A Receptor-binding Region in Escherichia coli α-Haemolysin

Escherichia coli α-hemolysin (HlyA) is a 107-kDa protein toxin with a wide range of mammalian target cells. Previous work has shown that glycophorin is a specific receptor for HlyA in red blood cells (Cortajarena, A. L., Goñi, F. M., and Ostolaza, H. (2001) J. Biol. Chem. 276, 12513–12519). The present study was aimed at identifying the glycophorin-binding region in the toxin. Data in the literature pointed to a short amino acid sequence near the C terminus as a putative receptor-binding domain. Previous sequence analyses of several homologous toxins that belong, like HlyA, to the so-called RTX toxin family revealed a conserved region that corresponded to residues 914–936 of HlyA. We therefore prepared a deletion mutant lacking these residues (HlyAΔ914–936) and found that its hemolytic activity was decreased by 10,000-fold with respect to the wild type. This deletion mutant was virtually unable to bind human and horse red blood cells or to bind pure glycophorin in an affinity column. The peptide Trp914–Arg936 had no lytic activity of its own, but it could bind glycophorin reconstituted in lipid vesicles. Moreover, the peptide Trp914–Arg936 protected red blood cells from hemolysis induced by wild type HlyA. It was concluded that amino acid residues 914–936 constitute a major receptor-binding region in α-hemolysin. Escherichia coli α-hemolysin (HlyA) is a 107-kDa protein toxin with a wide range of mammalian target cells. Previous work has shown that glycophorin is a specific receptor for HlyA in red blood cells (Cortajarena, A. L., Goñi, F. M., and Ostolaza, H. (2001) J. Biol. Chem. 276, 12513–12519). The present study was aimed at identifying the glycophorin-binding region in the toxin. Data in the literature pointed to a short amino acid sequence near the C terminus as a putative receptor-binding domain. Previous sequence analyses of several homologous toxins that belong, like HlyA, to the so-called RTX toxin family revealed a conserved region that corresponded to residues 914–936 of HlyA. We therefore prepared a deletion mutant lacking these residues (HlyAΔ914–936) and found that its hemolytic activity was decreased by 10,000-fold with respect to the wild type. This deletion mutant was virtually unable to bind human and horse red blood cells or to bind pure glycophorin in an affinity column. The peptide Trp914–Arg936 had no lytic activity of its own, but it could bind glycophorin reconstituted in lipid vesicles. Moreover, the peptide Trp914–Arg936 protected red blood cells from hemolysis induced by wild type HlyA. It was concluded that amino acid residues 914–936 constitute a major receptor-binding region in α-hemolysin. α-Hemolysin (HlyA) 1The abbreviations used are: HlyA, α-haemolysin; HlyA Δ914–936, a deletion mutant of HlyA lacking amino acid residues 914–936; WR peptide, a peptide including amino acids Trp914–Arg936 of α-haemolysin; WT, wild type.1The abbreviations used are: HlyA, α-haemolysin; HlyA Δ914–936, a deletion mutant of HlyA lacking amino acid residues 914–936; WR peptide, a peptide including amino acids Trp914–Arg936 of α-haemolysin; WT, wild type. is a 107-kDa protein toxin secreted by pathogenic strains of Escherichia coli. It is a member of the so-called “RTX family,” a group of proteins characterized by the presence of a Gly- and Asp-rich nonapeptide sequence repeated in tandem near the protein C terminus (for reviews, see Refs. 1Coote J.G. Rev. Med. Microbiol. 1996; 7: 53-62Crossref Scopus (21) Google Scholar, 2Goñi F.M. Ostolaza H. Braz. J. Med. Biol. Res. 1998; 31: 1019-1034Crossref PubMed Scopus (31) Google Scholar, 3Stanley P. Koronakis V. Hughes C. Microbiol. Mol. Biol. Rev. 1998; 62: 309-333Crossref PubMed Google Scholar, 4Young J. Holland I.B. Biochim. Biophys. Acta. 1999; 1461: 177-200Crossref PubMed Scopus (130) Google Scholar, 5Welch R.A. Curr. Top. Microbiol. Inmunol. 