Evidence That It Is a Bacterioferritin - Journal of Clinical Microbiology

JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1991, p. 1652-1658 0095-1137/91/081652-07$02.00/0 Copyright C 1991, American Society for Microbiology

Vol. 29, No. 8

Mycobacterium paratuberculosis Antigen D: Characterization and Evidence That It Is a Bacterioferritin BRIAN W. BROOKS,'* N. MARTIN YOUNG,2 DAVID C. WATSON,2 RUTH H. ROBERTSON,' EDWARD A. SUGDEN,1 KLAUS H. NIELSEN,' AND SUSI A. W. E. BECKER' Agriculture Canada, Animal Diseases Research Institute, Nepean, P.O. Box 11300, Station H, Nepean, Ontario, Canada K2H 8P9,' and Division of Biological Sciences, National Research Council of Canada,

Ottawa, Ontario, Canada KJA CR62 Received 28 November 1990/Accepted 23 May 1991

By using a combination of agarose and polyacrylamide gel electrophoresis, Mycobacterium paratuberculosis antigen D was resolved from a crude sonicated preparation of the organism and characterized as a component with a molecular mass of approximately 400,000 Da. While this component was composed mainly of protein, with unusually high proportions of glutamic acid and leucine, it was resistant to digestion with a number of proteolytic enzymes. Structural detail revealed by electron microscopy, amino acid sequence data, and the demonstration of a Soret band in its absorption spectrum indicated that antigen D was similar to an Escherichia coli bacterioferritin.

Diagnosis of paratuberculosis (Johne's disease), an enteric disease caused by infection with Mycobacterium paratuberculosis and with many of the immunopathological spectral manifestations observed in leprosy (23) and tuberculosis (17), can be difficult in the living animal. Fecal culture is presently recognized as the most reliable index of infection in live cattle, but current procedures are laborious, fail to detect animals shedding low numbers of organisms, require approximately 8 to 16 weeks of incubation for the development of M. paratuberculosis colonies, and frequently fail to prevent the overgrowth of culture media with more rapidly growing organisms (8, 24). In addition, certain M. paratuberculosis strains are not readily cultivated (9). Serological tests are potentially cheaper and much more rapid than culture, and a number of procedures have been evaluated for the detection of antibody which may develop during the course of infection with M. paratuberculosis (8, 24). Problems with sensitivity and specificity are common. These have been attributed to the spectrum of immunological response to M. paratuberculosis (10), to the high degree of antigenic cross-reactivity among the mycobacteria (25, 28) and between mycobacteria and other organisms (34), and to the use of crude, nonstandardized complex mixtures of components as antigens. A better understanding of the diversity and frequency of the immunological responses of animals to defined components or epitopes of M. paratuberculosis may lead to the development of improved serological tests. M. paratuberculosis antigen D has been demonstrated to be useful for the serological diagnosis of paratuberculosis in sheep (6). Antibodies to antigen D were detected in high frequency in sera from animals with paratuberculosis, and the magnitude of the serological response to this component correlated well with the acid-fast bacterial load. Antigen D was recognized as a precipitin arc on agar gel immunodiffusion and crossed immunoelectrophoresis (CIE). Further characterization of antigen D would facilitate the development of purification procedures which would allow its evaluation in other serological assays. *

Corresponding author. 1652

The objective of the present study was to further define antigen D. By agarose and polyacrylamide gel electrophoresis (PAGE), antigen D was characterized as a protein component with a molecular mass of approximately 400,000 Da. Electron microscopy and amino acid sequence data indicated that antigen D was similar to an Escherichia coli bacterioferritin (cytochrome bl). (Preliminary aspects of this work were presented at the 93rd Annual Meeting of the United States Animal Health Association, Las Vegas, Nev., 1989.)

