, e.g., mutants AL10-4 and AL10-9, and/or in their inability to form pores, e.g., mutants AL2 and AL9 (23-25). A common property of all four mutant hemolysins is the inability to interact with erythrocytes, as demonstrated by the failure of polyclonal anti-HlyA antibodies and all MAbs to form an erythrocyte-hemolysin-antibody complex detectable in the previously described FACS assay (29). Interestingly, there are four MAbs (A4G4, C7D1C1O, C7D8E11, and D11H1) which failed to bind in an ELISA to the latter four mutant hemolysins (Table 2), although the deletions of the four hemolysins are located in different regions of HlyA (Fig. 4). Two additional MAbs (C7D3D5 and C7D3F8) failed to bind to mutant hemolysins AL9, AL10-4,
FSourescwox
FIG. 3. FACS analysis of complex formation between mutant hemolysins and human erythrocytes and binding of two MAbs (C7D1A8 and A4G4) to the complex. Panels: a and d, binding of C7D1A8 (a) and A4G4 (d) to the erythrocyte-AL13 complex; b and e, binding of C7D1A8 (b) and A4G4 (e) to the erythrocyte-AL2 complex; c and f, binding of C7D1A8 (c) and A4G4 (f) to the erythrocyte-AL10-1 complex. ELISA+ and ELISA- indicate the abilities of the MAbs to bind to mutant hemolysin alone.
and AL10-9 but still bound to AL2. Except for MAb A4G4, all of these MAbs were raised against denatured hemolysin. Only two MAbs raised against denatured HlyA (C7D1A8 and D7B12) did not show this general lack of binding to the nonhemolytic mutant HlyA proteins. Whereas MAb C7D1A8 did not bind to mutant hemolysin AL9, which has lost the hydrophobic region involved in pore formation (24, 25), D7B12 did not bind to mutant hemolysin AL10-9, which
A4D11
MAb
A4F9 C7D1C1O C7D3D5
HlyA
D7G2
C7D1AO
~nn
n
UB U
L
A
ED A113 (9-37)
A4G2 A4G0 D7812
C7WDE11
A4F10 A4G4 foeo
CA12 (300-319)
Goo
oo
Too
E
D
O A110-1 (775-783) O
A19 (200-609)i
goo
,/- ,17 0
= AI10-4 (775-783; 014-822)
A10-9 (757-822)
slr AA
300
PepI
LSTSAAAAGL
Pep2 IASNTLA
LYGDKONDTLSOONGODD Pep3
Pep3-AA LYGODKOND LSOONODD POP3-B
FIG. 4. Summary of approximate epitope localizations of the MAbs mapped with various deletion mutants of HlyA and the CNBr fragments of wild-type hemolysin (A to E). The deletions of the mutants are indicated in the lower part (numbers in parentheses indicate deleted amino acids [AA]). The upper line represents the primary amino acid sequence of HlyA (14); the numbers above the line give the amino acid positions. The three open boxes between amino acids 230 and 410 indicate the hydrophobic region, and the hatched boxes represent the repeat domain of HlyA (amino acids 740 to 849) (23). The symbol A marks the CNBr cleavage sites. A to E represent the following CNBr fragments: A, 16.9 kDa; B, 26.0 kDa; C, 0.9 kDa; D, 51 kDa; E, 14.4 kDa. Peptides Pepl, Pep2, Pep3, Pep3-A, and Pep3-B were used for characterization of the epitopes recognized by MAbs A4F10 and A4G2.
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OROPEZA-WEKERLE ET AL.
INFECT. IMMUN. O.D. 405
A
10
0.51
0.62
1.25
2.5
5
10 MAb
(ng/mI)
0 62
1.25
2.5
5
10 MAb (
MAb (ng/mi)
FIG. 5. Binding of MAbs A4G2 and A4F1O to specific HlyA-derived peptides, determined by ELISA (30). (A) Binding of A4G2 to Pep3 (amino acids 758 to 774 of HlyA) (0), native hemolysin (0), and Pepl (amino acids to 300 to 309) as the control (V). (B) Binding of A4G2 to Pep3-A (0) and Pep3-B (0), with Pepl (V) as a control and unspecific serum with Pep3-A (v) as a control. (C) Binding of MAb A4F1O to Pep2 (0), native hemolysin (0), Pepl (v), and ovalbumin (V) as the control. The concentrations used to coat the wells were 2 ,uM for the peptides and 50 pM for hemolysin. O.D. 405, optical density at 405 nm.
lacks most of the repeat domain. This mutant hemolysin lacks Ca2"-dependent binding to the erythrocyte membrane but retains its full pore-forming activity when measured in an asolectin lipid bilayer system (23). Two other MAbs (A4G2 and A4G5) raised against native hemolysin also failed to recognize this mutant hemolysin specifically, whereas mutants AL10-1 and AL10-4, which lost only repeat 5 and repeats 5 and 8, respectively, still bound both MAbs (and also MAb D7B12). This suggests that the three MAbs recognize epitopes located in repeat 3, 4, 6, or 7, which are missing in mutant AL10-9. The other six MAbs raised against native hemolysin still bound to all of the mutant hemolysins tested. Mapping of HlyA epitopes recognized by the MAbs. As shown previously, the mutant HlyA proteins used in this investigation are produced and secreted in amounts similar to that of the wild-type hemolysin (23, 25, 30). To localize the binding sites for the MAbs on the HlyA polypeptide chain further, these mutant HlyA proteins (in denatured form) were immunoblotted with the MAbs. In addition, binding of these MAbs to the five CNBr fragments of hemolysin confirmed this rough epitope mapping. The data are summarized in Table 2 and Fig. 4. Previous studies (16, 31) have, by a similar approach, mapped MAbs raised against hemolysin. In this study, we used MAbs raised against native and denatured hemolysin. As shown in Fig. 4, the binding sites for the seven MAbs against native hemolysin seem to be more randomly distributed on the hemolysin molecule than the five MAbs against denatured hemolysin that we tested. A similar concentration of the epitopes for MAbs raised against denatured hemolysin on CNBr fragment D (51 kDa) was also found by the other investigators (16). Identification of the epitopes for MAbs A4F1O and A4G2 by using synthetic peptides. MAbs A4F10 and A4G2 inhibited hemolytic activity to different extents (80 and 50%, respectively, compared with polyclonal anti-HlyA serum). The binding studies described above indicated that A4F10 may recognize an epitope in the hydrophobic region of HlyA between amino acids 300 and 319 which is involved in the pore-forming structure of hemolysin (24). MAb A4G2, on the other hand, seemed to recognize an epitope in the repeat domain between amino acids 740 and 849 of HlyA, presumably, repeat unit 3 or 4 or an overlapping sequence. To localize the sequences recognized by A4F10 and A4G2 more precisely, we used synthetic peptides covering the presump-
tive epitopes and measured the binding of these MAbs to the peptides by using the ELISA described before. Peptide 3 (Pep3) contains amino acids 758 to 774 of HlyA and thus includes repeats 3 and 4, whereas peptides Pep3-A and Pep3-B represent the sequences for repeats 3 and 4 separately. As shown in Fig. 5, MAb A4G2 bound specifically to Pep3 (and hemolysin as a control). Moreover, MAb A4G2 bound more efficiently to Pep3-A than to Pep3-B, indicating that the epitope for A4G2 is either repeat 3 directly or a sequence overlapping repeats 3 and 4. The hydrophobic region between amino acids 300 and 317 is covered by peptides Pepl and Pep2 (Pepl from amino acids 300 to 309 and Pep2 from 310 to 317). While Pepl did not bind MAb A4F10, Pep2 exhibited efficient and specific binding to A4F10 (Fig. 5C). Furthermore, Pep3 inhibited binding of A4G2 to hemolysin when added in increasing amounts and Pep2 competed with hemolysin binding to A4F10 (Fig. 6). Unrelated peptides of lengths similar to those of Pepl, Pep2, and Pep3 or BSA did not bind to the MAbs or interfere with binding of hemolysin to the MAbs (data not shown). This further indicates that the epitopes which A4G2 and A4F10 recognize are very similar to the peptides tested. DISCUSSION We isolated MAbs raised against denatured gel-purified HlyA protein and native hemolysin and tested each MAb in three binding assays: (i) an ELISA which measures MAb binding to native, HlyC-modified hemolysin; (ii) a FACSassisted test which measures antibody binding to a preformed erythrocyte-hemolysin complex; and (iii) immunoblotting of the MAb to hemolysin which was separated on SDS-polyacrylamide gel electrophoresis and hence should represent predominantly denatured HlyA protein. In addition to wild-type hemolysin, we included in this study various HlyA mutants that carry deletions in regions of the HlyA polypeptide known to be involved in Ca2+-dependent binding of hemolysin to erythrocytes (AL10-1, AL10-4, and AL10-9) or pore formation (AL2, AL9, and AL13) (23-25). All 15 MAbs tested bound to hemolysin, but only three inhibited the hemolytic activity significantly. MAbs A4F10 and A4G4 blocked hemolysis by more than 70% in a liquid assay using human erythrocytes (compared with equal concentrations of polyclonal anti-HlyA antibodies). The two MAbs map within one of the three hydrophobic regions of
VOL. 59, 1991
MAbs AGAINST E. COLI ALPHA-HEMOLYSIN O..406
0.6
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A
.
