Isolation and Characterization of Recombinant Escherichia coli

Vol. 56, No. 5

INFECTION AND IMMUNITY, May 1988, P. 1135-1143 0019-9567/88/051135-09$02.00/0 Copyright C 1988, American Society for Microbiology

Isolation and Characterization of Recombinant Escherichia coli Clones Secreting a 24-Kilodalton Antigen of Treponema pallidum PEI-LING HSU,* MINDE QIN, STEVEN J. NORRIS, AND STEWART SELL Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77225 Received 6 October 1987/Accepted 5 February 1988

Escherichia coli clones containing Treponema pallidum DNA in the pUC8 vector and secreting a 24-kilodalton antigen of T. palidum have been isolated. Both syphilitic human and syphilis-immune rabbit sera reacted with the recombinant p24 antigen, indicating that an equivalent protein in T. pallidum is capable of eliciting antibody responses during natural infections. The p24 antigen of T. pailidum was identified by using two-dimensional gel electrophoresis and immunoblotting with monospecific anti-p24 serum. We tentatively concluded that this cloned antigen is a secreted protein or a labile or minor component of T. palidum because (i) p24 was secreted by the recombinant E. coli cells; (ii) recombinant p24 in E. coli cells was processed into several smaller species with molecular masses ranging from 12 to 20 kilodaltons, which correlate well with the masses of secreted antigens described by others; and (iii) p24 protein appeared to be highly antigenic during natural infections, but only a very small amount of this antigen was associated with or retained by the purified organisms. The possible role of the p24 protein in determining the growth characteristics of T. palldum is suggested by the ability of recombinant p24 to induce growth changes in E. coli cells. All E. coli colonies expressing the p24 polypeptide exhibited a flat and rough colony morphology and a tilamentous growth pattern that were different from those of other E. coli cells. The DNA sequence coding for the p24 polypeptide is located on a 1.7-kilobase-pair BamHI fragment of the T. paUidum genomic DNA and is absent in the nonpathogenic Treponema phagedenis DNA. However, any possible relationship between the p24 antigen and the virulence of T. paUidum remains to be determined. In preliminary studies, rabbits immunized with the purified p24 were not protected from the infection with live T. pallidum organisms.

culture media of the pallidum and pertenue subspecies of T. pallidum (36). These proteins could not be identified in the culture medium of the nonpathogenic Treponema phagedenis Reiter strain. The secreted proteins were specifically precipitated by sera from rabbits infected with T. pallidum or T. pertenue strains but not with the sera of rabbits immunized with T. phagedenis Reiter. In addition, most sera taken from human syphilitic patients beyond the primary stage of the disease also precipitated these secreted proteins. Thus, these extracellular antigens are synthesized during human infection and are capable of eliciting antibody responses. In this report, we describe the isolation and characterization of E. coli clones expressing a 24-kilodalton (kDa) antigen (p24) of T. pallidum. All p24-positive clones exhibited a unique morphology and growth pattern that were different from those of the other E. coli clones. In addition, p24 was secreted by the recombinant E. coli cells. The DNA sequence coding for p24 was present only in the genomic DNA of T. pallidum but not in the DNA of the nonpathogenic T. phagedenis or rabbit DNA. In T. pallidum purified by Percoll density gradient centrifugation (hereafter referred to as Percoll-purified T. pallidum), only a very small amount of p24 was detected. Several lines of evidence suggested that this p24 antigen represented a secreted protein of T. pallidum (or its precursor). Further studies of the secreted proteins of T. pallidum may contribute to the understanding of the pathogenesis of treponemal infections.