MATERIALS AND METHODS Preparation of sonic extract. M. paratuberculosis C-286 sonic extract was prepared as described elsewhere (6). In summary, growth was in modified Long's liquid medium (18) for 12 weeks at 37°C. The bacilli were collected on a Whatman no. 2 filter and washed once, and the pellet was weighed, and suspended in sterile saline, and sonicated for 22 min. The sonicated material was centrifuged at 20,000 x g (4°C) for 20 min, and the supernatant was concentrated to 3.5 mg of protein per ml. The protein concentration was determined by a dye-binding method (5). The concentrated material was aliquoted and stored at -20°C. CIE. CIE was performed according to standard procedures (6). For tandem CIE (15), two 4-mm-diameter wells with an 8-mm center-to-center spacing were used. A voltage gradient of 10 V/cm (measured at the center of the cooling plate) was applied for 1.5 h for first-dimension electrophoresis. A 2-cm-wide gel strip containing the separated antigens was cut and transferred to another glass plate, and a reference gel containing 7.1 ml of agarose and 0.9 ml of serum was cast on the remainder of the plate. The serum used in all CIE analyses was from a pool of serum samples obtained from a single herd of goats heavily infected with M. paratuberculosis. A voltage gradient of 2 V/cm was applied for 16 h for second-dimension electrophoresis. The plate was then placed in a humid chamber for 24 to 72 h at room temperature before pressing, washing, and staining. In some cases for first-dimension electrophoresis, a larger antigen well (4 by 8 mm) was cut and 40 ,ul of the M. paratuberculosis sonic extract was added. After electropho-

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CHARACTERIZATION OF M. PARATUBERCULOSIS ANTIGEN D

resis at 10 V/cm for 1.5 h, the agarose was cut to obtain a strip 0.8 cm wide extending from the antigen well to the anode side of the plate. The agarose strip was cut perpendicular to its long axis into 0.5-cm-wide slices. The slices were mascerated in 80 p1l of a sodium dodecyl sulfate (SDS)-PAGE sample buffer (see below). M. paratuberculosis components eluted from the gel slices were obtained by leaving the agarose suspended in the sample buffer for at least 16 h at 4°C and then centrifuging it at 16,000 x g (50C) for 5 min. The supernatant was removed and applied directly to a gel or stored at 4°C. SDS-PAGE. SDS-PAGE was performed by a modification of the discontinuous system of Laemmli (16) in 0.8-mm-thick slab gels. The stacking gel contained 6% acrylamide, and the separating gel was an 8 to 15% linear acrylamide gradient. SDS was not included in either the stacking or the separating gel. One volume of the M. paratuberculosis sonic extract was combined with an equal volume of an SDS-PAGE sample buffer containing 1.25% (wt/vol) SDS, 1.25% (vol/ vol) 2-mercaptoethanol, 12.5% (vol/vol) glycerol, 0.0625 M Tris base (pH 6.8), and 0.00125% (wt/vol) bromophenol blue and, with or without boiling for 5 min, was applied to the gel. Proteins were visualized with Coomassie blue. The silverstain method of Tsai and Frasch (32) which detects mainly carbohydrates was also used. Bands of interest in nonstained gels, located by comparison with stained gels, were cut out and mascerated in 80 ,ul of saline. After at least 16 h at 4°C, the saline-acrylamide mixture was centrifuged at 16,000 x g (5°C) for 5 min and the supernatant was removed and stored at 4°C. Molecular mass standards were purchased from Bio-Rad Laboratories Canada Ltd., Mississauga, Canada. Horse spleen ferritin (440,000 Da) was obtained from the Sigma Chemical Company, St. Louis, Mo. SDS-PAGE was also performed on 20% acrylamide gels with the Pharmacia PhastSystem gel apparatus (Pharmacia Canada Ltd., Baie D'Urfe, Quebec, Canada) by using silver staining, a standard protein mixture (Bio-Rad Laboratories), and a mixture of myoglobin (17,200 Da), and P-lactoglobulin (18,400 Da). NATIVE PAGE. Nondenaturing, discontinuous PAGE was performed as described elsewhere (lla) in 0.8-mm-thick slab gels. The stacking gel contained 3.1% acrylamide, and the separating gel contained 8% acrylamide. Molecular mass standards (thyroglobulin, 669,000 Da; ferritin, 440,000 Da; catalase, 232,000 Da; lactate dehydrogenase, 140,000 Da; and albumin, 67,000 Da) were purchased from Pharmacia Canada Ltd. Enzyme digestions. Proteinase K (E. Merck Biochemicals), trypsin (Difco Laboratories), pronase (Calbiochem), pepsin (BDH Chemicals), lysozyme (Worthington Biochemical Corp.), DNase (BDH Chemicals), RNase (ICN Biochemicals), chymotrypsin, lipase (Nutritional Biochemicals), and Staphylococcus aureus V8 protease (Sigma) were dissolved or suspended (lipase) at a concentration of 2.5 mg/ml in 0.85% NaCl. Papain (Nutritional Biochemicals) was dissolved at a concentration of 2.5 mg/ml in a buffer containing 1.1 mM EDTA (disodium salt; Fisher Scientific), 0.07 mM 2-mercaptoethanol, and 5.5 mM cysteine-HCl (BDH Chemicals). Enzyme solutions or suspensions were prepared within 2 h of use. The enzyme was mixed 1:2.5