2,6
B 2
0,6
1.6
0,4 0,3 0,2
0,6F
0.1 0E ELISA
w : . ,. ............... 7"
0
Hly
A 3
alone PeptId. 2 alone no 60 el
0.1
1.0
1Ou9/lm
Peptide CompetItIon
Nly A ELISA
a
aone
Peptid.
3
0 ug
alone
10
S0 Ou
I
Pptm. CompeUton
FIG. 6. (A) Inhibition of MAb A4F1O binding to hemolysin by Pep2. Increasing amounts of Pep2 were incubated overnight at 4°C with MAb A4F10 (10 ,ug/ml) and then added to wells precoated with 3 ng of hemolysin. Binding of the MAb to hemolysin was measured by a previously described ELISA (30). (B) Inhibition of MAb A4G2 binding to hemolysin by Pep3. This assay was performed as described above, but the hemolysin concentration was 8 ng/ml and the MAb A4G2 concentration was 15 ,ug/ml. O.D. 405, optical density at 405 nm.
HlyA which represent essential regions for the pore-forming structure of hemolysin (24). The third MAb (A4G2) binds to the repeat domain of HlyA. By using synthetic peptides derived from the presumptive regions of HlyA, the amino acid sequences which are probably very close to the recognized epitopes were determined for A4G2 and A4F10. We obtained not only specific binding of these two MAbs to the corresponding peptides but also specific inhibition of binding of these MAbs to hemolysin when increasing amounts of the competing peptide were added. When hemolysin is bound to erythrocytes, all MAbs have access to their epitopes, although there may be slight differences in accessibility, as demonstrated by the different efficiencies of hemolysin-erythrocyte-antibody complex formation (Fig. 2). This suggests that the domains of HlyA involved in binding to the erythrocyte membrane and pore formation are not fully protected by erythrocytes or that the interaction between hemolysin and erythrocytes is transient enough to allow binding of the MAbs to their epitopes even when hemolysin interacts with the membrane. Alternatively, the HlyA domains which interact with the membrane of the target cell may not be very immunogenic. This latter assumption has been proposed recently (31). These investigators could not identify MAbs which bound to the hydrophobic region and the repeat domain of HlyA. In the present study, we detected two MAbs that bound to the hydrophobic region and two that bound to the repeat region, but all four MAbs were still able to bind to hemolysin when complexed with erythrocytes. These data further support the assumption that binding of hemolysin to the erythrocyte membrane is transient so that a MAb can still recognize its epitope, even when hemolysin is in contact with the erythrocytes. In agreement with previous reports (16, 31),
we also observed clustering of the binding sites for the MAbs in the region between the hydrophobic region and the repeat domain of HlyA when MAbs raised against denatured hemolysin were analyzed. In contrast, the binding sites for the MAbs raised against native hemolysin were more evenly distributed over the HlyA polypeptide. Interestingly, some of the MAbs were unable to bind to native mutant hemolysins which carry deletions in regions remote from their actual binding sites. For instance, MAbs A4G4 and C7D1C1O no longer bound to mutant HlyA proteins with extended deletions in the hydrophobic or
repeat regions of HlyA, although their actual binding sites on
HlyA, determined by immunoblotting against denatured HlyA protein, are located in the hydrophobic region or in a region between the two domains. This suggests that extended deletions within these two regions alter the entire conformation of the mutant hemolysin so that the actual epitope is inaccessible to the MAb. Another interesting finding is the binding behavior of several MAbs to native mutant hemolysin AL13 when it is complexed with erythrocytes. This mutant hemolysin has lost amino acids 9 to 37 at the N-terminal end of HlyA. The consequences of this deletion are a significantly higher specific hemolytic activity and formation of a larger pore with a much longer lifetime than wild-type hemolysin (24). These altered properties suggest higher affinity of this mutant hemolysin for the erythrocyte membrane and may explain why some epitopes on AL13 are inaccessible for their MAbs when AL13 is complexed with erythrocytes. ACKNOWLEDGMENTS We thank M. Wuenscher for critically reading the manuscript and E. Appel for editorial help. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 165 B4). REFERENCES 1. Benz, R., A. Schmid, W. Wagner, and W. Goebel. 1989. Pore formation by the Escherichia coli hemolysin: evidence for an association-dissociation equilibrium of the pore-forming aggregates. Infect. Immun. 57:887-895. 2. Bhakdi, S., and J. Tranum-Jensen. 1987. Damage of mammalian cells by proteins that form transmembrane pores. Rev. Physiol. Biochem. Pharmacol. 107:147-223. 3. Bhakdi, S., and J. Tranum-Jensen. 1988. Damage to cell membranes by pore-forming bacterial cytolysin. Prog. Allergy 40:143. 4. Boehm, D. F., R. A. Welch, and I. S. Snyder. 1990. Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes. Infect. Immun. 58:19591964. 5. Erb, K., M. Vogel, W. Wagner, and W. Goebel. 1987. Alkaline phosphatase which lacks its own signal sequence becomes enzymatically active when fused to N-terminal sequences of E. coli haemolysin. Mol. Gen. Genet. 208:88-93. 6. Felmlee, T., S. Peilet, and R. A. Welch. 1985. Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J. Bacteriol. 163:94-105.
1852
OROPEZA-WEKERLE ET AL.