2001; 257: 85-111PubMed Google Scholar). These repeats constitute a Ca2+-binding domain whose structure has been solved at high resolution for a non-toxin member of the RTX family, the alkaline protease from Pseudomonas aeruginosa (6Baumann U. Wu S. Flaherty K. McKay D. EMBO J. 1993; 12: 3357-3364Crossref PubMed Scopus (421) Google Scholar). HlyA first binds a receptor on the cell surface, a β2-integrin in leukocytes (7Lally E.T. Kieba I.R. Sato A. Green C.L. Rosenbloom J. Korostoft J. Wang J.F. Shenker B.L. Ortlepp S. Robinson M.K. Billings P.C. J. Biol. Chem. 1997; 272: 30463-30469Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) or glycophorin in red blood cells (8Cortajarena A.L. Goñi F.M. Ostolaza H. J. Biol. Chem. 2001; 276: 12513-12519Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and then becomes inserted in the cell membrane. Recent data indicate that insertion may take place in the absence of Ca2+ (9Schindel C. Zitzer A. Schulte B. Gerhards A. Stanley P. Hughes C. Koronakis V. Bhakdi S. Palmer M. Eur. J. Biochem. 2001; 268: 800-808Crossref PubMed Scopus (48) Google Scholar, 10Hyland C. Vuillard Hughes C. Koronakis V. J. Bacteriol. 2001; 183: 5364-5370Crossref PubMed Scopus (45) Google Scholar), but Ca2+ binding to the nonapeptide repeat domain is essential for membrane lysis (9Schindel C. Zitzer A. Schulte B. Gerhards A. Stanley P. Hughes C. Koronakis V. Bhakdi S. Palmer M. Eur. J. Biochem. 2001; 268: 800-808Crossref PubMed Scopus (48) Google Scholar, 11Ostolaza H. Soloaga A. Goñi F.M. Eur. J. Biochem. 1995; 228: 39-44PubMed Google Scholar, 12Bakás L. Veiga M.P. Soloaga A. Ostolaza H. Goñi F.M. Biochim. Biophys. Acta. 1998; 1368: 225-234Crossref PubMed Scopus (48) Google Scholar). Note that the Ca2+-binding domain is located near the protein C terminus, whereas the membrane insertion domain is located near the N terminus (9Schindel C. Zitzer A. Schulte B. Gerhards A. Stanley P. Hughes C. Koronakis V. Bhakdi S. Palmer M. Eur. J. Biochem. 2001; 268: 800-808Crossref PubMed Scopus (48) Google Scholar, 10Hyland C. Vuillard Hughes C. Koronakis V. J. Bacteriol. 2001; 183: 5364-5370Crossref PubMed Scopus (45) Google Scholar, 13Soloaga A. Veiga M.P. García-Segura L.M. Ostolaza H. Brasseur R. Goñi F.M. Mol. Microbiol. 1999; 31: 1013-1024Crossref PubMed Scopus (49) Google Scholar). The present study is devoted to exploring the early stages of HlyA interaction with the target cell, namely its binding to the surface receptor. In particular, our investigation is aimed at the region(s) of the protein that bind(s) the receptor glycophorin on mammalian erythrocytes (8Cortajarena A.L. Goñi F.M. Ostolaza H. J. Biol. Chem. 2001; 276: 12513-12519Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). A number of previous studies suggest that a region between the repeat domain and the C terminus may be involved in binding this specific receptor. For example, Bejerano et al. (14Bejerano M. Nisan I. Ludwig A. Goebel W. Hanski E. Mol. Microbiol. 1999; 31: 381-392Crossref PubMed Scopus (35) Google Scholar) have described two amino acid “blocks,” located after the nonapeptide repeats, in the C terminus of the adenylate cyclase toxin (another member of the RTX family). Block A (15 amino acids) is essential for the toxic activity since it is required for the toxin binding and insertion into the membrane. Deletion of block B, however, does not affect the toxin activity. HlyA possesses homologous A and B blocks (Fig. 1), and it has been shown (14Bejerano M. Nisan I. Ludwig A. Goebel W. Hanski E. Mol. Microbiol. 