(vol/vol) with the M. paratuberculosis sonicated preparation for a final enzyme concentration of 0.7 mg/ml. Digestions were for 30 min or 24 h at room temperature or for 10 min at 70°C. After digestion the enzyme-sonic extract preparation was combined 1:1 (vol/vol) with the SDS-PAGE sample buffer and applied to an SDS-PAGE gel. As a control, saline was used in place of the enzyme solution. In addition, each

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of the enzyme preparations was mixed with saline and processed in the same manner as the enzyme-sonic extract mixture was. Purification of M. paratuberculosis antigen D. Ammonium sulfate (0.25 g) was added to 1 ml of the M. paratuberculosis sonicated preparation. The mixture was left for 4 h at room temperature and then centrifuged at 4,000 x g for 30 min at 4°C. The pellet was washed (4,000 x g, 30 min, 4°C) twice with 1.75 M ammonium sulfate. The pellet was resuspended in 1.0 ml of 0.01 M Tris buffer, pH 7.5, 0.4 ml of proteinase K (2.5 mg/ml in 0.01 M Tris buffer) was added, and the mixture was left at room temperature for 1 h and then dialyzed against 0.15 M NaCl at 4°C. The dialyzed material was precipitated and washed with ammonium sulfate as described above. The final pellet was resuspended in 0.01 M Tris buffer, pH 7.5, and dialyzed against 0.15 M NaCl at 4°C. Purified antigen D was also chromatographically prepared in larger amounts as described previously (30). Briefly, the ammonium sulfate-precipitated sonicated preparation was chromatographed on Sephacryl S-200 and the high-molecular-mass antigen D was collected in the exclusion peak. Antigen D was further purified by NaCl elution from DEAESephacel followed by passage through concanavalin A-Sepharose 4B to remove carbohydrate contamination. Amino acid analysis and N-terminal sequencing. Samples of antigen D, purified by chromatography, were hydrolyzed in 6 N HCl for 22 h at 110°C, and the hydrolysates were analyzed for amino acids with a Durrum D500 analyzer. Automatic gas-phase sequencing was performed on an Applied Biosystems 475A protein-sequencing system incorporating a model 470A gas phase sequencer equipped with an on-line model 120A PTH analyzer under the control of a model 900A control-data analysis module. Electron microscopy. A drop (approximately 50 ,ul) of an aqueous suspension of purified antigen D was placed on a 400-mesh Formvar carbon-coated electron microscope grid. Excess fluid was removed with filter paper. A drop of either 3% phosphotungstic acid, pH 7.1, or 2% uranyl acetate, pH 4.0, was applied for 25 s. Excess stain was removed with filter paper, and the grid was examined immediately with a Hitachi model HU-12A electron microscope operating at 75 kV. Spectrophotometry. UV-visible absorption spectra were measured with a Varian Superscan 3 scanning spectrophotometer. A 2-mm-pathlength cell was filled with a solution of antigen D (approximately 0.8 mg/ml) in 0.15 M sodium chloride-0.01 M phosphate buffer, pH 7.2. After the spectrum was recorded, the sample was reduced with a few crystals of sodium dithionite and the spectrum was redetermined. RESULTS The D precipitin arc and other arcs demonstrated by CIE using the M. paratuberculosis sonicated antigen preparation and pooled serum from goats are shown in Fig. 1A. With the same reagents and conditions, the area under each of the arcs and their location, relative to the antigen well were consistent between runs. The base of the D arc began approximately 4.5 cm from the antigen well and extended anodally approximately 1.5 cm. SDS-PAGE protein profiles of the M. paratuberculosis sonicated preparation obtained with different conditions of heating are shown in Fig. 2 (lanes 4, 5, and 6). Forty-five bands in the molecular mass range from approximately 10,000 to 400,000 Da were visible. One band which migrated