7. Felmlee, T., and R. A. Welch. 1988. Alterations of amino acid repeats in the Escherichia coli hemolysin affect cytolytic activity and secretion. Proc. Natl. Acad. Sci. USA 85:5269-5273. 7a.Garcia, F. M., T. Jarchau, and W. Goebel. Unpublished data. 8. Grimminger, F. D., R. Walmrath, G. Birkemly, S. Bhakdi, and W. Seeger. 1990. Leukotriene and hydroxyeicosatetraenoic acid generation elicited by low doses of Escherichia coli hemolysin in rabbit lungs. Infect. Immun. 58:2659-2663. 9. Gross, E., and B. Witkop. 1962. Nonenzymatic cleavage of peptide bonds: the methionine residue in bovine pancreatic ribonuclease. J. Biol. Chem. 237:1856-1862. 10. Gustafsson, B., A. Rosen, and T. Holme. 1982. Monoclonal antobodies against Vibrio cholerae lipopolysaccharide. Infect. Immun. 38:449-454. 11. Hacker, J., C. Hughes, H. Hof, and W. Goebel. 1983. Cloned hemolysin genes from Escherichia coli that cause urinary tract infection determine different levels of toxicity in mice. Infect. Immun. 42:57-63. 12. Hacker, J., E. Schroeter, A. Schrettenbrunner, C. Hughes, and W. Goebel. 1983. Hemolytic Escherichia coli strains in human fecal flora as potential urinary pathogens. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 254:370-378. 13. Heijne, G. 1985. Signal sequences. The limits of variation. J. Mol. Biol. 184:99-105. 14. Hess, J., W. Weis, M. Vogel, and W. Goebel. 1986. Nucleotide sequence of a plasmid-encoded hemolysin determinant and its comparison with a corresponding chromosomal hemolysin sequence. FEMS Microbiol. Lett. 34:1-11. 15. Hughes, C., J. Hacker, A. Roberts, and W. Goebel. 1983. Hemolysin production as a virulence marker in symptomatic and asymptomatic urinary tract infections caused by Escherichia coli. Infect. Immun. 39:546-552. 16. Ji, G. E., and P. 0. Hanley. 1990. Epitopes of Escherichia coli alpha-hemolysin: identification of monoclonal antibodies that prevent hemolysis. Infect. Immun. 58:3029-3035. 17. Kearny, J. F. 1984. Hybridomas and monoclonal antibodies. p. 751-756. In W. E. Paul (ed.), Fundamental immunology. Raven Press, New York. 18. Konig, B., W. Konig, J. Scheffer, J. Hacker, and W. Goebel. 1986. Role of E. coli hemolysin and bacterial adherence in infection: requirement for release of inflammatory mediators from granulocytes and mast cells. Infect. Immun. 54:886-892. 19. Kraig, E., T. Dailey, and D. Kolodrubetz. 1990. Nucleotide sequence of the leukotoxin gene from Actinobacillus actinomycetemcomitans: homology to the alpha-hemolysin/leukotoxin gene family. Infect. Immun. 58:920-929. 20. Laemmli, U. K. 1970. Cleavage of structural proteins during the
INFECT. IMMUN.
assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 21. Lally, E. T., I. R. Kieba, D. R. Demuth, J. Rosenbloom, E. E. Golub, N. S. Taichman, and C. W. Gibson. 1989. Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene. Biochem. Biophys. Res. Commun. 159:256-
262. 22. Lo, R. Y. C., C. A. Strathdee, and P. E. Shewen. 1987. Nucleotide sequence of the leukotoxin genes of Pasteurella haemolytica Al. Infect. Immun. 55:1987-1996. 23. Ludwig, A., T. Jarchau, R. Benz, and W. Goebel. 1988. The repeat domain of Escherichia coli haemolysin is responsible for its Ca2+-dependent binding to erythrocytes. Mol. Gen. Genet. 214:553-561. 24. Ludwig, A., A. Schmid, R. Benz, and W. Goebel. Mutations affecting pore formation of haemolysin from Escherichia coli. Mol. Gen. Genet., in press. 25. Ludwig, A., M. Vogel, and W. Goebel. 1987. Mutation affecting activity and transport of haemolysin in Escherichia coli. Mol. Gen. Genet. 206:238-245. 26. Menestrina, G., N. Mackman, I. B. Holland, and S. Bhakdi. 1987. Escherichia coli haemolysin forms voltage dependent ion channels in lipid membranes. Biochim. Biophys. Acta 905:109117. 27. Nicaud, J. M., N. Mackman, L. Gray, and I. B. Holland. 1985. Characterization of HlyC and mechanism of activation and secretion of hemolysin from E. coli 2001. FEBS Lett. 187:339344. 28. Noegel, A., U. Rdest, and W. Goebel. 1981. Determination of the functions of hemolytic plasmid pHlylS2 of Escherichia coli. J. Bacteriol. 145:233-247. 29. Oropeza-Wekerle, R. L., E. Muller, P. Kern, R. Meyermann, and W. Goebel. 1989. Synthesis, inactivation, and location of extracellular and intracellular Escherichia coli hemolysins. J. Bacteriol. 171:2783-2788. 30. Oropeza-Wekerle, R. L., V. Speth, B. Imhof, I. Gentschev, and W. Goebel. 1990. Translocation and compartmentalization of Escherichia coli hemolysin (HlyA). J. Bacteriol. 172:3711-3717. 31. Pellet, S., D. F. Boehm, I. S. Snyder, G. Rowl, and R. A. Welch. 1990. Characterization of monoclonal antibodies against the Escherichia coli hemolysin. Infect. Immun. 58:822-827. 32. Wagner, W., M. Kuhn, and W. Goebel. 1988. Active and inactive forms of hemolysin (HlyA) from E. coli. Biol. Chem. Hoppe-Seyler 369:39-46. 33. Welch, R. A., E. P. Dellinger, B. Minshew, and S. Falkow. 1981. Haemolysin contributes to virulence of extra-intestinal E. coli infections. Nature (London) 294:665-667.
Vol. 59, No. 5
0019-9567/91/051846-07$02.00/0 Copyright X) 1991, American Society for Microbiology
Characterization of Monoclonal Antibodies against Alpha-Hemolysin of Escherichia coli OROPEZA-WEKERLE,l P. KERN,' D. SUN,2 S. MULLER,3 J. P. BRIAND,3 AND W. GOEBEL'* Institute for Microbiology, University of Wurzburg,' and Clinical Research Unit for Multiple Sclerosis, Max Planck Society,2 D-8700 Wurzburg, Federal Republic of Germany, and Biologie Moleculaire et Cellulaire, 67084 Strasbourg Cedex, France3
R. L.
Received 15 November 1990/Accepted 26 February 1991
Monoclonal antibodies (MAbs) were raised against native and denatured alpha-hemolysin (HlyA) of Escherichia coli. Binding of the MAbs to native, denatured, and erythrocyte-complexed active wild-type hemolysin and mutant derivatives was tested. All 15 MAbs analyzed bound to native hemolysin, even when the toxin was complexed with human erythrocytes. While some MAbs were unable to bind to a specific native mutant hemolysin, others could not even bind to mutant hemolysin carrying deletions remote from their actual binding sites. A rough determination of the binding sites of 15 MAbs on HlyA was performed by Western immunoblot analysis using CNBr fragments of HlyA and mutant hemolysin proteins. Interestingly, the binding sites of the MAbs against native hemolysin seem to be more randomly distributed on HlyA than are those of MAbs against denatured hemolysin. Three MAbs inhibited the hemolytic activity significantly. Two of these MAbs bound to the hydrophobic region, and the other one bound to the repeat domain of HlyA. The use of synthetic peptides from these regions allowed determination of the linear epitopes for two of these MAbs.