1999; 31: 381-392Crossref PubMed Scopus (35) Google Scholar) that deletion of a region that includes the last two residues of block A, the connection between the blocks, and the first nine amino acids of block B abolishes the hemolytic activity. Previously, Chervaux and Holland (15Chervaux C. Holland I.B. J. Bacteriol. 1996; 178: 1232-1236Crossref PubMed Google Scholar) had shown that five point mutations in that region (residues 918, 920, 921, 928, and 932) inhibited hemolysis without affecting protein export. Leukotoxin from Pasteurella hemolytica, also a RTX toxin, can be neutralized by incubation with a specific antibody whose epitope is located in the C-terminal region of leukotoxin between residues 841–872 (16Lainson F.A. Murray J. Davies R.C. Donachie W. Microbiology (Read.). 1996; 142: 2499-2507Crossref PubMed Scopus (25) Google Scholar). This epitope appears to be related to the binding of the toxin to the β2-integrin receptor in the target cell (17Sun Y. Clinkenbeard K.D. Cudd L. Clarke C. Clinkenbeard P. Infect. Immun. 1999; 67: 6264-6269Crossref PubMed Google Scholar). The sequence of the epitope overlaps partially blocks A and B: 841WFREADFAKEVPNYKATKDEK IEEIIGQNGER872. Another RTX toxin, leukotoxin from Actinobacillus actinomycetemcomitans, contains an epitope, recognized by neutralizing antibodies, that begins 78 residues after the last repeat and overlaps partially with the sequence given above (18Lally E.T. Golub E.E. Kieba I.R. J. Biol. Chem. 1994; 269: 31289-31295Abstract Full Text PDF PubMed Google Scholar). Finally, in the adenylate cyclase toxin, deletion of the last 75 residues abolishes both the lytic and cell binding activities (19Iwaki M. Ullmann A. Sebo P. Mol. Microbiol. 1995; 17: 1015-1024Crossref PubMed Scopus (61) Google Scholar). The deleted region contains a sequence homologous to the leukotoxin epitope mentioned above. In view of the above findings, we performed a sequence analysis on members of the RTX family, searching for peptides homologous to the 841–872 epitope of leukotoxin, with the result that this sequence was found to be highly conserved among members of the family, including HlyA (residues 914–936). A mutant HlyA lacking this peptide (HlyA Δ914–936) was prepared and found to be incapable of binding erythrocytes or purified glycophorin. However, the 914–936 peptide (WR peptide) did bind pure glycophorin and protected red blood cells from HlyA hemolysis. Thus, residues 914–936 of HlyA appear to be essential for the toxin to bind its erythrocyte receptor, glycophorin. Materials—Horse red blood cells were supplied by Biomedics (Alcobendas, Spain). Human erythrocytes were obtained from a local blood bank. Glycophorin from horse or human erythrocyte ghosts was purified as described previously (8Cortajarena A.L. Goñi F.M. Ostolaza H. J. Biol. Chem. 2001; 276: 12513-12519Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Egg phosphatidylcholine was Grade I from Lipid Products (South Nutfield, England). Rhodamine-phosphatidylethanolamine was purchased from Avanti Polar Lipids (Alabaster, AL). Hi-Trap desalting, Superdex HR-200, and N-hydroxysuccinimideactivated Hi-Trap columns were supplied by Amersham Biosciences. Oligonucleotides were synthesized by Amersham Biosciences. Taq polymerase was purchased from Bioline (London, UK). Restriction enzymes NcoI, PacI, and the T4 DNA ligase were from New England Biolabs (Hertforshire, UK). The restriction enzyme BseA1 was supplied by Roche Applied Science. WR peptide was synthesized, with >80% purity, by the Instituto de Inmunología de Colombia (Bogotá, Colombia). Mutagenesis of HlyA—The deletion mutant, lacking residues 914–936 of HlyA, was generated using the polymerase chain reaction. In the first