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FIG. 1. CIE of M. paratuberculosis C-286 sonic extract and a pool of sera from goats with paratuberculosis. (A) 5 ,ul of sonic extract plus 5 ,ul of saline; (B) 5 p.l of sonic extract plus 5 ,ul of SDS-PAGE sample buffer; (C) tandem CIE-5 p.l of sonic extract plus 5 p.l of saline (left well) and 10 RI of purified antigen D (right well). Antigen wells are at the lower left. Arrows indicate the D precipitin arc. The anode is to the right in the first-dimension electrophoresis and at the top in the second-dimension electrophoresis in all plates. Plates were stained with Coomassie blue.

through a 3.15% acrylamide gel but only slightly entered a 6% acrylamide gel was also observed (results not shown). A component with a molecular mass of approximately 400,000 Da was seen when the sonicated preparation-SDS-PAGE sample buffer mixture was not heated (Fig. 2, lane 4) and when the sonicated preparation was boiled for 5 min and then combined with the SDS-PAGE sample buffer and the mixture was not heated (Fig. 2, lane 5). However, when the sonicated preparation-sample buffer mixture was boiled for 5

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FIG. 2. Coomassie blue-stained SDS-PAGE protein profiles of M. paratuberculosis sonic extract after various heat treatments and digestion with proteinase K. Lanes 1 and 2, molecular mass markers (molecular mass at left in thousands); lane 3, horse spleen ferritin (440,000 Da); lane 4, sonic extract plus SDS-PAGE sample buffer (not heated); lane 5, sonic extract (boiled for 5 min) plus SDS-PAGE sample buffer; lane 6, sonic extract plus SDS-PAGE sample buffer (mixture boiled for 5 min); lane 7, sonic extract digested with proteinase K; lane 8, proteinase K and no sonic extract.

min, this high-molecular-mass component was not detected (Fig. 2, lane 6). On native PAGE the molecular mass of the large-size component was also estimated to be approximately 400,000 Da (results not shown). The components in the M. paratuberculosis sonicated preparation were also resolved by a combination of CIE and SDS-PAGE. The sonicated preparation was mixed with the SDS-PAGE sample buffer and without heating was resolved by CIE (Fig. 1B). Under native conditions and in the presence of the SDS-PAGE sample buffer, the location and the area enclosed by the D arc were similar (compare Fig. 1A and B). Also, the components in the sonicated preparation were separated under the conditions of first-dimension CIE, the agarose strip containing the separated components was cut into 0.5-cm-wide slices, and the components in each of the slices were eluted with the SDS-PAGE sample buffer and without heating were resolved by SDS-PAGE. A 400,000-Da component was observed with the material eluted from the four slices 4.0 to 6.0 cm from the antigen well, which is the same region in which the D arc was visualized with the pooled goat sera. The effect of various enzymes on the M. paratuberculosis sonicated preparation was investigated. After treatment with pronase, papain, proteinase K, trypsin, chymotrypsin, S. aureus V8 protease, and pepsin for 1 or 24 h at room temperature or for 10 min at 70°C followed by SDS-PAGE, the gels were stained with Coomassie blue and only a

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CHARACTERIZATION OF M. PARATUBERCULOSIS ANTIGEN D

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FIG. 3. SDS-PAGE (Phast System) of antigen D, with silver staining. Lane 1, myoglobulin and 1-lactoglobulin; lane 2, antigen D, boiled in SDS-buffer for 5 min; lane 3, protein standards (molecular mass in thousands).