Escherichia coli alpha-hemolysin (HlyA) is an extracellular protein toxin which is produced predominantly by E. coli strains isolated from patients with pyelonephritis. Hemolysin appears to be an essential virulence factor of such uropathogenic E. coli isolates (11, 12, 15, 33). The cytolytic 110-kDa protein forms pores in erythrocyte membranes of various origins and in artificial lipid bilayer systems (1-3, 26). The diameter of the pores introduced into erythrocyte membranes and lipid bilayers is about 2 nm (1, 26). The average single-channel conductance is 500 pS in 0.15 M KCl, and the mean lifetime of the pore is 2 s at 20 mV (1). The amino acid sequence of HlyA (1,024 amino acids) allows the prediction of several interesting structural domains: (i) an amphiphilic, a-helical N-terminal sequence (amino acids 1 to 40); (ii) a hydrophobic region from amino acids 238 to 410; and (iii) a repeat domain of 11 tandemly arranged repeats of the glycine-rich nine-amino-acid sequence XLXGGXGDD (6, 14). This repeat region is localized in the HlyA protein between amino acids 736 and 849. Recent investigations (7, 19, 21, 22) have indicated that repeat domains with rather conserved basic repeat units but various repeat numbers are common to a collection of different toxins, which is designated the RTX (repeats in toxin) protein family. By using site-specific mutagenesis and monoclonal antibodies (MAbs) against hemolysin (16, 31), part of the functional domains of HlyA was recently unravelled. It was shown (4, 23) that the repeat domain is involved in Ca2+-dependent binding of hemolysin to erythrocytes but not required for pore formation in a lipid bilayer (23). These data suggest that the repeat domain does not directly participate in the pore formation leading to hemolysis but rather may represent the recognition site for an unknown receptor on the erythrocyte membrane. Association of hemolysin with erythrocytes or artificial lipid bilayers is dependent upon modification of HlyA by HlyC (27, 28). Although the *
chemical nature of the component which is covalently linked to a defined amino acid of HlyA (7a, 32) is not known, this component appears to represent an anchor which fixes the modified but not the unmodified HlyA protein to the membrane (29). The function of the a-helical, amphiphilic N-terminal sequence is unclear. Removal of this region, which possesses membrane-seeking properties (13), leads to a mutant hemolysin with increased specific hemolytic activity (24, 25). We have recently proposed a model for the structure of the hemolysin pore (24) which anticipates the possibility that four amphipathic and four hydrophobic a-helical segments of the hydrophobic region of HlyA are inserted into the membrane. On the basis of this model, we have suggested that the amphiphilic N-terminal sequence can compete with the amphipathic transmembrane segments of the pore-forming structure, thereby restricting the lifetime of the hemolysin pore to a short period. The short lifetime may be an important factor for the in vivo function(s) of E. coli hemolysin during infection, since this hemolysin not only exhibits cytolytic activity but also triggers induction of synthesis and release of inflammatory substances, such as leukotrienes, from granulocytes, mast cells, and macrophages (8, 18). The latter activity, caused in vitro by sublytic doses of hemolysin, may require a short lifetime of the pore. Here we report on the isolation and characterization of MAbs raised against native and denatured hemolysin and the binding of these MAbs to wild-type and mutant hemolysins which carry deletions at the N terminus, the hydrophobic region, or the repeat domain of hemolysin. It is shown that some MAbs fail to bind to a specific mutant hemolysin, whereas other MAbs are unable to bind to several mutant hemolysins with extended deletions in various regions of the HlyA protein. By using synthetic peptides, we localized the epitopes of two MAbs which inhibit hemolytic activity. Our data confirm and extend those of previous studies done with HlyA-specific MAbs (16, 31).
Corresponding author. 1846
MAbs AGAINST E. COLI ALPHA-HEMOLYSIN
VOL. 59, 1991
MATERIALS AND METHODS Bacterial strains and plasmids. The E. coli strains and the plasmids that encode the mutant hemolysins used in this study have already been described (23, 24, 30). Isolation of extracellular HlyA protein. An overnight culture of the respective E. coli strains was inoculated into 20 ml of 2x YT medium and incubated with shaking at 37°C. Cells in the logarithmic growth phase were pelleted by centrifugation and discarded. The cell-free supernatant was mixed with 75% ammonium sulfate, and complete precipitation was achieved by incubation of the mixture on ice for 1 h. The pellet was suspended in 10 ml of TCU buffer (20 mM Tris, 150 mM NaCl, 6 M urea, pH 7.0) and dialyzed overnight against TCU buffer. After 24 h, the hemolytic activity was tested and the preparation was stored at -70°C. Equal volumes of hemolysin and sample buffer containing 50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% mercaptoethanol, and 0.5% bromophenol blue were used for SDS-polyacrylamide gel electrophoresis as described by Laemmli (20). Immunoblotting. Hemolysin separated on SDS-10% polyacrylamide gels was transferred to 0.2-pum-pore-size nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, N.H.). Electrophoretic transfer was accomplished overnight at 4°C at 70 mA in 0.025 M Tris buffer (pH 8.3, containing 0.19 M glycine) by using a TE 52 Transphor unit (Hoefer Scientific Instruments, San Francisco, Calif.). Residual protein-binding sites on the membrane were blocked by incubation in 50 mM Tris-buffered saline (pH 7.5) containing 5% bovine serum albumin (BSA) for 60 min at room temperature. This nitrocellulose was transferred to an antibody test solution containing either mouse polyclonal antiserum or mouse monoclonal ascitic fluid, each diluted 1:1,000 in Tris-buffered saline-BSA. The membrane was incubated with the antibody probe for 90 min and then washed four times (10 min each time) in Tris-buffered saline. Reaction of the antibody probes with hemolysin was detected by incubation of the nitrocellulose in horseradish peroxidase-conjugated rabbit anti-mouse antibodies (DAKO PATTS, Roskilde, Denmark) with specificity for either immunoglobulin G or M. After incubation and washing, the nitrocellulose was transferred to a peroxidase substrate solution containing 6 ml of 0.3% 4-chloro-1-naphthol (Sigma), 42 ,ul of 30% H202 (Fischer Scientific Co.), and 94 ml of Tris-buffered saline for detection of horseradish peroxidase on the membrane. Determination of hemolytic activity. Hemolytic activity was determined essentially as described before (29). Antibody production. Male BALB/c mice (8 weeks old) obtained from the Max-Planck-Institut fur Immunbiologie, Freiburg, Federal Republic of Germany, or from the Zentralinstitut fur Versuchstierzucht, Hannover, Federal Republic of Germany, were used as a source of immune lymphocytes for hybridoma production. BALB/c mice were injected with three doses of hemolysin at weekly intervals. The first dose, administered intraperitoneally, contained 10 ,ug of hemolysin per ml in Freund incomplete adjuvant. The second 10-,ug/ml dose of hemolysin in 6 M urea was administered intraperitoneally and subcutaneously. The final injection (5 R.g of hemolysin per ml) was given by tail vein. The mice were killed 2 days postimmunization by cervical dislocation. Lymphocytes were obtained from their axillary, inguinal, brachial, and popliteal lymph nodes and fused with X63-Ag8.653 mouse myeloma cells as previously described (17). For ascites production, mice were sensitized with an intraperitoneal injection of pristane (Aldrich Chemical Co.,