step, fragments upstream and downstream of the deletion were amplified, in two separate reactions, using the flanking primers and the primers A and B, which flank the region to be deleted and introduce a BseA1 new restriction site. The two fragments were then digested with BseA1 and ligated. The resulting fragment was digested with NcoI and PacI and cloned into pSU124 restricted with the same restriction enzymes. The resulting plasmid contained an inserted fragment lacking the 914–936 region and with the new restriction endonuclease site. Deletion was confirmed by DNA sequencing as follows: flanking primers, 5′-GATATCTTCCATGGCGCGG-3′ and 5′-GATTTCATTAATTAATGGATTA-3′; primers (Δ914–936) A, 5′-ACCTGAATCCGGAGTGATTATGTTCCTGAATGTAATACCAT-3′, and B, 5′-ATAATCACTCCGGATTCCCTTAA-3′. The newly introduced restriction site is printed in bold. Protein Purification and Storage—Wild type HlyA and HlyA Δ914–936 were expressed in an E. coli D1210 strain containing plasmid pSU124 and purified as described by Ostolaza et al. (20Ostolaza H. Bartolomé B. Serra J.L. de la Cruz F. Goñi F.M. FEBS Lett. 1991; 280: 195-198Crossref PubMed Scopus (45) Google Scholar) for the wild type. The proteins were stored at –20°C in 150 mm NaCl, 6 m urea, 20 mm Tris-HCl, pH 7.0 buffer. Hemolysis Assays—A standard red blood cell suspension was used, obtained by diluting the erythrocytes with saline so that 37.5 μl of the mixture in 3 ml of distilled water gave an absorbance of 0.6 at 412 nm. Equal volumes of the standard suspension of washed human or horse erythrocytes were added to serial 2-fold dilutions of hemolysin in hemolysis buffer (150 mm NaCl, 10 mm CaCl2, 20 mm Tris-HCl, pH 7.0) in a microtiter plate. The mixtures were incubated at room temperature for a few hours so that erythrocyte sedimentation occurred. The absorbance of the supernatants, appropriately diluted with distilled water, was measured at 412 nm. The blank (zero hemolysis) consisted of a mixture of equal volumes of buffer and erythrocytes. Toxin Binding to Erythrocytes—Erythrocytes were washed and resuspended in hemolysis buffer at 2 × 108 cells/ml. The appropriate amounts of WT (wild type) or mutant HlyA were added, and the mixture was incubated at 37 °C for 30 min. The cells were then centrifuged at 14,000 × g, for 10 min at room temperature. The pelleted cells were lysed at 4 °C with 5 mm phosphate buffer, pH 8.0, and washed in the same buffer by centrifugation (14,000 × g, 10 min, 4 °C). The red blood cell membranes were resuspended in the same volume of 4% (w/v) SDS, 4% (w/v) glycerol, 0.02% (w/v) bromphenol blue, 100 mm 1,4-dithiothreitol, 50 mm Tris-HCl, pH 6.8 and boiled for 5 min. These samples were subjected to SDS-PAGE and then transferred to nitrocellulose by the method of Towbin et al. (21Towbin H. Staehlin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4359Crossref PubMed Scopus (44841) Google Scholar). Blots were blocked with 10% skim milk in TBST buffer (150 mm NaCl, 0.05% Tween 20 (w/v), 10 mm Tris-HCl, pH 7.5) for 2 h at room temperature. They were then incubated with a solution containing a polyclonal rabbit anti-hemolysin antibody (1:1,000) in 5% skim milk/TBST overnight at 4 °C, washed with TBST buffer, and finally reacted with peroxidase-conjugated anti-rabbit Ig antibody (Sigma) (1:2,000) in TBST buffer with 5% skim milk for 1 h at room temperature. Immunoblots were developed by a chemiluminescent method (ECL, Amersham Biosciences). Toxin Binding to Phospholipid Vesicles—Toxin binding to large unilamellar vesicles composed of egg phosphatidylcholine was assayed by the flotation method of Pereira et al. (22Pereira F.B. Goñi F.M. Nieva J.L. FEBS Lett. 1995; 362: 243-246Crossref PubMed Scopus (75) Google Scholar). The