400,000-Da band and several very low molecular weight bands were seen. As an example, the results obtained following treatment of the sonicated preparation with proteinase K are shown in Fig. 2, lane 7 (compare with Fig. 2, lane 4). A band with a molecular mass of approximately 92,000 Da was also seen with proteinase K alone (Fig. 2, lane 8). Carbohydrate components, including the lipoarabinomannan antigen (31), were detected in the sonicated preparation before and after treatment with the various proteases. A 400,000-Da band was also observed after treatment of the sonicated preparation with lysozyme, lipase, RNase, or DNase (results not shown). The 400,000-Da component was purified for further study by three procedures: (i) elution from the top 0.5 cm of the SDS-PAGE separating gel on which the M. paratuberculosis sonicated preparation had been resolved, (ii) digestion of the sonicated preparation with proteinase K for 24 h at room temperature followed by SDS-PAGE and elution from the top 0.5 cm of the separating gel, and (iii) ammonium sulfate precipitation of the sonicated preparation, digestion with proteinase K, and extensive dialysis. Purified preparations of antigen D were reddish in color. When the purity of the material obtained was assessed by SDS-PAGE, only the 400,000-Da band was observed after staining with Coomassie blue and no components were detected with the Tsai and Frasch modified silver stain. A reaction of identity between the purified 400,000-Da component obtained with each of the three procedures and the D component in the M. paratuberculosis sonicated preparation was demonstrated by tandem CIE (Fig. 1C; compare with Fig. 1A).

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SDS-PAGE analysis using the Phast System on samples of purified antigen D that were boiled for at least 5 min in SDS buffer (1% SDS [final concentration]) with mercaptoethanol showed three major bands of similar apparent amounts with molecular masses close to those of myoglobin (17,000 Da) and 3-lactoglobulin (18,400 Da) (Fig. 3). There were also traces of minor components with molecular masses of 30,000 and 10,000 Da. The amino acid composition of antigen D (Table 1) included unusual proportions of glutamic acid and leucine. The protein gave an excellent N-terminal amino acid sequence run, fully interpretable to residue 42 (Fig. 4). Only one sequence was found with a yield consistent with the molecular masses of the major bands. This suggested that the major bands seen in SDS-PAGE (Fig. 3) represent processing variants of the same protein. The N-terminal sequence included several glutamic acid, glutamine, and leucine residues consistent with the amino acid analysis, and a search of the National Biomedical Research Foundation data bank revealed significant homology to an E. coli bacterioferritin (1) (Fig. 4). Examination of negatively stained preparations of antigen D by electron microscopy showed the presence of an electron-dense core approximately 5.1 nm in diameter within an approximately spherical electron-transmitting shell about 9.8 nm in diameter (Fig. 5). In preparations stained with phosphotungstic acid, the cores appeared to be more electron dense than in those stained with uranyl acetate. The absorption spectrum of antigen D showed a prominent Soret band at 417 nm and weaker bands above 500 nm (Fig. 6). Reduction with sodium dithionite shifted the bands and intensified the longer-wavelength ones. The A28JA417 ratio was 3.14 for the nonreduced sample, and the A557/A425 ratio was 6.17 for the reduced sample.

TABLE 1. Comparison of amino acid compositions of M. paratuberculosis antigen D and E. coli bacterioferritin Amino acid

Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Trp

M.

E. coli bacterioferritin

paratuberculosis antigen D (% of protein)

Gene (no. of residues/100

10.7 6.6

15.2 1.3 2.5 15.4 0.6 7.0 5.1 0 3.8 4.4 7.0 15.2 7.0 1.9 3.2 5.7 5.7 1.3

residues)'

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18.3 3.7 4.9 8.6 NDc 2.2 ND 4.9 13.9 1.6 4.3 4.5 4.1 5.7 ND

Data from the work of Andrews et al. (1). Data from the work of Tsugita and Yariv (33). ' ND, not determined.

a

b

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proteinb rti 15.2 2.9 4.3 15.2 0.7 8.0 5.8 2.9 3.6 2.9 5.8 13.0 4.3 1.4 2.2 5.1 5.1 1.4

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