1847
Inc., Milwaukee, Wis.) and 24 h later they were injected with 107 hybridoma cells. After 2 to 3 weeks, ascitic fluid was harvested. Polyclonal sera were prepared by an immunization protocol similar to that described above. ELISA. Mouse sera and ascitic fluid were tested for antibody reactivity by a direct enzyme-linked immunosorbent assay (ELISA). Wells of polyvinyl assay plates (Costar, Cambridge, Mass.) were coated with hemolysin and BSA as previously described (10). Activity of mouse polyclonal or monoclonal antibodies with hemolysin was detected with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins (DAKO PATTS). Binding of the conjugated antibody was detected with ABTS (Serva). Monoclonal antibody subclass determination was done by a similar assay, using a panel of peroxidase-conjugated rabbit immunoglobulins specific for individual mouse antibody subclasses (Miles). When ELISA was used in inhibition experiments with synthetic peptides, the peptides were first incubated overnight at 4°C with 10 ,ug of MAb per ml and the mixture was then added to wells precoated with 3 jig of hemolysin. Binding of MAbs to hemolysin was measured by a previously described ELISA (30). Binding of wild-type and mutant hemolysins to human erythrocytes. Wild-type hemolysin and mutant hemolysins AL2, AL9, AL13, AL10-1, AL10-4, and AL10-9 (23, 24, 29) were isolated from the supernatant of E. coli 5K strains carrying the corresponding plasmids, which all carry hlyC+ hlyB+ hlyD+. Binding of hemolysin was determined in a fluorescence-activated cell sorter (FACS) using mouse polyclonal antibodies or MAbs as the first reagent and fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin antibodies as the second reagent (29). CNBr cleavage. Chemical cleavage of hemolysin with CNBr was performed by the method of Gross and Witkop (9). The protein was dissolved in 1 ml of 70% formic acid containing 100 mg of CNBr (Pierce Chemical Co., Rockford, Ill.). The reaction mixture was dried under vacuum in a Speed Vac centrifuge. The pellet was suspended in water and dried again to remove traces of CNBr and formic acid. Peptide synthesis. Synthetic peptides corresponding to residues 300 to 309 (peptide 1), 310 to 317 (peptide 2), and 758 to 774 (peptide 3) of the hemolysin of E. coli were synthesized by the solid-phase method using an NPS 4000 peptide synthesizer. Peptides 1, 2, 3A, and 3B were coupled to BSA by using glutaraldehyde, and the peptide/carrier ratio was determined by amino acid analysis. To facilitate coupling to the carrier protein in a defined orientation, an additional cysteine was added to peptide 1. Synthesis of peptides was performed at Neosystem (Strasbourg, France).
RESULTS Isolation and characterization of MAbs against native and denatured hemolysin. Eight hemolysin-specific hybridoma clones producing MAbs A4D3, A4D11, A4F9, A4F10, A4G2, A4G4, A4G5, and D7G2 were isolated by fusing mouse myeloma cell line X63-Ag8-653 with lymphocytes from BALB/c mice which were immunized against native hemolysin obtained from E. coli 5K carrying plasmid pANN202-812 (29). In a second fusion, denatured hemolysin, isolated as a 110-kDa protein by SDS-polyacrylamide gel electrophoresis, was used as the antigen. The hemolysinspecific MAbs obtained in this experiment are designated C7D1A8, C7D1C1O, C7D3D5, D7D8E11, C7D3F8, D7B12, and DllH1. A total of about 2,000 hybrids from both fusions were screened. The isolated MAbs belonged to five isotypes
1848
OROPEZA-WEKERLE ET AL.
INFECT. IMMUN.
TABLE 1. MAbs raised against native and denatured E. coli hemolysin Antigen and
A. B.
Class or subclass
MAb
Native hemolysin A4D3 .............. A4D11 ............... A4F9 .............. A4F1O .............. A4G2 ............... A4G4 .............. A4G5 .............. D7G2 ..............
Denatured hemolysin C7D1A8 .............. C7D1C1O .............. C7D3D5 ............... C7D8E11 .............. C7D3F8 .............. D7B12 ............... DllH1 ..............
110-
110-.-
--
.
IgGl IgGl
IgG2b IgM IgM IgGl IgM IgM
1
2
3
4
5
6
S
7
1
2
3
45
6
9
7
FIG. 1. Immunoblots of MAbs raised against native hemolysin (A) and denatured HlyA protein (B) with the 110-kDa HlyA protein. Lane 1 (A and B), binding of polyclonal anti-HlyA antibodies. Lanes in panel A: 2, A4G2; 3, A4G5; 4, D7G2; 5, A4F1O; 6, A4D11; 7, A4D3; 8, A4G4. Lanes in panel B: 2 and 3, D7B12; 3, D7B1O; 4, C7D3D5; 5, C7D1C1O; 6, C7D1A8; 7, C7D3F8; 8, C7D8E11; 9, DllHl.
IgGl IgM IgA IgG2a IgG2a IgGl IgGl
80%, compared with polyclonal anti-HlyA serum at the same immunoglobulin G [IgG] concentration), indicating that the epitopes recognized by most of the MAbs are not directly involved in hemolytic activity, e.g., in binding to erythrocytes and/or pore formation. This assumption is in line with the observation that all MAbs still were able to bind to hemolysin which was bound to human erythrocytes. The assay for measuring antibody binding to the erythrocytehemolysin complex was performed as previously described (29), by using a FACS after coupling the erythrocytehemolysin-MAb complex with rabbit anti-mouse immunoglobulin antibodies conjugated with fluorescein isothiocyanate (Table 2; Fig. 2).
(Table 1). The MAbs were selected by binding to activated hemolysin (HlyA modified by HlyC) in an ELISA. All selected MAbs also reacted in Western blots with the denatured 110-kDa HlyA protein (Table 2; Fig. 1), albeit with different binding efficiencies, indicating that most MAbs recognize linear epitopes on the HlyA protein. Only three of the isolated MAbs (A4G2, A4G4, and A4F10) were able to inhibit hemolytic activity to an appreciable extent (50 to
TABLE 2. MAb binding to native or denatured hemolysin and preformed hemolysin-erythrocyte complexesa Binding to: Wild-type
Antibody
NH
Polyclonal antibody MAbs A4D3 A4D11 A4F9 A4F1O A4G2 A4G4 A4G5 D7G2 C7D1A8 C7D1C1O C7D3D5 C7D8E11 C7D3F8 D7B12 DllHl
Mutant AL13
hemolysin N NHEDH E
-
Mutant AL2
EDH E
Mutant AL9
Mutant
Mutant AL10-4
AL10-1
CNBr
Mutant 10-9
fragment:
NH NH DH DH~NH ~~~ DH~NHH NH DH NH + E DH -E DH NH ~~~~~~ -E ED DH AA B ~~~~~~~~~~~~~~+E
+
+
+
+
+
+
+
-
+
+
-
+
+
+
+
+
-
+
+
-
+
+ + +
+ + + + + + + +
+ + +
+ + +
+ + +
+ + +
+ + +
-
+ + +
+ + +
-
+ + +
+
+
+
+ .
+ + + + + + + + + + +
+ + + + + + + + + + +
+ + + + + +
+ + + + +
+ + + + + +.+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + +
+ + + + +
-
+ + + + -
-
-
-
+ + + + + + + + + + + + + + +
-
-
+ + -
-
+ + + + + + + + + + + + + +
+
+ + + + + + + + + + +
+ + + + + + +
.
.
.
.
.
