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Analysis of the functional domains of Arcanobacterium pyogenes pyolysin using monoclonal antibodies
Veterinary Microbiology 81 (2001) 235–242
Analysis of the functional domains of Arcanobacterium pyogenes pyolysin using monoclonal antibodies Keisuke Imaizumi, Asami Serizawa, Nozomu Hashimoto, Toshio Kaidoh, Shotaro Takeuchi* Faculty of Biotechnology, Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima Matsuoka, Fukui 910-1195, Japan Received 31 October 2000; received in revised form 20 February 2001; accepted 20 February 2001
Abstract Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY) family of bacterial toxins. Four monoclonal antibodies (mAbs) to PLO were prepared for the analysis of functional domains of this toxin. Two (mAbs S and H) of these markedly inhibited the hemolytic activity of PLO, but the inhibiting activity of the other two antibodies (mAbs C and G) was weaker. Subsequently, nine truncated PLOs were derived from recombinant Escherichia coli by various deletions from the N-terminus. Strong hemolytic activity was recognized in truncates of PLO following the deletion of 30 or 55 amino acids, but not in the truncate with deletion of 74 residues. Truncated PLOs were used in immunoblotting experiments to locate the epitopes for the mAbs. The epitope for mAbs C and G lies within the undecapeptide region (amino acids 487–505) of the C-terminus of PLO, which seems to be the binding site to erythrocytes. In contrast, the epitopes for mAbs S and H, which showed strong neutralizing activity, were found to lie in the N-terminal regions of the PLO ranging from 55 to 73 and 123 to 166 amino acids, respectively. From these results, it seems that the N-terminal region of PLO, in particular, the region of amino acids 55–74 is important for hemolytic activity. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Arcanobacterium pyogenes; Pyolysin; Monoclonal antibody
*
Corresponding author. Tel.: þ81-7766-16000; fax: þ81-7766-16015. E-mail address: [email protected] (S. Takeuchi). 0378-1135/01/$ – see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 1 ) 0 0 3 4 2 - X
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1. Introduction Arcanobacterium pyogenes was formerly classified under the genera Corynebacterium or Actinomyces and termed Corynebacterium pyogenes or Actinomyces pyogenes. This organism is the causative agent of various pyogenic diseases in pigs, such as subcutaneous abscesses, suppurative arthritis and vertebral abscesses (Takeuchi et al., 1979a). In cows, it has frequently been isolated in cases of heifer mastitis, hepatic abscesses and spontaneous abortion (Hinton, 1974; Kanoe et al., 1976). A. pyogenes produces several extracellular toxins, including hemolysin, protease and neuraminidase in vitro, which may all contribute to the pathogenicity of this organism. The hemolysin of A. pyogenes is hemolytic for erythrocytes of a number of animal species, and lethal for mice and dermonecrotic for guinea pigs (Lovell, 1944). This hemolysin has been partially purified and characterized by some scientists (Roberts, 1968; Katsaras and Zeller, 1978; Takeuchi et al., 1979b; Funk et al., 1996). Recently, Ding and La¨mmler (1996) purified and characterized a hemolysin from A. pyogenes ATCC 8164 and designated it as pyolysin (PLO). Subsequently, Billington et al. (1997) cloned and sequenced the gene encoding PLO of A. pyogenes. This gene possessed undecapeptide and complement activation regions, which are highly conserved by the thiol-activated cytolysins (TACYs) family in a number of gram-positive bacteria. In the undecapeptide of PLO, however, the cysteine residue required for thiol activation has been replaced with alanine. We also cloned the plo gene from A. pyogenes and recognized this substitution of the cysteine residue in the undecapeptide (Ikegami et al., 2000). Therefore, PLO of A. pyogenes seems to be a novel member of the TACY family of bacterial toxins. Jost et al. (1999) reported that insertional inactivation of the A. pyogenes plo gene results in decreased virulence of the plo mutant and that PLO, like other TACYs, is cytotoxic for phagocytic cells. However, little is known about the functional domains of PLO from A. pyogenes. In this study, we prepared four monoclonal antibodies (mAbs) against PLO by fusing myeloma with spleen cells from immunized mice. In addition, truncated PLOs were produced by recombinant E. coli with N-terminal deletion and analyzed for mAbs epitopes by immunoblotting.
