Expression and Immunogenicity of Proteins Encoded by Sequences Specific to Mycobacterium avium subsp. paratuberculosis

Bacterial strains.M. avium subsp. paratuberculosis K-10, a bovine clinical isolate from a dairy herd in Wisconsin, was cultured in Middlebrook 7H9 broth (pH 6.0) supplemented with oleic acid-albumin-dextrose-catalase (Becton Dickinson Microbiology, Sparks, Md.), 0.05% Tween 80, and ferric mycobactin J (2 mg/liter; Allied Monitor Inc., Fayette, Mo.). This strain served as the source of the template for all PCR amplifications. Escherichia coli DH5α was routinely grown in Luria-Bertani broth (LB) and maintained on LB agar plates. All maltose binding protein (MBP) fusions were constructed in this strain. E. coli TOP10F′ and E. coli BL21(DE3) (both from Invitrogen Life Technologies, Carlsbad, Calif.) cells were cultured in SOC medium prepared according to Sambrook et al. (31a). When appropriate, ampicillin (50 or 100 μg/ml) was added to the medium for selection. Frozen stocks were prepared by harvesting mid-log-phase cells in fresh LB supplemented with 20% glycerol and storing at −80°C.

Cloning of M. avium subsp. paratuberculosis-specific sequences.MBP fusions of 21 M. avium subsp. paratuberculosis predicted coding sequences were produced in E. coli by using the pMAL-c2 vector (New England Biolabs, Beverly, Mass.). These coding sequences and their nucleotide accession numbers have been described previously (2) and are designated genes 10, 11, 38, 56, 57, 135, 159, 217, 218, 219, 228, 240, 241, 250, 251, 252, 253, 254, 255, 256, and 257. Primers were designed from the reading frame of each coding sequence and contained an XbaI site in the 5′ primer and a HindIII site in the 3′ primer for cloning purposes. Amplifications were performed by using Pwo polymerase (Roche Diagnostics Corp., Indianapolis, Ind.) and M. avium subsp. paratuberculosis genomic DNA as the template under conditions described previously (2). The vector and amplification product were digested with XbaI and HindIII. Ligation of these restricted DNA fragments resulted in an in-frame fusion between the malE gene in the vector and the reading frame of interest. Following ligation, the products were transformed into E. coli DH5α and selected on LB agar plates containing 100 μg of ampicillin/ml. The pCRT7/CT-TOPO vector (Invitrogen Life Technologies) was used to produce polyhistidine (six-His)-tagged M. avium subsp. paratuberculosis proteins. No restriction sites were engineered in primers used in these amplifications, because each gel-purified PCR product was cloned into the vector by using topoisomerase. The construct was transformed into E. coli TOP10F′ cells and then subcloned into the BL21(DE3)/pLysS cells (Invitrogen Life Technologies). Transformed cells were plated on LB agar containing 50 μg of ampicillin/ml. Inserts in all plasmid constructions were confirmed by DNA sequencing. Table 1 lists the primers used to clone each gene in the E. coli expression vectors pMAL-c2 and pCRT7/CT-TOPO.

Protein expression.Each MBP fusion protein (e.g., MBP-gene 218) was overexpressed in E. coli by induction with 0.3 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Sigma, St. Louis, Mo.) and purified by affinity chromatography using an amylose resin supplied by New England Biolabs. A detailed protocol has been published previously (1). Expression of mycobacterial fusion proteins was monitored by GelCode Blue (Pierce Biotechnology Inc., Rockford, Ill.)-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and by immunoblot analysis with a monoclonal antibody against MBP (α-MBP) developed at the National Animal Disease Center (NADC). The same approach was used for production and purification of all MBP fusion proteins. E. coli DH5α harboring the parental plasmid pMAL-c2 was expressed, purified, and used as a control in all experiments. Purified protein from this control strain consists of an MBP fusion of the LacZ alpha peptide. Expression of six-His-tagged M. avium subsp. paratuberculosis proteins was similarly induced with 0.3 mM IPTG. Three hours following induction, the cells were lysed by sonication and the recombinant protein was purified on TALON resin (Clontech, Palo Alto, Calif.) columns under denaturing conditions. Mycobacterial genes were expressed as soluble proteins with a C-terminal six-His tag downstream of the gene of interest. Expression of mycobacterial protein was monitored by immunoblot analysis with a horseradish peroxidase-conjugated monoclonal antibody to the six-His tag (Clontech). Purified fractions were pooled and dialyzed by using Slide-A-Lyzer cassettes (Pierce Biotechnology Inc.) in 1 liter of phosphate-buffered saline (PBS, comprising 150 mM NaCl and 10 mM NaPO₄ [pH 7.4]) with three exchanges at 4°C.