Treponema pallidum is the etiologic agent of venereal syphilis (subsp. pallidum), endemic syphilis (subsp. endemicum), and yaws (subsp. pertenue). These diseases exhibit multiple stages of infection, latency, and an extremely complex pattern of pathogenesis and immunological responses (1, 29, 30, 33, 34). Questions about the components of the T. pallidum have been difficult to answer because of the limited multiplication of the organism in vitro (6, 25). Their propagation in rabbit testicles creates the possibility of contamination with host proteins. To circumvent some of the problems associated with the cultivation of T. pallidum, several laboratories have established T. pallidum gene banks in Escherichia coli. The availability of cloned antigens would clearly facilitate the study of the structure, physiology, pathology, and immunology of T. pallidum infections. Up to two dozen genes encoding the antigens of treponemes have been cloned in E. coli cells. Recombinant E. coli clones express polypeptides which are recognized by the immune sera from infected animals or monoclonal antibodies directed against T. pallidum (for a list of these genes, see reference 26). Whereas much interest has been focused on studying the genes encoding the membrane or other cellular components of T. pallidum (5-8, 10, 13-15, 17, 23, 24, 26, 29-32, 38-41), little is known about the secreted proteins of the organisms. The secreted proteins may interact with the infected host and contribute to the virulence of the organisms. Therefore, they are particularly important from the standpoint of pathology and immunology. Using in vitro radiolabeling and immunoprecipitation, Stamm and Bassford identified four secreted polypeptides with molecular weights ranging from 10,500 to 17,000 in the *

MATERIALS AND METHODS Propagation and purification of T. paUidum. T. pallidum Nichols, obtained from James N. Miller, was maintained by intratesticular passage in adult male New Zealand White

Corresponding author. 1135

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rabbits. Animals were inoculated with 2 x 107 to 5 x 107 T. pallidum organisms which were either freshly extracted from infected rabbit testes or stored frozen at -70°C in saline containing 50% normal rabbit serum, 15% glycerol, and 1 mM dithiothreitol. In some experiments, the animals were given intramuscular injections of the corticosteroid triamcinolone acetonide (4 mg/kg of body weight; Sigma Chemical Co., St. Louis, Mo.) on days 4 and 9 postinfection to increase the yield of T. pallidum. The infected testes were harvested aseptically at the time of peak orchitis, which occurred on days 12 to 14 postinoculation. Percoll density gradient centrifugation was used to separate T. pallidum from host tissue constituents as described before (11). The approximate yield of T. pallidum was between 4 x 109 to 3 x 1010 organisms per rabbit. DNA isolation and restriction enzyme digestion. Highmolecular-weight DNA was isolated from Percoll-purified T. pallidum by the lysozyme-sodium dodecyl sulfate (SDS)proteinase K method. Approximately 1010 cells were suspended in 10 ml of TE buffer (10 mM Tris [pH 8.0], 5 mM EDTA) and then treated with 1 mg of lysozyme per ml on ice for 30 min. Proteinase K (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and SDS were added to a final concentration of 50 jig/ml and 0.5%, respectively, and the mixture was incubated at 37°C for 2 h. NaCl was added to a final concentration of 1 N, and the mixture was phenol extracted, ethanol precipitated, dried, and resuspended in TE buffer. The DNA was treated with pancreatic RNase A (50 jig/ml; Worthington Diagnostics, Freehold, N.J.) and then purified by phenol extraction and ethanol precipitation. High-molecular-weight rabbit liver DNA was isolated according to the standard SDS-proteinase K method (20). Plasmid DNA was isolated from E. coli cells after overnight amplification in chloramphenicol (170 jig/ml). The alkaline lysis method of Birnboim and Doly (2) was used to prepare plasmid DNA. The plasmid DNA was digested with enzyme directly or further purified on low-melting-point agarose gel and recovered by passage through an NACS column (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). Large amounts of plasmid DNA were prepared by CsCl gradient centrifugation according to the standard method (20). Restriction enzymes were purchased from Bethesda Research Laboratories. The DNA fragments were analyzed on agarose or polyacrylamide gels by using the standard Tris-acetate or Tris-borate buffer as described elsewhere (20). Establishment of T. pallidum DNA bank in E. coli. E. coli DH1 (F- recAl endAl gyrA96 thi-J hsdRJ7 [rK- MK+] supE44) was used as the host for DNA clones. The cloning vectors were pUC8 and pUC19 (12, 42). T. pallidum DNA was partially digested with BamHI to an average size of 8 to 10 kilobase-pairs (kb) and then purified by phenol extraction and ethanol precipitation. Digested DNA (5 jig) was ligated to 2 jig of the BamHI-cut

pUC8 vector without further size selection. Before ligation, the vector DNA was treated with calf intestine alkaline phosphatase (Boehringer Mannheim) to prevent self circularization. The ligated DNA was used to transform E. coli DH1 cells according to the procedure of Kushner (18). Transformants were plated directly on BA85 nitrocellulose filters (Schleicher & Schuell, Inc., Keene, N.H.) overlaid on LB plates containing 30 jig of ampicillin per ml. Replica plating was carried out as described by Hanahan and Meselson (10). Additional banks were constructed by using Sau3A partially digested DNA cloned into the BamHI site of pUC8 and pUC19 for other reading frames.