2. Materials and methods 2.1. Bacteria and vector A. pyogenes strain No. 42 (Ikegami et al., 2000), isolated from an abscess lesion in a pig, was used for the purification of PLO and genomic DNA. PinPointTM Xa-1 T-Vector (Promega Corporation, Madison, WI, USA) was used for cloning and expression of the plo gene. E. coli JM109 was used as host for the recombinant plasmid. 2.2. Purification of PLO PLO was purified from the culture supernatant of A. pyogenes by the procedures described previously (Ikegami et al., 2000). Purity of the purified PLO was
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determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. 2.3. Production of mAbs Purified PLO was inactivated by incubating at 48C overnight in 0.5% formalin. Subsequently, 0.5 ml of the toxoid-PLO (200 mg/ml) was emulsified in an equal amount of Freund’s complete adjuvant and injected intraperitonealy into two BALB/c mice twice at intervals of 2 weeks. Three weeks after the second injection, 0.2 ml of the toxoid-PLO was injected intravenously as a booster. One week after the booster, spleen cells from the immunized mice were fused with myeloma NS-1 (Japanese Cancer Resources Bank) using 50% polyethylene glycol (molecular weight 1,300–1,800). The cells were washed, resuspended in HAT (hypoxanthine–aminopterin–thymidine) medium and plated in 96-well culture plates. Hybridomas selected in HAT medium were screened by an enzyme-linked immunosorbent assay (ELISA), and the positive hybridomas were cloned twice by limiting dilution, using thymocyte feeder layers. The cloned hybridoma cells were injected intraperitonealy into the BALB/c mice pretreated with 2,6,10,14-tetramethylpentadecane, and about 2 weeks later, ascitic fluid was collected. mAbs were purified from the collected ascitic fluid by Protein G Superose (Amersham Pharmacia Biotech, Uppsala, Sweden) column chromatography. Isotypes of the purified mAbs were determined by using a mouse monoclonal isotyping kit (Serotec, UK). 2.4. Cloning and expression of truncated plo gene Cloning and expression of the truncated plo gene was performed using PinPointTM Xa1 T-Vector systems (Promega), which carries a segment encoding a peptide (13 kDa) that becomes biotinylated in E. coli and subsequently functions as a purification tag. Briefly, the DNA fragment (1.6 kb) of the plo open reading frame (ORF) was amplified from genomic DNA of A. pyogenes by polymerase chain reaction (PCR) and cloned into PinPointTM Xa-1 T-Vector according to the manufactures instructions. Subsequently, various DNA fragments of truncated plo gene were amplified by PCR using the above recombinant plasmid as a template and cloned into the same vector. All the primers used were designed and synthesized from the sequence of the plo gene described previously (Ikegami et al., 2000). These recombinant plasmids were transformed into E. coli JM109 and the resulting recombinants were cultured on LB/ampicillin plates. After incubation at 378C for 18 h, these recombinant clones were screened by the colony PCR method with the vector and insert DNA primers. In addition, the insert DNA in the screened recombinants was confirmed by sequencing using the vector and the insert DNA sequencing primers. The recombinants obtained were cultured in LB broth containing 2 mM biotin and ampicillin with shaking at 378C for 1 h, then 100 mM IPTG was added for the expression of various truncated PLOs. After centrifugation, the cells were sonicated and examined for various truncated PLOs by SDS-PAGE and immunoblotting assays.
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2.5. ELISA ELISA was used for the screening of hybridoma supernatants. Flat-bottomed 96-well polystrene enzyme immunoassay plates (Falcon 3915) were coated with 100 ml of purified PLO (20 mg/ml) in 50 mM carbonate buffer (pH 9.6) and incubated at 48C for 16 h. After washing with 20 mM phosphate-buffered saline solution (pH 7.2, 0.15 M NaCl) containing 0.05% Tween-20 (PBS-Tween), 100 ml of hybridoma supernatant was added to each well of the plates and incubated at 378C for 30 min. After washing with PBS-Tween, goat anti-mouse IgG (H þ L)-horseradish peroxidase (HRP) conjugate (BioRad Laboratories, Alfred Nobel Drive, Hercules, CA, USA) was added and the plates were incubated at 378C for 30 min. After washing, ELISA was developed using an HRP substrate kit (Bio-Rad Laboratories). 2.6. SDS-PAGE and immunoblotting SDS-PAGE was performed by the procedure of Laemmli (1970) with a 12.5% separating gel and 3% stacking gel. Samples of purified PLO and various truncated PLOs were treated with SDS-PAGE loading buffer at 1008C for 5 min. After electrophoresis, the separated components of the samples were electrophoretically transferred from gels to nitrocellulose membranes. Subsequently, an immunoblotting assay of the membranes was performed using an immunoblot assay kit (Bio-Rad Laboratories). In the immunoblotting assay, four mAbs prepared as described above and anti-PLO rabbit polyclonal serum (Ikegami et al., 2000) were used as the primary serum. As the secondary serum, goat antimouse IgG (H þ L)-HRP conjugate and Protein A — HRP conjugate (Bio-Rad Laboratories) were used. Streptavidin alkaline phosphatase (Promega Corporation, Madison, USA) was used for the detection of biotinylated proteins. 2.7. Assay of hemolytic activity Hemolytic activity was determined as previously described (Ikegami et al., 2000). A 50 ml aliquot of purified PLO and various truncated PLOs was diluted two-fold with 0.85% NaCl in V-bottomed 96-well microtiter plates. An equal volume of a 1% suspension of sheep erythrocytes in saline solution was added to each well of the microtiter plates, and they were incubated at 378C for 1 h. One hemolytic unit was expressed as the reciprocal of the highest dilution that showed complete hemolysis. 2.8. Inhibition of hemolytic activity A 25 ml aliquot of mAb samples (280 nm, 0.3 OD) was diluted two-fold with 0.85% saline solution in V-bottomed 96-well microtiter plates. An equal volume of PLO (4 hemolytic units), purified from A. pyogenes, was added to each well of the microtiter plates, and the plates were incubated at 378C for 30 min. Subsequently, 50 ml of 1% sheep erythrocytes in the saline solution was added, and the plates were incubated at 378C for 30 min. The titer of antibody was expressed as the reciprocal of highest dilution which completely inhibited hemolysis.