SDS-PAGE and immunoblotting.E. coli protein lysates were prepared as previously described (30). PAGE was performed using 12% (wt/vol) polyacrylamide gels. Proteins were electrophoretically transferred onto pure nitrocellulose filters (Schleicher & Schuell, Keene, N.H.) with the Trans Blot Cell (Bio-Rad Laboratories, Hercules, Calif.) in sodium phosphate buffer (25 mM; pH 7.8) at 0.8 amps for 90 min. After transfer, filters were blocked with PBS plus 2% bovine serum albumin (BSA) and 0.1% Tween 20 (PBS-BSA). Antisera were diluted as listed in Table 2 in PBS-BSA and were incubated on the blot at room temperature for 2 h. After three washes in PBS plus 0.1% Tween 20, blots were incubated for 1.5 h in either protein A-peroxidase (Pierce Biotechnology Inc.) or peroxidase-conjugated antibodies that detect mouse and cow antibodies (Pierce Biotechnology Inc.). All secondary antibodies were diluted 1:20,000 in PBS-BSA. The blots were again washed three times as described above and were developed for chemiluminescence by using Supersignal detection reagents (Pierce Biotechnology Inc.).

Sera from M. avium subsp. paratuberculosis-exposed rabbits and mice and from cattle with Johne's disease.New Zealand White rabbits were immunized with heat-killed (n = 3) or live (n = 1) preparations of M. avium subsp. paratuberculosis in earlier studies (35). BALB/c mice (n = 2) were immunized with a sonicated lysate of M. avium subsp. paratuberculosis K-10. This lysate was prepared by harvesting M. avium subsp. paratuberculosis at an optical density at 540 nm of 0.4, resuspending in cold PBS (1/100 of culture volume), and sonicating three times for 10 min each time, with a 10-min incubation on ice in between sonications. Cell debris was centrifuged out at 10,000 × g.

The NADC is a national leader in large animal biocontainment. There are currently more than 20 head of cattle (holsteins and Brown Swiss) housed at the NADC for Johne's disease research activities. Control cattle are allowed to graze on pastures, whereas clinical cows are held in BL2 animal barns at NADC. Sera from 10 well-characterized cattle in the clinical stage of Johne's disease (shedding more than 100 bacilli per g of feces as determined by plate counts on Middlebrook 7H9 slopes) and 9 control cattle were used in immunoblot analysis for detection of antibodies that bind unique M. avium subsp. paratuberculosis proteins. Table 2 lists all sera used in these studies. All cattle sera were diluted 1:500 in immunoblot assays.

Cloning, expression, and purification of M. avium subsp. paratuberculosis sequences in E. coli.Twenty-one predicted coding sequences that are present uniquely in M. avium subsp. paratuberculosis have been identified previously (2). The deduced polypeptides produced from these coding sequences have no known peptide motifs or sequence similarity to other proteins in public databases such as GenBank. Each of these coding sequences was amplified from purified M. avium subsp. paratuberculosis genomic DNA and cloned into an E. coli expression vector as described in Materials and Methods. Because affinity purification tags coexpressed with the protein of interest can profoundly influence stability, solubility, and expression levels of recombinant protein, we evaluated two different affinity tags with each protein of interest. Predicted coding sequences were expressed in conjunction with either a six-His tag (5) or the 42-kDa MBP tag (31) for affinity purification. Efforts to clone and express each of the coding regions yielded various degrees of success with each tag. All of the MBP fusion proteins were successfully expressed and purified, although protein yields were very different (Fig. 1A). Coding sequences designated genes 253 (Fig. 1A, lane 3), 57 (lane 11), 252 (lane 16), 135 (lane 19), 217 (lane 20), 257 (lane 21), and 254 (lane 23) and the C-terminal half of gene 218 (218-C) (lane 25) were all expressed to high levels in E. coli. Other coding sequences including genes 219 (Fig. 1, lane 8), 256 (lane 18), and 250 (lane 22) were only marginally produced by E. coli. Still other proteins showed multiple bands (gene 159 [lane 5] and gene 251 [lane 4]), possibly due to proteolytic cleavage. Conversely, only 5 of the 21 six-His tag clones produced recombinant M. avium subsp. paratuberculosis protein, and those that did, produced a relatively small yield that could be detected only by immunoblotting (Fig. 2). Therefore, this study focused only on the MBP fusion proteins that were expressed and purified from E. coli. Table 3 lists the number of deduced amino acids, predicted antigenicity, calculated molecular size, and purification yield for each MBP fusion protein.

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FIG. 1.