+ + + + -
-
+ +
C
D
E
+
+
+
ND + + +
+ +
+ +
+ + + +
ND +
ND
a Binding of MAbs to native, HlyC-modified HlyA proteins (NH) was measured in the ELISA described in Materials and Methods. Constant concentrations (50 ng/ml) of the mutant hemolysins were used in the binding assay, and the concentration of the MAb was adjusted to 20 ,ug of protein per ml of ascitic fluid. Binding of hemolysin to a MAb was considered positive at an optical density higher than 0.03. The background optical density was 0.01 or lower. NH-E indicates binding of the MAb to a preformed hemolysin-erythrocyte complex. Formation of hemolysin-erythrocyte-antibody complexes was determined by FACS analysis (29). DH indicates binding of the MAbs to denatured HlyA protein determined by Western blot analysis. Binding of MAbs to CNBr fragments A (16.9 kDa), B (26.0 kDa), C (0.9 kDa), D (51 kDa), and E (14.4 kDa) was also determined by immunoblotting of the separated CNBr fragments with the MAbs. The mutants carried the following deletions: AL13, amino acids 9 to 37; AL2, amino acids 300 to 319; AL9, amino acids 200 to 599; AL10-1, amino acids 775 to 783 and repeat 5; AL10-4, amino acids 775 to 783 and 814 to 822 and repeats 5 and 8; 10-9, amino acids 757 to 822 and repeats 3 to 8. ND, not done.
MAbs AGAINST E. COLI ALPHA-HEMOLYSIN
VOL. 59, 1991 100F
C,D,As
a
C7D,As
b
f LISA
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C7D,A
C
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400
.
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400
400
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.400
Fluorescenc
FIG. 2. FACS analysis of complex formation between wild-type hemolysin and human erythrocytes after binding of polyclonal anti-HlyA antibodies (a), MAb A4G4 (b), MAb A4F10 (c), MAb A4G5 (d), MAb C7D3D5 (e), and MAb D7B12 (f).
Binding of MAbs to native mutant hemolysins. We have recently constructed and characterized several mutant hemolysins which are altered in their pore-forming properties and/or binding to erythrocytes. Some of these mutant hemolysins were tested for binding to the MAbs described. Mutant hemolysin AL13, encoded by pANN202-312AL13* (23), lacks amino acids 9 to 37 from the N-terminal end of HlyA. This hemolysin has threefold higher specific hemolytic activity and forms pores with a much longer lifetime than those formed by wild-type hemolysin (23, 24). As shown in Table 2, all MAbs bound to this mutant hemolysin (in the native or denatured form), but interestingly, five MAbs (A4G2, A4G4, C7D1C1O, C7D8E11, and D7B12) no longer bound to this protein when it was complexed with erythrocytes (Table 2). This suggests that binding of mutant hemolysin AL13 to the erythrocyte membrane protects or alters the epitopes recognized by these five MAbs. Mutant hemolysin AL10-1, produced by E. coli carrying plasmid pANN202-312AL10-1*, lacks repeat 5 within the repeat region of hemolysin (23). This hemolysin has reduced hemolytic activity which can be restored to the wild-type level by higher Ca2+ concentrations (23). As shown in Table 2 and Fig. 3, all MAbs bound to this mutant hemolysin, even when it was complexed with erythrocytes. While the former two mutant hemolysins still possess hemolytic activity, which implies that they are able to bind to the erythrocyte membrane and form pores, a second group of hemolysins, described below, have a loss of hemolytic activity in common. The reason for this lies either in the lack of Ca2+-dependent binding of the mutant hemolysins to the erythrocyte membrane, e.g., mutants AL10-4 and AL10-9, and/or in their inability to form pores, e.g., mutants AL2 and AL9 (23-25). A common property of all four mutant hemolysins is the inability to interact with erythrocytes, as demonstrated by the failure of polyclonal anti-HlyA antibodies and all MAbs to form an erythrocyte-hemolysin-antibody complex detectable in the previously described FACS assay (29). Interestingly, there are four MAbs (A4G4, C7D1C1O, C7D8E11, and D11H1) which failed to bind in an ELISA to the latter four mutant hemolysins (Table 2), although the deletions of the four hemolysins are located in different regions of HlyA (Fig. 4). Two additional MAbs (C7D3D5 and C7D3F8) failed to bind to mutant hemolysins AL9, AL10-4,
FSourescwox
FIG. 3. FACS analysis of complex formation between mutant hemolysins and human erythrocytes and binding of two MAbs (C7D1A8 and A4G4) to the complex. Panels: a and d, binding of C7D1A8 (a) and A4G4 (d) to the erythrocyte-AL13 complex; b and e, binding of C7D1A8 (b) and A4G4 (e) to the erythrocyte-AL2 complex; c and f, binding of C7D1A8 (c) and A4G4 (f) to the erythrocyte-AL10-1 complex. ELISA+ and ELISA- indicate the abilities of the MAbs to bind to mutant hemolysin alone.
and AL10-9 but still bound to AL2. Except for MAb A4G4, all of these MAbs were raised against denatured hemolysin. Only two MAbs raised against denatured HlyA (C7D1A8 and D7B12) did not show this general lack of binding to the nonhemolytic mutant HlyA proteins. Whereas MAb C7D1A8 did not bind to mutant hemolysin AL9, which has lost the hydrophobic region involved in pore formation (24, 25), D7B12 did not bind to mutant hemolysin AL10-9, which
A4D11
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FIG. 4. Summary of approximate epitope localizations of the MAbs mapped with various deletion mutants of HlyA and the CNBr fragments of wild-type hemolysin (A to E). The deletions of the mutants are indicated in the lower part (numbers in parentheses indicate deleted amino acids [AA]). The upper line represents the primary amino acid sequence of HlyA (14); the numbers above the line give the amino acid positions. The three open boxes between amino acids 230 and 410 indicate the hydrophobic region, and the hatched boxes represent the repeat domain of HlyA (amino acids 740 to 849) (23). The symbol A marks the CNBr cleavage sites. A to E represent the following CNBr fragments: A, 16.9 kDa; B, 26.0 kDa; C, 0.9 kDa; D, 51 kDa; E, 14.4 kDa. Peptides Pepl, Pep2, Pep3, Pep3-A, and Pep3-B were used for characterization of the epitopes recognized by MAbs A4F10 and A4G2.
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OROPEZA-WEKERLE ET AL.
INFECT. IMMUN. O.D. 405
A
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0.62
1.25
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FIG. 5. Binding of MAbs A4G2 and A4F1O to specific HlyA-derived peptides, determined by ELISA (30). (A) Binding of A4G2 to Pep3 (amino acids 758 to 774 of HlyA) (0), native hemolysin (0), and Pepl (amino acids to 300 to 309) as the control (V). (B) Binding of A4G2 to Pep3-A (0) and Pep3-B (0), with Pepl (V) as a control and unspecific serum with Pep3-A (v) as a control. (C) Binding of MAb A4F1O to Pep2 (0), native hemolysin (0), Pepl (v), and ovalbumin (V) as the control. The concentrations used to coat the wells were 2 ,uM for the peptides and 50 pM for hemolysin. O.D. 405, optical density at 405 nm.