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2.9. Inhibition of binding of PLO to erythrocytes Inhibition test of binding to erythrocytes was performed essentially as described by Toyos et al. (1996). A 25 ml aliquot of mAb samples (280 nm, 0.3 OD) was added to an equal volume of purified PLO (100 mg/ml), and tubes were incubated at 378C for 1 h, then 1 ml of 1% sheep erythrocytes was added. After incubation at 48C for 30 min, erythrocytes were washed three times and then lysed in water. Erythrocyte membrane ghosts were harvested, washed, and then resuspended in SDS-PAGE loading buffer. After SDS-PAGE, immunobloting assay was performed with a 1:1000 dilution of anti-PLO rabbit polyclonal serum, followed with Protein A — HRP conjugate (Bio-Rad Laboratories).
3. Results 3.1. Production and characterization of mAbs Four mAbs (C, G, H and S) against PLO of A. pyogenes were obtained by the procedures described in Materials and Methods. As shown in Fig. 1, all of the mAbs reacted strongly with PLO purified from the culture supernatant of A. pyogenes in immunoblotting assay. mAbs H and S markedly inhibited the hemolytic activity of purified PLO, but the inhibiting activity of mAbs C and G was weaker. The inhibiting titers of mAbs H, S, C and G were 128, 128, 4 and 2, respectively. These mAbs did not inhibit the binding of purified PLO to erythrocytes. mAbs C and G were of the IgG1 isotype, while the mAbs H and S were of the IgG2b isotype. 3.2. Production and characterization of truncated PLO Nine truncates with N-terminal deletions of PLO were made with PinPointTM Xa-1 TVector systems (Fig. 2). All of these truncated PLOs formed bands of the expected
Fig. 1. Immunoblotting assay of mAbs against purified PLO. Lane 1: anti-PLO rabbit polyclonal serum; lane 2: mAb H; lane 3: mAb S; lane 4: mAb G; lane 5: mAb C.
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Fig. 2. Recombinant designation, molecular size, hemolytic activity, and binding to erythrocytes of truncated PLOs: (a) molecular size of truncated PLOs sized with biotinylated tag peptide (13 kDa); (b) one hemolytic unit is expressed as the reciprocal of the highest dilution showing complete hemolysis; (c) binding of truncated PLOs to erythrocytes analyzed by immunoblotting.
molecular size with a biotinylated tag peptide (13 kDa) in the immunoblotting assay using an anti-PLO rabbit polyclonal serum and streptavidin alkaline phosphatase. Two truncates (L30 and L55) of PLO with 30 and 55 amino acids deleted from the N-terminus showed strong hemolytic activity, but one truncate (E74) with deletion of 74 residues lost remarkable hemolytic activity, suggesting that the region of 55–74 residues of PLO is important for hemolytic activity. Other truncates (K95, F123, V167, K375, A482, and K506) showed no hemolytic activity. Binding to erythrocytes was recognized in all of the truncates, except K506 with deletion of the undecapeptide region (amino acids 487–505) of PLO, which seems to contain the binding site to erythrocytes. Table 1 Reaction of mAbs with truncated PLO Recombinations
r-PLO L30 L55 E74 K95 F123 V167 K375 A482 K506 Expected epitipec a
PLO residuesa
3–534 30–534 55–534 74–534 95–534 123–534 167–534 375–534 482–534 506–534
Reaction of the following mAbsb mAb H
mAb S
mAb G
mAb C
þ þ þ 55–73
þ þ þ þ þ þ 123–166
þ þ þ þ þ þ þ þ þ 482–506
þ þ þ þ þ þ þ þ þ 482–506
Numbers represent amino acids from the native molecule present in truncates. Reaction of mAbs with truncated PLO analyzed by immunoblotting. c Numbers represent amino acids which might be epitopes of mAbs as shown by immunoclotting. b
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3.3. Reaction of mAbs with truncated PLO Reaction of mAbs with truncated PLO was examined by immunoblotting assay. As shown in Table 1, mAb H, which showed strong neutralizing activity, reacted with truncate L55 which had 55 amino acids deleted from N-terminus, but did not react with truncate E74 with a deletion of 74 residues, indicating that the epitope for mAb H lies between amino acids 55 and 73 of PLO. While mAb S had strong neutralizing activity similar to mAb H, this mAb reacted with truncate F123 with a deletion of 123 residues, but not with truncate V167 with a deletion of 167 residues. This suggested that the mAb S epitope differs from the epitope of mAb H and lies between amino acids 123 and 166. mAbs C and G reacted with all of the truncates, except truncate K506 which had a deletion in the undecapeptide region (487–505 resides), suggesting that the epitope for mAbs C and G lies within the undecapeptide region.