INFECT. IMMUN.

Immunological screening of T. pallidum DNA bank. Replicas of the bacterial colonies were grown on nitrocellulose filters overlaid on LB plates containing 30 ,ug of ampicillin per ml. Filter-bound colonies were lysed by chloroform vapor and then treated with DNase I and lysozyme according to the screening procedure described before (16). The filters were first blocked for 30 min with a 1:200 dilution of normal goat serum in the presence of 3% nonfat dried milk in TS buffer (20 mM Tris [pH 7.6], 0.15 N NaCl) and then incubated for 2 h with antiserum of rabbit or human origin (1:200 dilution preadsorbed with a boiled lysate of E. coli). After three washes with TST buffer (TS buffer with 0.1% Triton X-100), a biotin-labeled goat anti-rabbit or goat antihuman immunoglobulin (1:200 dilution from the Vectorstain ABC kit; Vector Laboratories, Inc., Burlingame, Calif.) was added for 30 min. The filters were washed again three times with TST buffer and then reacted with preformed avidinbiotin-peroxidase complex for 30 min. After three more washes with TST buffer, the filters were developed in 4-chloro-1-naphthol (0.6 mg/ml of phosphate-buffered saline with 20% methanol) and 0.06% hydrogen peroxide. Positive colonies were identified by a purple appearance. The control filters were treated with normal human or rabbit serum instead of immune serum. Partial purification of the p24 protein. An overnight culture of E. coli cells was pelleted, washed one time with buffer A containing 20 mM Tris-10 mM EDTA, and then resuspended in 1/50 of the original volume of buffer A. The cells were treated with 1 mg of lysozyme per ml on ice for 30 min and then centrifuged at 100,000 x g for 15 min. The supernatant was adjusted to 50 mM in NaCl and then loaded onto a DE52 ion exchange column (Whatman, Inc., Clifton, N.J.). After the column was washed with Tris-EDTA-50 mM NaCl, it was eluted with a stepwise gradient containing increasingly higher NaCl concentrations (0.1 to 1.5 N). The fractions containing p24 protein were further purified by preparative gel electrophoresis on an SDS-12% polyacrylamide gel, followed by transfer to nitrocellulose membranes. Regions corresponding to the p24 protein were identified by immunoperoxidase staining using stripes cut from the left and right edges of the protein blots. The middle portion of the blot containing the p24 protein was cut out, dissolved in dimethyl sulfoxide, and used directly for immunization. Immunization and challenge. In a preliminary experiment, two adult New Zealand White rabbits were immunized with nitrocellulose-bound p24. Before injection, the nitrocellulose was dissolved in 0.5 ml of dimethyl sulfoxide and then injected subcutaneously into three skin sites per rabbit (one with and one without the addition of complete Freund adjuvant). Approximately 2 to 6 ,ug of p24 was used for each injection. The rabbits were boosted again subcutaneously with filter-bound protein (no adjuvant) on days 30 and 90. Bleedings were done periodically to monitor the titer of antibody. One week after the final injection, the rabbits were challenged at eight skin sites with 103 viable T. pallidum organisms per site. Two normal rabbits were also inoculated with the organisms at the same time. The development of lesions was visually inspected periodically for 3 months. Gel electrophoresis and immunoblotting. Percoll-purified T. pallidum or fresh overnight cultures of E. coli cells were lysed by boiling in Laemmli sample buffer. Supernatants from cultures of E. coli clones were filtered (filter pore size, 0.45 ,im; Millipore Corp., Bedford, Mass.), concentrated 20-fold by centrifugation with Centricon-10 (Amicon Corp., Lexington, Mass.), and mixed with sample buffer before