lacks most of the repeat domain. This mutant hemolysin lacks Ca2"-dependent binding to the erythrocyte membrane but retains its full pore-forming activity when measured in an asolectin lipid bilayer system (23). Two other MAbs (A4G2 and A4G5) raised against native hemolysin also failed to recognize this mutant hemolysin specifically, whereas mutants AL10-1 and AL10-4, which lost only repeat 5 and repeats 5 and 8, respectively, still bound both MAbs (and also MAb D7B12). This suggests that the three MAbs recognize epitopes located in repeat 3, 4, 6, or 7, which are missing in mutant AL10-9. The other six MAbs raised against native hemolysin still bound to all of the mutant hemolysins tested. Mapping of HlyA epitopes recognized by the MAbs. As shown previously, the mutant HlyA proteins used in this investigation are produced and secreted in amounts similar to that of the wild-type hemolysin (23, 25, 30). To localize the binding sites for the MAbs on the HlyA polypeptide chain further, these mutant HlyA proteins (in denatured form) were immunoblotted with the MAbs. In addition, binding of these MAbs to the five CNBr fragments of hemolysin confirmed this rough epitope mapping. The data are summarized in Table 2 and Fig. 4. Previous studies (16, 31) have, by a similar approach, mapped MAbs raised against hemolysin. In this study, we used MAbs raised against native and denatured hemolysin. As shown in Fig. 4, the binding sites for the seven MAbs against native hemolysin seem to be more randomly distributed on the hemolysin molecule than the five MAbs against denatured hemolysin that we tested. A similar concentration of the epitopes for MAbs raised against denatured hemolysin on CNBr fragment D (51 kDa) was also found by the other investigators (16). Identification of the epitopes for MAbs A4F1O and A4G2 by using synthetic peptides. MAbs A4F10 and A4G2 inhibited hemolytic activity to different extents (80 and 50%, respectively, compared with polyclonal anti-HlyA serum). The binding studies described above indicated that A4F10 may recognize an epitope in the hydrophobic region of HlyA between amino acids 300 and 319 which is involved in the pore-forming structure of hemolysin (24). MAb A4G2, on the other hand, seemed to recognize an epitope in the repeat domain between amino acids 740 and 849 of HlyA, presumably, repeat unit 3 or 4 or an overlapping sequence. To localize the sequences recognized by A4F10 and A4G2 more precisely, we used synthetic peptides covering the presump-
tive epitopes and measured the binding of these MAbs to the peptides by using the ELISA described before. Peptide 3 (Pep3) contains amino acids 758 to 774 of HlyA and thus includes repeats 3 and 4, whereas peptides Pep3-A and Pep3-B represent the sequences for repeats 3 and 4 separately. As shown in Fig. 5, MAb A4G2 bound specifically to Pep3 (and hemolysin as a control). Moreover, MAb A4G2 bound more efficiently to Pep3-A than to Pep3-B, indicating that the epitope for A4G2 is either repeat 3 directly or a sequence overlapping repeats 3 and 4. The hydrophobic region between amino acids 300 and 317 is covered by peptides Pepl and Pep2 (Pepl from amino acids 300 to 309 and Pep2 from 310 to 317). While Pepl did not bind MAb A4F10, Pep2 exhibited efficient and specific binding to A4F10 (Fig. 5C). Furthermore, Pep3 inhibited binding of A4G2 to hemolysin when added in increasing amounts and Pep2 competed with hemolysin binding to A4F10 (Fig. 6). Unrelated peptides of lengths similar to those of Pepl, Pep2, and Pep3 or BSA did not bind to the MAbs or interfere with binding of hemolysin to the MAbs (data not shown). This further indicates that the epitopes which A4G2 and A4F10 recognize are very similar to the peptides tested. DISCUSSION We isolated MAbs raised against denatured gel-purified HlyA protein and native hemolysin and tested each MAb in three binding assays: (i) an ELISA which measures MAb binding to native, HlyC-modified hemolysin; (ii) a FACSassisted test which measures antibody binding to a preformed erythrocyte-hemolysin complex; and (iii) immunoblotting of the MAb to hemolysin which was separated on SDS-polyacrylamide gel electrophoresis and hence should represent predominantly denatured HlyA protein. In addition to wild-type hemolysin, we included in this study various HlyA mutants that carry deletions in regions of the HlyA polypeptide known to be involved in Ca2+-dependent binding of hemolysin to erythrocytes (AL10-1, AL10-4, and AL10-9) or pore formation (AL2, AL9, and AL13) (23-25). All 15 MAbs tested bound to hemolysin, but only three inhibited the hemolytic activity significantly. MAbs A4F10 and A4G4 blocked hemolysis by more than 70% in a liquid assay using human erythrocytes (compared with equal concentrations of polyclonal anti-HlyA antibodies). The two MAbs map within one of the three hydrophobic regions of
VOL. 59, 1991
MAbs AGAINST E. COLI ALPHA-HEMOLYSIN O..406
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1851
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FIG. 6. (A) Inhibition of MAb A4F1O binding to hemolysin by Pep2. Increasing amounts of Pep2 were incubated overnight at 4°C with MAb A4F10 (10 ,ug/ml) and then added to wells precoated with 3 ng of hemolysin. Binding of the MAb to hemolysin was measured by a previously described ELISA (30). (B) Inhibition of MAb A4G2 binding to hemolysin by Pep3. This assay was performed as described above, but the hemolysin concentration was 8 ng/ml and the MAb A4G2 concentration was 15 ,ug/ml. O.D. 405, optical density at 405 nm.
HlyA which represent essential regions for the pore-forming structure of hemolysin (24). The third MAb (A4G2) binds to the repeat domain of HlyA. By using synthetic peptides derived from the presumptive regions of HlyA, the amino acid sequences which are probably very close to the recognized epitopes were determined for A4G2 and A4F10. We obtained not only specific binding of these two MAbs to the corresponding peptides but also specific inhibition of binding of these MAbs to hemolysin when increasing amounts of the competing peptide were added. When hemolysin is bound to erythrocytes, all MAbs have access to their epitopes, although there may be slight differences in accessibility, as demonstrated by the different efficiencies of hemolysin-erythrocyte-antibody complex formation (Fig. 2). This suggests that the domains of HlyA involved in binding to the erythrocyte membrane and pore formation are not fully protected by erythrocytes or that the interaction between hemolysin and erythrocytes is transient enough to allow binding of the MAbs to their epitopes even when hemolysin interacts with the membrane. Alternatively, the HlyA domains which interact with the membrane of the target cell may not be very immunogenic. This latter assumption has been proposed recently (31). These investigators could not identify MAbs which bound to the hydrophobic region and the repeat domain of HlyA. In the present study, we detected two MAbs that bound to the hydrophobic region and two that bound to the repeat region, but all four MAbs were still able to bind to hemolysin when complexed with erythrocytes. These data further support the assumption that binding of hemolysin to the erythrocyte membrane is transient so that a MAb can still recognize its epitope, even when hemolysin is in contact with the erythrocytes. In agreement with previous reports (16, 31),
we also observed clustering of the binding sites for the MAbs in the region between the hydrophobic region and the repeat domain of HlyA when MAbs raised against denatured hemolysin were analyzed. In contrast, the binding sites for the MAbs raised against native hemolysin were more evenly distributed over the HlyA polypeptide. Interestingly, some of the MAbs were unable to bind to native mutant hemolysins which carry deletions in regions remote from their actual binding sites. For instance, MAbs A4G4 and C7D1C1O no longer bound to mutant HlyA proteins with extended deletions in the hydrophobic or
repeat regions of HlyA, although their actual binding sites on
HlyA, determined by immunoblotting against denatured HlyA protein, are located in the hydrophobic region or in a region between the two domains. This suggests that extended deletions within these two regions alter the entire conformation of the mutant hemolysin so that the actual epitope is inaccessible to the MAb. Another interesting finding is the binding behavior of several MAbs to native mutant hemolysin AL13 when it is complexed with erythrocytes. This mutant hemolysin has lost amino acids 9 to 37 at the N-terminal end of HlyA. The consequences of this deletion are a significantly higher specific hemolytic activity and formation of a larger pore with a much longer lifetime than wild-type hemolysin (24). These altered properties suggest higher affinity of this mutant hemolysin for the erythrocyte membrane and may explain why some epitopes on AL13 are inaccessible for their MAbs when AL13 is complexed with erythrocytes. ACKNOWLEDGMENTS We thank M. Wuenscher for critically reading the manuscript and E. Appel for editorial help. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 165 B4). REFERENCES 1. Benz, R., A. Schmid, W. Wagner, and W. Goebel. 1989. Pore formation by the Escherichia coli hemolysin: evidence for an association-dissociation equilibrium of the pore-forming aggregates. Infect. Immun. 57:887-895. 2. Bhakdi, S., and J. Tranum-Jensen. 1987. Damage of mammalian cells by proteins that form transmembrane pores. Rev. Physiol. Biochem. Pharmacol. 107:147-223. 3. Bhakdi, S., and J. Tranum-Jensen. 1988. Damage to cell membranes by pore-forming bacterial cytolysin. Prog. Allergy 40:143. 4. Boehm, D. F., R. A. Welch, and I. S. Snyder. 1990. Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes. Infect. Immun. 58:19591964. 5. Erb, K., M. Vogel, W. Wagner, and W. Goebel. 1987. Alkaline phosphatase which lacks its own signal sequence becomes enzymatically active when fused to N-terminal sequences of E. coli haemolysin. Mol. Gen. Genet. 208:88-93. 6. Felmlee, T., S. Peilet, and R. A. Welch. 1985. Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J. Bacteriol. 163:94-105.