4. Discussion The family of thiol-activated cytolysins (TACYs) includes alveolysin (Bacillus alvei), listeriolysin O (Listeria monocytogenes), perfringolysin O (Clostridium perfringens), pneumolysin (Streptococcus pneumoniae) and streptolysin O (Streptococcus pyogenes). Neutralizing mAbs against these TACYs have been prepared for the analysis of the functional domains of the toxins by various investigators. Jacobs et al. (1999) reported that the neutralizing mAb PLY-5 against pneumolysin recognized all members of the TACY family, and peptide mapping assigned its epitope to the undecapeptide motif, which seems to be the binding site to erythrocytes. In the present study, however, the non/ weak-neutralizing mAbs C and G against PLO recognized the undecapeptide region of Cterminus of this toxin. This finding is in agreement with the data of Nato et al. (1991), who showed that mAbs raised against the undecapeptide (ETCGLAWEWWR) did not neutralize the hemolytic activity of listeriolysin O. Darji et al. (1996) reported that the epitopes for the neutralizing mAbs against listeriolysin O lie between positions 59 and 279 of the N-terminus. Also in our study, the epitopes recognized by the strong-neutralizing mAbs H and S were found on the Nterminal regions of the PLO ranging from 55 to 73 and 123 to 166 amino acids, respectively. In addition, two truncates (L30 and L55) of PLO with 30 and 55 amino acids deleted from the N-terminus showed strong hemolytic activity, but one truncate (E74) with 74 residues lost remarkable hemolytic activity. From these results, it seems that the region of 55–74 resides of PLO is important for hemolytic activity. On the other hand, Shimada et al. (1999) reported that the C-terminal region of perfringolysin O has two distinct roles; the last 21 amino acids are involved in maintaining an ordered overall structure, while the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities. Therefore, we are now attempting to make a series of truncated versions of PLO in which varying numbers of amino acids are deleted from the C-terminus in order to examine the roles of amino acids of the C-terminus.
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References Billington, S.J., Jost, B.H., Cuevas, W.E., Bright, K.R., Songer, J.G., 1997. The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family. J. Bacteriol. 179, 6100–6106. Darji, A., Niebuhr, K., Hense, M., Wehland, J., Chakraborty, T., Weiss, S., 1996. Neutralizing monoclonal antibodies against listeriolysin: mapping of epitopes involved in pore formation. Infect. Immun. 64, 2356– 2358. Ding, H., La¨ mmler, C., 1996. Purification and further characterization of a hemolysin of Actinomyces pyogenes. J. Vet. Med. Ser. B 43, 179–188. Funk, P.G., Staats, J.J., Howe, M., Nagaraja, T.G., Chengappa, M.M., 1996. Identification and partial characterization of an Actinomyces pyogenes hemolysin. Vet. Microbiol. 50, 129–142. Hinton, M., 1974. Corynebacterium pyogenes and bovine abortion. J. Hyg. (Camb.) 72, 365–368. Ikegami, M., Hashimoto, N., Kaidoh, T., Sekizaki, T., Takeuchi, S., 2000. Genetic and biochemical properties of a hemolysin (pyolysin) produced by a swine isolate of Arcanobacterium (Actinomyces) pyogenes. Microbiol. Immunol. 44, 1–7. Jacobs, T., Cima-Cabal, M.D., Darji, A., Me´ ndez, F.J., Va´ zquez, F., Jacobs, A.A.C., Shimada, Y., Ohno-Iwashita, Y., Weiss, S., Toyos, J.R., 1999. The conserved undecapeptide shared by thiol-activated cytolysins is involved in membrane binding. FEBS Letters 459, 463–466. Jost, B.H., Songer, J.G., Billington, S.J., 1999. An Arcanobacterium (Actinomyces) pyogenes mutant deficient in production of the pore forming cytolysin pyolysin has reduced virulence. Infect. Immun. 67, 1723–1728. Kanoe, K., Imagawa, H., Toda, M., Sato, A., Inoue, M., Yoshida, Y., 1976. Bacteriology of bovine hepatic abscesses. Jpn. J. Vet. Sci. 38, 263–268. Katsaras, K., Zeller, U.P., 1978. Studies on the exotoxin of Corynebacterium pyogenes after purification with gel filtration and DEAE ion exchanger. Zbl. Vet. Med. B 25, 596–604. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lovell, R., 1944. Further studies on the toxin of Corynebacterium pyogenes. J. Pathol. Bacteriol. 56, 525–529. Nato, F., Reich, K., Lhopital, S., Rouyre, S., Geoffroy, C., Mazie, J.C., Cossart, P., 1991. Production and characterization of neutralizing and nonneutralizing monoclonal antibodies against listeriolysin. Infect. Immun. 59, 4641–4646. Roberts, R.J., 1968. A study of the hemolysin of Corynebacterium pyogenes. Res. Vet. Sci. 9, 350–353. Shimada, Y., Nakamura, M., Naito, Y., Nomura, K., Ohno-Iwahita, Y., 1999. C-terminal amino acid residues are required for the folding and cholesterol binding property pf perfringolysin O, a pore-forming cytolysin. J. Biol. Chem. 274, 18536–18542. Takeuchi, S., Azuma, R., Nakajima, Y., Suto, T., 1979a. Diagnosis of Corynebacterium pyogenes infection in pigs by immunodiffusion test with protease antigen. Nat. Inst. Anim. Health Quart. 19, 77–82. Takeuchi, S., Azuma, R., Suto, T., 1979b. Purification and some properties of hemolysin produced by Corynebacterium pyogenes. Jpn. J. Vet. Sci. 41, 511–516. Toyos, J.R., Me´ ndez, F.J., Aparicio, J.F., Va´ zquez, F., Sua´ rez, M.M.G., Fleites, A., Hardisson, C., Morgan, P.J., Andrew, P.W., Mitchell, T.J., 1996. Functional analysis of pneumolysin by use of monoclonal antibodies. Infect. Immun. 64, 480–484.