T. PALLIDUM ANTIGEN p24

VOL. 56, 1988

they were loaded on the SDS-polyacrylamide slab gels. Electrophoresis was done according to the standard Laemmli method (19). Two-dimensional gel electrophoresis was done as described previously (27). Before transfer, the gels were soaked in transfer buffer (20% methanol, 14.4 g of glycine per liter, 3 g of Tris per liter) for 20 min to remove SDS. A BA83 nitrocellulose filter (0.2 ,um; Schleicher and Schuell) was used as the blotting membrane. For the detection of transferred proteins, one stripe of the nitrocellulose filter was stained with 0.5% fast green in 20% methanol-5% acetic acid. The identification of antigens on the blot was carried out as described above by using immune or specific anti-p24 serum. All antisera were preadsorbed with a lysate of E. coli cells to reduce the nonspecific background staining. The tetramethyl benzidine color reagent was used as described by Hindersson et al. (15) to detect antigens on two-dimensional electrophoresis electroblots. Southern blot hybridization. Genomic and plasmid DNAs were digested with the indicated restriction enzyme, electrophoresed on a 1% agarose gel, and then transferred to a nitrocellulose filter (BA83) according to the procedure of Southern (35). The filter was baked for 2 h at 80°C in the vacuum oven and then prehybridized in a solution containing 6x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5x Denhart solution, 0.5% SDS, 0.01 mM EDTA, and 100 ,ug of denatured herring sperm DNA per ml at 42°C for at least 4 h. 32P-labeled nick-translated probes were added, and the hybridization was continued at 42°C overnight. The filters were washed at room temperature with two changes of 2x SSC-0.01% SDS and then washed at 55°C with two changes of 0.lx SSC-0.1% SDS. The filters were dried and exposed to XAR-5 film at -70°C with or without intensifying screens.

The amounts of p24 in the lysates of these seven clones (for example, clone pPH21) were approximately 0.2 to 0.5% of the total E. coli proteins estimated from Coomassie blue staining of the gels (Fig. 1). On immunoblots, p24 reacted with both SHS and IRS but not with pooled normal sera. A control E. coli clone, pPH9, bearing an unrelated DNA insert on the same vector, did not express the p24 antigen. The p24 antigen did not cross-react with normal E. coli proteins, since extensive adsorption of IRS or SHS with the lysate of E. coli cells did not abolish the anti-p24 reactivity. The molecular mass and antigenicity of the p24 antigen was not affected by heat or reducing agents (data not shown). In most immunoblots, the p24 antigen-expressing clones (e.g., pPH21) also expressed several smaller reactive antigens with molecular masses ranging from 12 to 20 kDa. These lowermolecular-mass antigens are likely to be the proteolytic cleavage products of the original p24 polypeptide, since they also reacted with monospecific antisera directed against purified and intact recombinant p24 (see below and Fig. 2). Interestingly, the colony morphologies of all p24-producing E. coli clones were characterized by a flat and rough surface texture, in contrast to the raised and smooth appearance of other E. coli colonies. On microscopic examination, the 24-kDa antigen-producing E. coli clones appeared as filamentous cells in contrast to the rod-shaped cells of normal E. coli (data not shown). A filamentous growth pattern was not observed in other E. coli cells carrying an unrelated plasmid. The seven p24 antigen-expressing clones

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RESULTS Identification of E. coli clones expressing antigens of T. pallidum. A DNA library was constructed by inserting T. pallidum DNA partially digested with BamHI into the plasmid vector pUC8 and transformation of E. coli cells. We chose pUC8 as the cloning vector because of the presence of multiple cloning sites and lac promoter activity which could facilitate the manipulation of the restriction fragments and promote the expression of the inserted genes (providing that the foreign DNA was inserted in the correct reading frame). This pUC8 library contained an estimated 2 x 106 transformants. Approximately 95% of the colonies contained plasmids with DNA inserts. By using an in situ immunoscreening method, 2.5% of the E. coli colonies reacted with the immune sera of rabbits infected with T. pallidum (IRS) and approximately 0.5 to 1% of colonies reacted with the syphilitic human sera (SHS) from patients with primary or secondary syphilis. From the initial screening of 20,000 colonies with SHS, 39 very strongly positive colonies were selected and subjected to further analysis with (i) pooled IRS, (ii) SHS, (iii) pooled normal human sera, and (iv) pooled normal rabbit sera. All 39 colonies reacted with SHS and IRS but not with the normal sera (data not shown; see Fig. 5 for examples of immunoscreening). Expression of the p24 antigen in E. coli cels. The antigens expressed by the E. coli clones were characterized by using SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting. Both SHS and IRS reacted with a 24-kDa antigen in 7 of the 39 strongly positive clones, whereas normal sera showed no reactivity with the p24 antigen.