SDS-PAGE and immunoblot analysis of affinity-purified M. avium subsp. paratuberculosis gene products. (A) Shown are two SDS-12% PAGE gels loaded with affinity-purified MBP fusion proteins that were stained with GelCode Blue. Size standards (in kilodaltons) are indicated on the left. (B through E) Corresponding immunoblots were probed with a monoclonal antibody to MBP (B) or with serum from rabbit 275 (C), mouse 47 (D), and clinical cow 67 (E). Note that MBP is not detected in panel C, D, or E. Lanes: 1, protein size standards; 2, gene 255; 3, gene 253; 4, gene 251; 5, gene 159; 6, gene 228; 7, gene 10; 8, gene 219; 9, gene 38; 10, gene 56; 11, gene 57; 12, MBP-MMP; 13, gene 218 (N-terminal half); 14, protein size standards; 15, gene 241; 16, gene 252; 17, gene 240; 18, gene 256; 19, gene 135; 20, gene 217; 21, gene 257; 22, gene 250; 23, gene 254; 24, gene 11; 25, gene 218 (C-terminal half); 26, MBP. Arrows point to the five subspecies-specific antigens identified in this study.

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FIG. 2.

Expression of M. avium subsp. paratuberculosis six-His tag clones in E. coli. An immunoblot containing IPTG-induced E. coli BL21(DE3) lysates was probed with a monoclonal antibody that detects the polyhistidine tag. Size standards (in kilodaltons) are indicated on the left. Lanes: 1, protein size standards; 2, gene 254; 3, gene 159; 4, gene 56; 5, gene 38; 6, gene 228; 7, E. coli BL21(DE3) cells.

Serological analysis of recombinant M. avium subsp. paratuberculosis proteins with rabbit and mouse sera.Each protein was next examined by immunoblot analysis with rabbit and mouse sera containing α-M. avium subsp. paratuberculosis antibodies. Rabbits and mice that were immunized with various preparations of M. avium subsp. paratuberculosis are listed in Table 2. All fusion proteins were detected by a monoclonal antibody to the MBP tag (Fig. 1B); however, only five of the purified proteins were detected by α-M. avium subsp. paratuberculosis antibodies present in both rabbit and mouse sera (Fig. 1C and D, respectively). These antigenic proteins are MBP-gene 56 (Fig. 1C and D, lanes 10), MBP-gene 218 (lanes 13 and 25), MBP-gene 241 (lanes 15), MBP-gene 135 (lanes 19), and MBP-gene 254 (lanes 23). Serological reactivity associated with the two fusion proteins encoded by genes 56 (lanes 10) and 135 (lanes 19) was readily visible in mouse (Fig. 1D) but not rabbit (Fig. 1C) serum. Additional immunoblotting experiments showed that the same five proteins were also detected by sera from mouse 48 and rabbits 273 and 274 (data not shown). One MBP fusion protein detected by the mouse serum only (Fig. 1D, lane 8) was not of the expected size and therefore was not considered further. In addition, a known M. avium subsp. paratuberculosis protein, termed the major membrane protein (MMP) (3), included as a control, was detected by both the rabbit and mouse sera (Fig. 1C and D, lanes 12), whereas the MBP control protein was detected by neither (Fig. 1C and D, lanes 26). These data suggest that at least five subspecies-specific proteins are produced by M. avium subsp. paratuberculosis and are immunogenic.

Reactivity to sera from control and Johne's disease cattle.To determine whether M. avium subsp. paratuberculosis fusion proteins will specifically react with antibodies from Johne's disease cattle, sera from 10 cattle in the clinical stage of Johne's disease were used in immunoblot experiments with the same set of purified proteins described above. Results of a typical experiment using serum from clinical cow 67 are shown in Fig. 1E. As was seen in the immunoblots with rabbit and mouse sera, both the N- and C-terminal halves of protein 218 were detected by serum from this clinical cow. In addition, products from genes 241 (lane 15), 135 (lane 19), and 254 (lane 23) were detected. The antigen produced from gene 56 (lane 10) may also be detected; however, this is not conclusive due to background present in that region of the immunoblot. We base this conclusion on the fact that the nonspecific binding occurs at a size range that is inconsistent with the predicted and observed size values of the recombinant purified proteins (as shown in Fig. 1A). However, this background (primarily a 50-kDa band) was not observed with the mouse or rabbit serum.

Serum from clinical cow 67 did not detect the MMP control protein (Fig. 1E, lane 12), although this protein was detected in sera from other clinical cattle (data not shown). Sera from nine additional cattle showing overt clinical disease were also evaluated against the five antigenic proteins listed in Table 4; however, four sera had antibodies to the MBP protein, making interpretation difficult (Table 4). Sera from cows 47, 167, and 598 detected all five immunogenic proteins, whereas sera from cows 14, 67, and Kay did not have antibodies to all five proteins. Only the MBP-gene 241 antigen was detected by all MBP-negative clinical sera tested. Control sera from five of nine healthy cows had antibodies to MBP, and only serum from animal 181 failed to react with any of the five proteins (Table 4).