Analysis of the functional domains of Arcanobacterium pyogenes pyolysin using monoclonal antibodies Keisuke Imaizumi, Asami Serizawa, Nozomu Hashimoto, Toshio Kaidoh, Shotaro Takeuchi* Faculty of Biotechnology, Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima Matsuoka, Fukui 910-1195, Japan Received 31 October 2000; received in revised form 20 February 2001; accepted 20 February 2001
Abstract Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY) family of bacterial toxins. Four monoclonal antibodies (mAbs) to PLO were prepared for the analysis of functional domains of this toxin. Two (mAbs S and H) of these markedly inhibited the hemolytic activity of PLO, but the inhibiting activity of the other two antibodies (mAbs C and G) was weaker. Subsequently, nine truncated PLOs were derived from recombinant Escherichia coli by various deletions from the N-terminus. Strong hemolytic activity was recognized in truncates of PLO following the deletion of 30 or 55 amino acids, but not in the truncate with deletion of 74 residues. Truncated PLOs were used in immunoblotting experiments to locate the epitopes for the mAbs. The epitope for mAbs C and G lies within the undecapeptide region (amino acids 487–505) of the C-terminus of PLO, which seems to be the binding site to erythrocytes. In contrast, the epitopes for mAbs S and H, which showed strong neutralizing activity, were found to lie in the N-terminal regions of the PLO ranging from 55 to 73 and 123 to 166 amino acids, respectively. From these results, it seems that the N-terminal region of PLO, in particular, the region of amino acids 55–74 is important for hemolytic activity. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Arcanobacterium pyogenes; Pyolysin; Monoclonal antibody
*
Corresponding author. Tel.: þ81-7766-16000; fax: þ81-7766-16015. E-mail address: [email protected] (S. Takeuchi). 0378-1135/01/$ – see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 1 ) 0 0 3 4 2 - X
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1. Introduction Arcanobacterium pyogenes was formerly classified under the genera Corynebacterium or Actinomyces and termed Corynebacterium pyogenes or Actinomyces pyogenes. This organism is the causative agent of various pyogenic diseases in pigs, such as subcutaneous abscesses, suppurative arthritis and vertebral abscesses (Takeuchi et al., 1979a). In cows, it has frequently been isolated in cases of heifer mastitis, hepatic abscesses and spontaneous abortion (Hinton, 1974; Kanoe et al., 1976). A. pyogenes produces several extracellular toxins, including hemolysin, protease and neuraminidase in vitro, which may all contribute to the pathogenicity of this organism. The hemolysin of A. pyogenes is hemolytic for erythrocytes of a number of animal species, and lethal for mice and dermonecrotic for guinea pigs (Lovell, 1944). This hemolysin has been partially purified and characterized by some scientists (Roberts, 1968; Katsaras and Zeller, 1978; Takeuchi et al., 1979b; Funk et al., 1996). Recently, Ding and La¨mmler (1996) purified and characterized a hemolysin from A. pyogenes ATCC 8164 and designated it as pyolysin (PLO). Subsequently, Billington et al. (1997) cloned and sequenced the gene encoding PLO of A. pyogenes. This gene possessed undecapeptide and complement activation regions, which are highly conserved by the thiol-activated cytolysins (TACYs) family in a number of gram-positive bacteria. In the undecapeptide of PLO, however, the cysteine residue required for thiol activation has been replaced with alanine. We also cloned the plo gene from A. pyogenes and recognized this substitution of the cysteine residue in the undecapeptide (Ikegami et al., 2000). Therefore, PLO of A. pyogenes seems to be a novel member of the TACY family of bacterial toxins. Jost et al. (1999) reported that insertional inactivation of the A. pyogenes plo gene results in decreased virulence of the plo mutant and that PLO, like other TACYs, is cytotoxic for phagocytic cells. However, little is known about the functional domains of PLO from A. pyogenes. In this study, we prepared four monoclonal antibodies (mAbs) against PLO by fusing myeloma with spleen cells from immunized mice. In addition, truncated PLOs were produced by recombinant E. coli with N-terminal deletion and analyzed for mAbs epitopes by immunoblotting.