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FIG. 1. Identification of the 24-kDa antigen in recombinant clone pPH21. Total protein lysates of E. coli clones pPH21 and pPH9 and T. pallidum (T.P.) were electrophoresed on an SDS-12% polyacrylamide gel and then stained with Coomassie blue R-250 (A) or

transferred to a BA83 nitrocellulose filter and then immunoblotted with a 1:200 dilution of SHS (B), a 1:50 dilution of IRS (C), or a 1:50 dilution of normal rabbit serum (NRS) (D). Arrows indicate the positions of the p24 antigen.

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(pPH20 to pPH26) were chosen for further study because of their ability to express the antigen and the abnormal E. coli growth patterns. As confirmed in subsequent experiments (see below), this unique growth pattem appeared to be directly related to production of the p24 polypeptide. Immunogenicity of recombinant p24 and identification of the equivalent antigen in T. pallidum cells. The data in Fig. 1C indicate that although IRS reacted intensely with recombinant p24, only a very small amount of antigen with a 24-kDa molecule mass was detected in Percoll-purified T. pallidum. In addition, since the IRS used in this immunoblotting contained very heterogeneous populations of antibodies, these data did not permit the conclusion that an identical p24 is present in T. pallidum. Thus, to identify the T. pallidum protein that is equivalent to cloned p24, we decided to raise monospecific antisera to the recombinant p24 in rabbits. These immunized rabbits were also preliminarily tested to see whether they could be protected from infection with live T. pallidum organisms. Recombinant p24 was purified from E. coli clone pPH21 by lysozyme lysis, DEAE chromatography, and gel electrophoresis as described in Materials and Methods. This gentle lysis procedure was chosen because of its high efficiency in releasing the protein and producing less background contamination. From the DEAE column, the protein eluted at 0.5 N NaCl. This preparation was further purified by electrophoresis and then transferred to the nitrocellulose membrane. The protein spot corresponding to p24 was cut out, dissolved in dimethyl sulfoxide, and used directly to immunize the rabbits. One rabbit developed anti-p24 antibody by 14 days after a single injection of the filter-bound antigen. The antibody titer became higher after booster injections. The second rabbit

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INFECT. IMMUN.

HSU ET AL.

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also developed a high anti-p24 response after one booster injection. Three months after initial immunization (1 week after the last booster), the rabbits were challenged with live T. pallidum at eight skin sites. In this preliminary test, the immunized rabbits showed accelerated development of lesions compared with that of the unimmunized animals. However, both immunized and unimmunized animals recovered completely by 3 months postinfection. The newly generated anti-p24 sera (1:200 dilution) reacted intensely with the recombinant p24 in pPH21 cells (Fig. 2A and C). The sera also recognized several pPH21 polypeptides with sizes ranging from 12 to 20 kDa, suggesting that these smaller antigens may be the proteolytic degradation products of the original p24 polypeptide (Fig. 2A and C; also, see Discussion). A higher concentration of normal rabbit sera (1:50 dilution) did not react with recombinant p24 or the smaller polypeptides (Fig. 2B and E). Both monospecific anti-p24 sera (1:200 dilution) recognized a protein of exactly the same molecular size in Percoll-purified T. pallidum (Fig. 2A and C). However, the amount of p24 antigen in T. pallidum cells appeared to be very low, since at least 5 x 107 organisms were needed to show this reactivity. The low abundancy of p24 in T. pallidum suggested that this antigen is a secreted protein or that the protein is a highly antigenic but minor component of the organism, since both SHS and IRS contained high titers of anti-p24 activity. Analysis of p24 protein in E. coli and T. paUidum cells by two-dimensional gel electrophoresis and immunoblotting. The p24 in E. coli and T. pallidum cells was further characterized by using two-dimensional gel electrophoresis and immunoblotting (Fig. 3). The T. pallidum p24 polypeptide was identified as a smear at the basic side of the gel (Fig. 3B),

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VOL. 56, 1988

T. PALLIDUM ANTIGEN p24

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