vesicles were prepared (23Mayer L.D. Hope M.J. Cullis P.R. Biochim. Biophys. Acta. 1986; 858: 161-168Crossref PubMed Scopus (1565) Google Scholar) containing 0.6 mole percentage of rhodamine-phosphatidylethanolamine and diluted to 250 μm in a D2O buffer (150 mm NaCl, 10 mm CaCl2,20mm Tris-HCl, pH 7.0). Liposomes were incubated with protein for 1 h at 25 °C at 1:2,500 and 1:5,000 protein:lipid molar ratios. Then liposome-bound and non-bound proteins were separated by ultracentrifugation in a TLA 120.2 Beckman rotor × g, 2 20 °C). Liposomes containing protein on of the buffer. This was were in and lipid and protein were by Lipid was measured at and nm. Protein was measured at and nm. Toxin Binding to Hi-Trap from Amersham was used to bind purified glycophorin. The was first washed × 2 with 1 mm to Then the purified glycophorin solution prepared in the buffer m NaCl, m pH was added and overnight at 4 The of that had not to the protein and the of the were performed the described by the Binding of HlyA and 914–936 was measured by to the affinity 1 ml of protein in hemolysis buffer. 1 h for the was washed with the same buffer The was first with a pH buffer (150 mm NaCl, m pH and then with a high buffer m NaCl, 20 mm Tris-HCl, pH 7.0). The were and by WR Binding to Glycophorin in Lipid unilamellar vesicles of egg phosphatidylcholine were prepared by the method of et al. (23Mayer L.D. Hope M.J. Cullis P.R. Biochim. Biophys. Acta. 1986; 858: 161-168Crossref PubMed Scopus (1565) Google Scholar), using Glycophorin was reconstituted in lipid vesicles by the method of and R.C. J. Biol. Chem. Scholar) at a protein:lipid mole of as described previously (8Cortajarena A.L. Goñi F.M. Ostolaza H. J. Biol. Chem. 2001; 276: 12513-12519Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), that no were in the vesicles. WR peptide binding was assayed in the of the in the the peptide becomes was added to the to a of 10 min for was at and were in the for and were of 5 and 10 The expressed as in the the absence of Hemolysis by WR red blood cell were used, prepared as above The cells were incubated in with amounts of WR peptide for 30 min at room temperature with and then hemolysis was by HlyA. The of hemolysis was measured at as described above. The of hemolysis by WR peptide was as in the absence of WR in the presence of WR in the absence of WR of RTX toxin to the repeat domain revealed a conserved region to the 841–872 epitope of leukotoxin (Fig. The that high with This may be related to the that RTX toxins a range of target whereas specific P. Koronakis V. Hughes C. Microbiol. Mol. Biol. Rev. 1998; 62: 309-333Crossref PubMed Google Scholar). The homologous region of HlyA, including residues was the of our It was as the WR peptide of its and amino acid The Deletion was expressed in E. coli in the same amounts as the WT and could be purified the same (20Ostolaza H. Bartolomé B. Serra J.L. de la Cruz F. Goñi F.M. FEBS Lett. 1991; 280: 195-198Crossref PubMed Scopus (45) Google Scholar). It was recognized by the same polyclonal WT HlyA (8Cortajarena A.L. Goñi F.M. Ostolaza H. J. Biol. Chem. 2001; 276: 12513-12519Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). However, its lytic activity on horse or human red blood cells was of the WT (Fig. This is in with the that amino acids 914–936 involved in HlyA binding to its erythrocyte receptor. this were the the hemolysis at high of be to receptor-binding in the toxin, in with the by and A. Infect. Immun. 1996; PubMed Google Scholar) that several in the protein may be involved in cell have been L. S. J. D. E. Ludwig A. Goebel W. R.