2. Materials and methods 2.1. Bacteria and vector A. pyogenes strain No. 42 (Ikegami et al., 2000), isolated from an abscess lesion in a pig, was used for the purification of PLO and genomic DNA. PinPointTM Xa-1 T-Vector (Promega Corporation, Madison, WI, USA) was used for cloning and expression of the plo gene. E. coli JM109 was used as host for the recombinant plasmid. 2.2. Purification of PLO PLO was purified from the culture supernatant of A. pyogenes by the procedures described previously (Ikegami et al., 2000). Purity of the purified PLO was
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determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. 2.3. Production of mAbs Purified PLO was inactivated by incubating at 48C overnight in 0.5% formalin. Subsequently, 0.5 ml of the toxoid-PLO (200 mg/ml) was emulsified in an equal amount of Freund’s complete adjuvant and injected intraperitonealy into two BALB/c mice twice at intervals of 2 weeks. Three weeks after the second injection, 0.2 ml of the toxoid-PLO was injected intravenously as a booster. One week after the booster, spleen cells from the immunized mice were fused with myeloma NS-1 (Japanese Cancer Resources Bank) using 50% polyethylene glycol (molecular weight 1,300–1,800). The cells were washed, resuspended in HAT (hypoxanthine–aminopterin–thymidine) medium and plated in 96-well culture plates. Hybridomas selected in HAT medium were screened by an enzyme-linked immunosorbent assay (ELISA), and the positive hybridomas were cloned twice by limiting dilution, using thymocyte feeder layers. The cloned hybridoma cells were injected intraperitonealy into the BALB/c mice pretreated with 2,6,10,14-tetramethylpentadecane, and about 2 weeks later, ascitic fluid was collected. mAbs were purified from the collected ascitic fluid by Protein G Superose (Amersham Pharmacia Biotech, Uppsala, Sweden) column chromatography. Isotypes of the purified mAbs were determined by using a mouse monoclonal isotyping kit (Serotec, UK). 2.4. Cloning and expression of truncated plo gene Cloning and expression of the truncated plo gene was performed using PinPointTM Xa1 T-Vector systems (Promega), which carries a segment encoding a peptide (13 kDa) that becomes biotinylated in E. coli and subsequently functions as a purification tag. Briefly, the DNA fragment (1.6 kb) of the plo open reading frame (ORF) was amplified from genomic DNA of A. pyogenes by polymerase chain reaction (PCR) and cloned into PinPointTM Xa-1 T-Vector according to the manufactures instructions. Subsequently, various DNA fragments of truncated plo gene were amplified by PCR using the above recombinant plasmid as a template and cloned into the same vector. All the primers used were designed and synthesized from the sequence of the plo gene described previously (Ikegami et al., 2000). These recombinant plasmids were transformed into E. coli JM109 and the resulting recombinants were cultured on LB/ampicillin plates. After incubation at 378C for 18 h, these recombinant clones were screened by the colony PCR method with the vector and insert DNA primers. In addition, the insert DNA in the screened recombinants was confirmed by sequencing using the vector and the insert DNA sequencing primers. The recombinants obtained were cultured in LB broth containing 2 mM biotin and ampicillin with shaking at 378C for 1 h, then 100 mM IPTG was added for the expression of various truncated PLOs. After centrifugation, the cells were sonicated and examined for various truncated PLOs by SDS-PAGE and immunoblotting assays.