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7. Felmlee, T., and R. A. Welch. 1988. Alterations of amino acid repeats in the Escherichia coli hemolysin affect cytolytic activity and secretion. Proc. Natl. Acad. Sci. USA 85:5269-5273. 7a.Garcia, F. M., T. Jarchau, and W. Goebel. Unpublished data. 8. Grimminger, F. D., R. Walmrath, G. Birkemly, S. Bhakdi, and W. Seeger. 1990. Leukotriene and hydroxyeicosatetraenoic acid generation elicited by low doses of Escherichia coli hemolysin in rabbit lungs. Infect. Immun. 58:2659-2663. 9. Gross, E., and B. Witkop. 1962. Nonenzymatic cleavage of peptide bonds: the methionine residue in bovine pancreatic ribonuclease. J. Biol. Chem. 237:1856-1862. 10. Gustafsson, B., A. Rosen, and T. Holme. 1982. Monoclonal antobodies against Vibrio cholerae lipopolysaccharide. Infect. Immun. 38:449-454. 11. Hacker, J., C. Hughes, H. Hof, and W. Goebel. 1983. Cloned hemolysin genes from Escherichia coli that cause urinary tract infection determine different levels of toxicity in mice. Infect. Immun. 42:57-63. 12. Hacker, J., E. Schroeter, A. Schrettenbrunner, C. Hughes, and W. Goebel. 1983. Hemolytic Escherichia coli strains in human fecal flora as potential urinary pathogens. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 254:370-378. 13. Heijne, G. 1985. Signal sequences. The limits of variation. J. Mol. Biol. 184:99-105. 14. Hess, J., W. Weis, M. Vogel, and W. Goebel. 1986. Nucleotide sequence of a plasmid-encoded hemolysin determinant and its comparison with a corresponding chromosomal hemolysin sequence. FEMS Microbiol. Lett. 34:1-11. 15. Hughes, C., J. Hacker, A. Roberts, and W. Goebel. 1983. Hemolysin production as a virulence marker in symptomatic and asymptomatic urinary tract infections caused by Escherichia coli. Infect. Immun. 39:546-552. 16. Ji, G. E., and P. 0. Hanley. 1990. Epitopes of Escherichia coli alpha-hemolysin: identification of monoclonal antibodies that prevent hemolysis. Infect. Immun. 58:3029-3035. 17. Kearny, J. F. 1984. Hybridomas and monoclonal antibodies. p. 751-756. In W. E. Paul (ed.), Fundamental immunology. Raven Press, New York. 18. Konig, B., W. Konig, J. Scheffer, J. Hacker, and W. Goebel. 1986. Role of E. coli hemolysin and bacterial adherence in infection: requirement for release of inflammatory mediators from granulocytes and mast cells. Infect. Immun. 54:886-892. 19. Kraig, E., T. Dailey, and D. Kolodrubetz. 1990. Nucleotide sequence of the leukotoxin gene from Actinobacillus actinomycetemcomitans: homology to the alpha-hemolysin/leukotoxin gene family. Infect. Immun. 58:920-929. 20. Laemmli, U. K. 1970. Cleavage of structural proteins during the
INFECT. IMMUN.
assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 21. Lally, E. T., I. R. Kieba, D. R. Demuth, J. Rosenbloom, E. E. Golub, N. S. Taichman, and C. W. Gibson. 1989. Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene. Biochem. Biophys. Res. Commun. 159:256-
262. 22. Lo, R. Y. C., C. A. Strathdee, and P. E. Shewen. 1987. Nucleotide sequence of the leukotoxin genes of Pasteurella haemolytica Al. Infect. Immun. 55:1987-1996. 23. Ludwig, A., T. Jarchau, R. Benz, and W. Goebel. 1988. The repeat domain of Escherichia coli haemolysin is responsible for its Ca2+-dependent binding to erythrocytes. Mol. Gen. Genet. 214:553-561. 24. Ludwig, A., A. Schmid, R. Benz, and W. Goebel. Mutations affecting pore formation of haemolysin from Escherichia coli. Mol. Gen. Genet., in press. 25. Ludwig, A., M. Vogel, and W. Goebel. 1987. Mutation affecting activity and transport of haemolysin in Escherichia coli. Mol. Gen. Genet. 206:238-245. 26. Menestrina, G., N. Mackman, I. B. Holland, and S. Bhakdi. 1987. Escherichia coli haemolysin forms voltage dependent ion channels in lipid membranes. Biochim. Biophys. Acta 905:109117. 27. Nicaud, J. M., N. Mackman, L. Gray, and I. B. Holland. 1985. Characterization of HlyC and mechanism of activation and secretion of hemolysin from E. coli 2001. FEBS Lett. 187:339344. 28. Noegel, A., U. Rdest, and W. Goebel. 1981. Determination of the functions of hemolytic plasmid pHlylS2 of Escherichia coli. J. Bacteriol. 145:233-247. 29. Oropeza-Wekerle, R. L., E. Muller, P. Kern, R. Meyermann, and W. Goebel. 1989. Synthesis, inactivation, and location of extracellular and intracellular Escherichia coli hemolysins. J. Bacteriol. 171:2783-2788. 30. Oropeza-Wekerle, R. L., V. Speth, B. Imhof, I. Gentschev, and W. Goebel. 1990. Translocation and compartmentalization of Escherichia coli hemolysin (HlyA). J. Bacteriol. 172:3711-3717. 31. Pellet, S., D. F. Boehm, I. S. Snyder, G. Rowl, and R. A. Welch. 1990. Characterization of monoclonal antibodies against the Escherichia coli hemolysin. Infect. Immun. 58:822-827. 32. Wagner, W., M. Kuhn, and W. Goebel. 1988. Active and inactive forms of hemolysin (HlyA) from E. coli. Biol. Chem. Hoppe-Seyler 369:39-46. 33. Welch, R. A., E. P. Dellinger, B. Minshew, and S. Falkow. 1981. Haemolysin contributes to virulence of extra-intestinal E. coli infections. Nature (London) 294:665-667.