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. Mol. Microbiol. PubMed Scopus Google Scholar) the region the repeat domain in cell However, the in 3 point to the WR peptide as the major involved in HlyA binding to red blood cells. Note from the data in horse erythrocytes appear to be human erythrocytes WT or mutant HlyA. In toxin hemolysis are: WT, × mutant, × WT × and mutant × In view of these binding to erythrocytes and lipid vesicles was measured in with that of the The proteins were incubated at with red blood cells. The cells were washed and and membrane proteins were separated by WT and mutant HlyA were revealed by with a polyclonal antibody (8Cortajarena A.L. Goñi F.M. Ostolaza H. J. Biol. Chem. 2001; 276: 12513-12519Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). 4 that WT HlyA, but not could bind horse or human erythrocyte The antibody was to the deletion mutant, as in The toxin used in this were that in the of the WT, cell Thus, it can be concluded that the of the mutant to hemolysis (Fig. is to its cell binding Deletion of amino acids 914–936 the specific binding of HlyA to the cell surface binding L. Ostolaza H. Goñi F.M. Biophys. J. 1996; Full Text PDF PubMed Scopus Google Scholar) to lipid is the same as that of the For an protein:lipid of the binding of WT was and that of the deletion mutant was For an protein:lipid of the were and that the of binding of to erythrocytes was to a in the glycophorin receptor, the binding of WT and mutant HlyA to pure glycophorin in an affinity was using glycophorin purified from horse or human erythrocytes were mutant or WT HlyA was the in separate The was washed with buffer to HlyA. HlyA was with a pH buffer. The were by and the were revealed with a (Fig. In the of WT HlyA, a of the was in the first (Fig. whereas no was in the with (Fig. The protein in the with human glycophorin is in the of horse glycophorin. This was in several and may be related to the of human horse red blood cells HlyA that was in Thus, the from 5 is that the deletion mutant was unable to bind and this its to bind erythrocytes (Fig. and its to (Fig. The WR WR peptide, residues 914–936 of HlyA, was synthesized and used in lysis and binding These were as a to performed with the proteins lacking this The WR peptide had no hemolytic activity of its own, assayed at high (Fig. that the region deleted in in receptor binding of the deleted WR peptide to glycophorin reconstituted in was For this vesicles were prepared containing as described in R.C. J. Biol. Chem. A of these vesicles as a from a was for protein and lipid and found to phosphatidylcholine and glycophorin at a mole large unilamellar vesicles composed of pure egg were used as a Binding of WR peptide to reconstituted glycophorin was in the of the peptide as a of lipid the peptide in solution binds the membrane The in that WR peptide binds large unilamellar vesicles containing but does not bind to unilamellar vesicles not containing glycophorin. by WR of the absence of a of specific binding of WR peptide to glycophorin in we have that of red blood cells with the peptide the of hemolysis induced by HlyA. The of hemolysis by the WR peptide is shown in These data suggest that the peptide is binding the erythrocytes and the specific binding of HlyA. Thus, this peptide appear to be binding the specific receptor glycophorin. the above we that the HlyA region to amino acids 914–936 is a major in the specific binding of HlyA to the red blood cell surface glycophorin. The that a peptide containing amino acids 914–936 an HlyA may suggest a of this peptide in the E. coli in which HlyA has a pathogenic Moreover, homologous of the WR peptide in RTX the in this study can be to toxins as The to the Instituto de Inmunología de Colombia for the of the WR peptide and to R. of for and the

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