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2.5. ELISA ELISA was used for the screening of hybridoma supernatants. Flat-bottomed 96-well polystrene enzyme immunoassay plates (Falcon 3915) were coated with 100 ml of purified PLO (20 mg/ml) in 50 mM carbonate buffer (pH 9.6) and incubated at 48C for 16 h. After washing with 20 mM phosphate-buffered saline solution (pH 7.2, 0.15 M NaCl) containing 0.05% Tween-20 (PBS-Tween), 100 ml of hybridoma supernatant was added to each well of the plates and incubated at 378C for 30 min. After washing with PBS-Tween, goat anti-mouse IgG (H þ L)-horseradish peroxidase (HRP) conjugate (BioRad Laboratories, Alfred Nobel Drive, Hercules, CA, USA) was added and the plates were incubated at 378C for 30 min. After washing, ELISA was developed using an HRP substrate kit (Bio-Rad Laboratories). 2.6. SDS-PAGE and immunoblotting SDS-PAGE was performed by the procedure of Laemmli (1970) with a 12.5% separating gel and 3% stacking gel. Samples of purified PLO and various truncated PLOs were treated with SDS-PAGE loading buffer at 1008C for 5 min. After electrophoresis, the separated components of the samples were electrophoretically transferred from gels to nitrocellulose membranes. Subsequently, an immunoblotting assay of the membranes was performed using an immunoblot assay kit (Bio-Rad Laboratories). In the immunoblotting assay, four mAbs prepared as described above and anti-PLO rabbit polyclonal serum (Ikegami et al., 2000) were used as the primary serum. As the secondary serum, goat antimouse IgG (H þ L)-HRP conjugate and Protein A — HRP conjugate (Bio-Rad Laboratories) were used. Streptavidin alkaline phosphatase (Promega Corporation, Madison, USA) was used for the detection of biotinylated proteins. 2.7. Assay of hemolytic activity Hemolytic activity was determined as previously described (Ikegami et al., 2000). A 50 ml aliquot of purified PLO and various truncated PLOs was diluted two-fold with 0.85% NaCl in V-bottomed 96-well microtiter plates. An equal volume of a 1% suspension of sheep erythrocytes in saline solution was added to each well of the microtiter plates, and they were incubated at 378C for 1 h. One hemolytic unit was expressed as the reciprocal of the highest dilution that showed complete hemolysis. 2.8. Inhibition of hemolytic activity A 25 ml aliquot of mAb samples (280 nm, 0.3 OD) was diluted two-fold with 0.85% saline solution in V-bottomed 96-well microtiter plates. An equal volume of PLO (4 hemolytic units), purified from A. pyogenes, was added to each well of the microtiter plates, and the plates were incubated at 378C for 30 min. Subsequently, 50 ml of 1% sheep erythrocytes in the saline solution was added, and the plates were incubated at 378C for 30 min. The titer of antibody was expressed as the reciprocal of highest dilution which completely inhibited hemolysis.
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2.9. Inhibition of binding of PLO to erythrocytes Inhibition test of binding to erythrocytes was performed essentially as described by Toyos et al. (1996). A 25 ml aliquot of mAb samples (280 nm, 0.3 OD) was added to an equal volume of purified PLO (100 mg/ml), and tubes were incubated at 378C for 1 h, then 1 ml of 1% sheep erythrocytes was added. After incubation at 48C for 30 min, erythrocytes were washed three times and then lysed in water. Erythrocyte membrane ghosts were harvested, washed, and then resuspended in SDS-PAGE loading buffer. After SDS-PAGE, immunobloting assay was performed with a 1:1000 dilution of anti-PLO rabbit polyclonal serum, followed with Protein A — HRP conjugate (Bio-Rad Laboratories).
3. Results 3.1. Production and characterization of mAbs Four mAbs (C, G, H and S) against PLO of A. pyogenes were obtained by the procedures described in Materials and Methods. As shown in Fig. 1, all of the mAbs reacted strongly with PLO purified from the culture supernatant of A. pyogenes in immunoblotting assay. mAbs H and S markedly inhibited the hemolytic activity of purified PLO, but the inhibiting activity of mAbs C and G was weaker. The inhibiting titers of mAbs H, S, C and G were 128, 128, 4 and 2, respectively. These mAbs did not inhibit the binding of purified PLO to erythrocytes. mAbs C and G were of the IgG1 isotype, while the mAbs H and S were of the IgG2b isotype. 3.2. Production and characterization of truncated PLO Nine truncates with N-terminal deletions of PLO were made with PinPointTM Xa-1 TVector systems (Fig. 2). All of these truncated PLOs formed bands of the expected
Fig. 1. Immunoblotting assay of mAbs against purified PLO. Lane 1: anti-PLO rabbit polyclonal serum; lane 2: mAb H; lane 3: mAb S; lane 4: mAb G; lane 5: mAb C.
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Fig. 2. Recombinant designation, molecular size, hemolytic activity, and binding to erythrocytes of truncated PLOs: (a) molecular size of truncated PLOs sized with biotinylated tag peptide (13 kDa); (b) one hemolytic unit is expressed as the reciprocal of the highest dilution showing complete hemolysis; (c) binding of truncated PLOs to erythrocytes analyzed by immunoblotting.
molecular size with a biotinylated tag peptide (13 kDa) in the immunoblotting assay using an anti-PLO rabbit polyclonal serum and streptavidin alkaline phosphatase. Two truncates (L30 and L55) of PLO with 30 and 55 amino acids deleted from the N-terminus showed strong hemolytic activity, but one truncate (E74) with deletion of 74 residues lost remarkable hemolytic activity, suggesting that the region of 55–74 residues of PLO is important for hemolytic activity. Other truncates (K95, F123, V167, K375, A482, and K506) showed no hemolytic activity. Binding to erythrocytes was recognized in all of the truncates, except K506 with deletion of the undecapeptide region (amino acids 487–505) of PLO, which seems to contain the binding site to erythrocytes. Table 1 Reaction of mAbs with truncated PLO Recombinations
r-PLO L30 L55 E74 K95 F123 V167 K375 A482 K506 Expected epitipec a
PLO residuesa
3–534 30–534 55–534 74–534 95–534 123–534 167–534 375–534 482–534 506–534
Reaction of the following mAbsb mAb H
mAb S
mAb G
mAb C
þ þ þ 55–73
þ þ þ þ þ þ 123–166
þ þ þ þ þ þ þ þ þ 482–506
þ þ þ þ þ þ þ þ þ 482–506
Numbers represent amino acids from the native molecule present in truncates. Reaction of mAbs with truncated PLO analyzed by immunoblotting. c Numbers represent amino acids which might be epitopes of mAbs as shown by immunoclotting. b
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3.3. Reaction of mAbs with truncated PLO Reaction of mAbs with truncated PLO was examined by immunoblotting assay. As shown in Table 1, mAb H, which showed strong neutralizing activity, reacted with truncate L55 which had 55 amino acids deleted from N-terminus, but did not react with truncate E74 with a deletion of 74 residues, indicating that the epitope for mAb H lies between amino acids 55 and 73 of PLO. While mAb S had strong neutralizing activity similar to mAb H, this mAb reacted with truncate F123 with a deletion of 123 residues, but not with truncate V167 with a deletion of 167 residues. This suggested that the mAb S epitope differs from the epitope of mAb H and lies between amino acids 123 and 166. mAbs C and G reacted with all of the truncates, except truncate K506 which had a deletion in the undecapeptide region (487–505 resides), suggesting that the epitope for mAbs C and G lies within the undecapeptide region.
4. Discussion The family of thiol-activated cytolysins (TACYs) includes alveolysin (Bacillus alvei), listeriolysin O (Listeria monocytogenes), perfringolysin O (Clostridium perfringens), pneumolysin (Streptococcus pneumoniae) and streptolysin O (Streptococcus pyogenes). Neutralizing mAbs against these TACYs have been prepared for the analysis of the functional domains of the toxins by various investigators. Jacobs et al. (1999) reported that the neutralizing mAb PLY-5 against pneumolysin recognized all members of the TACY family, and peptide mapping assigned its epitope to the undecapeptide motif, which seems to be the binding site to erythrocytes. In the present study, however, the non/ weak-neutralizing mAbs C and G against PLO recognized the undecapeptide region of Cterminus of this toxin. This finding is in agreement with the data of Nato et al. (1991), who showed that mAbs raised against the undecapeptide (ETCGLAWEWWR) did not neutralize the hemolytic activity of listeriolysin O. Darji et al. (1996) reported that the epitopes for the neutralizing mAbs against listeriolysin O lie between positions 59 and 279 of the N-terminus. Also in our study, the epitopes recognized by the strong-neutralizing mAbs H and S were found on the Nterminal regions of the PLO ranging from 55 to 73 and 123 to 166 amino acids, respectively. In addition, two truncates (L30 and L55) of PLO with 30 and 55 amino acids deleted from the N-terminus showed strong hemolytic activity, but one truncate (E74) with 74 residues lost remarkable hemolytic activity. From these results, it seems that the region of 55–74 resides of PLO is important for hemolytic activity. On the other hand, Shimada et al. (1999) reported that the C-terminal region of perfringolysin O has two distinct roles; the last 21 amino acids are involved in maintaining an ordered overall structure, while the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities. Therefore, we are now attempting to make a series of truncated versions of PLO in which varying numbers of amino acids are deleted from the C-terminus in order to examine the roles of amino acids of the C-terminus.
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