Abstract
Three recombinant proteins, Map10, Map39, and Map41, produced based on nucleotide sequences obtained from the screening of Mycobacterium avium subsp. paratuberculosis genomic library expressed in Escherichia coli significantly elicited gamma interferon production in peripheral blood mononuclear cells from infected cattle. Two of these proteins were members of the PPE protein family.
The gamma interferon (IFN-γ) assay, which detects the cell-mediated immunity (CMI) response by measuring IFN-γ release from sensitized lymphocytes, has been used as an early diagnostic test for Mycobacterium avium subsp. paratuberculosis infection (11, 13). Although the IFN-γ assay is considered to be a sensitive diagnostic tool for the detection of paratuberculosis, the antigen preparation usually employed for the induction of IFN-γ is the PPD (purified protein derivative), which is the crude protein fraction prepared from the culture supernatant of M. avium subsp. paratuberculosis or M. avium subsp. avium. Because mycobacterial species contain a large amount of cross-reacting antigens, analysis of the CMI-inducing antigens of M. avium subsp. paratuberculosis and the use of those antigens with higher specificity for IFN-γ assay may further optimize the test. Furthermore, identifying the IFN-γ-inducing antigens of M. avium subsp. paratuberculosis would be useful not only for improving the CMI-based diagnostic test but also for gaining a better understanding of the host immune responses against this organism, because IFN-γ is considered to be one of the essential cytokines that play a number of important roles in achieving protective immunity against mycobacterial infection (5, 14).
In this study, an M. avium subsp. paratuberculosis expression library was constructed and screened for the identification and isolation of IFN-γ-inducing antigens by IFN-γ assay using peripheral blood mononuclear cells (PBMC) from infected cattle. Five male Holstein calves (5 to 7 days of age) were orally inoculated three times on consecutive days with 25 ml of the emulsion of the ileac portion obtained from a clinically infected cow. The emulsion of the ileum was prepared with half-strength brain heart infusion broth (Difco) containing 0.75% hexadecylpyridinium chloride (Kodak). Enumeration of M. avium subsp. paratuberculosis was carried out by serial dilution plating on Herrold's egg yolk medium containing mycobactin. The challenge inoculum contained 6.9 × 108 CFU/ml of M. avium subsp. paratuberculosis, and the total number of organisms used for the inoculation was approximately 5.2 × 1010 CFU per calf. PBMC from infected cattle were isolated by gradient centrifugation of heparinized blood on Ficoll-Paque Plus (Amersham Biosciences), and the samples were diluted in RPMI 1640 medium (Gibco BRL) with 10% fetal calf serum, penicillin (50 U/ml), and streptomycin (50 μg/ml), giving a concentration of 2 × 106 cells/ml. One milliliter of diluted PBMC was spread among the wells of 24-well tissue culture plates. The cells were stimulated with transfected E. coli or purified recombinant proteins at the appropriate concentration and then incubated at 37°C with 5% CO2 for 1 to 3 days. The IFN-γ concentrations of the culture supernatants were determined by a double-sandwich enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibody (MAb) against bovine IFN-γ (clone cc302; Serotec) (12). ELISA was performed in 96-well plates (MaxiSorp; Nunc) coated with anti-bovine IFN-γ MAb. Samples diluted with 1% skim milk in Tris-HCl-buffered saline containing 0.05% Tween 20 were added to the coated plates, incubated for 2 h at room temperature, and incubated with biotin-labeled anti-recombinant bovine IFN-γ rabbit immunoglobulin G (IgG) and with streptavidin-horseradish peroxidase conjugates (ELISA grade; Biosource). After incubation with peroxidase substrate (TMB Microwell Peroxidase Substrate System; KPL), the optical density at 450 nm was measured and the concentrations of IFN-γ were calculated according to the formula of the dose-response curve obtained from the same ELISA system using a recombinant bovine IFN-γ (18).
Genomic DNA from M. avium subsp. paratuberculosis strain ATCC 19698 was isolated according to an enzymatic method (23). Purified M. avium subsp. paratuberculosis genomic DNA was partially digested with Sau3AI (Toyobo) and fractionated by sucrose density gradient centrifugation. Fractions containing DNA fragments of average size (4 to 5 kbp) were pooled and ligated to ZAP Express vector digested with BamHI (Stratagene), and recombinant phage DNA was packaged in vitro by Gigapack Gold Packaging Extract (Stratagene) according to the manufacturer's procedures. The obtained phage library was amplified and titrated with E. coli strain XL-1blue, and the inserts cloned into the phage vector were excised out of the phage in the form of the phagemid vector with E. coli strain XLOLR. We divided 1,200 colonies of phagemid-transfected bacteria into 60 groups to obtain 20 transfectants per group. The colonies of each group were pooled and resuspended in 800 μl of RPMI 1640 medium with 10% fetal calf serum, and 25 μl of the resulting mixture was added to the wells of 24-well plates containing 2 × 106 PBMC from cattle that had been experimentally infected with M. avium subsp. paratuberculosis. The PBMC were cultured for 1 day at 37°C with 5% CO2 in the presence of polymyxin B sulfate (Sigma Chemical) at 25 ng/ml to reduce the influence of nonspecific production of IFN-γ by bacterial endotoxin, after which the culture supernatant was removed for the measurement of IFN-γ by ELISA. Individual colonies from the IFN-γ-positive groups were assayed until single positive clones were identified. Consequently, two recombinant clones (#37-16 and #60-3) were obtained (Fig. 1A and B). The DNA sequencing data indicated that these two clones (#37-16 and #60-3) contained 5,493-bp and 4,607-bp DNA fragments of M. avium subsp. paratuberculosis, respectively. These sequences corresponded to positions 157,279 to 162,771 of section 6 and 200,197 to 204,803 of section 12 of the M. avium subsp. paratuberculosis strain K-10 genomic sequences, respectively (GenBank accession no. AE017232 for section 6 and AE017238 for section 12).
FIG. 1.
Relative positions of recombinant clones in the genome of M. avium subsp. paratuberculosis K-10. Panels: A, section 6; B, section 12 of the genome. The regions expressed as proteins fused with β-galactosidase are shown as white arrows, and the deduced open reading frame for the PPE protein family of M. avium subsp. paratuberculosis is indicated by white shaded arrows.
The M. avium subsp. paratuberculosis DNA sequences that could be expressed as a portion of the fusion protein were a 264-bp fragment of clone #37-16 and a 1,248-bp fragment of clone #60-3. The homology searches using these nucleotide sequences and deduced amino acid sequences suggested that the sequence of clone #60-3 showed high homology with the PPE family protein of M. tuberculosis, and the complementary sequence of the 264-bp fragment of clone #37-16 was also similar to that of the PPE family protein (Fig. 1A). Because it has been reported that the PPE family proteins may play important roles as T-cell antigens in M. tuberculosis and/or other mycobacterial species (19, 22), we attempted to produce three types of recombinant six-histidine-tagged proteins in E. coli. Recombinant Map10 is a peptide of 88 amino acids which may be expressed as a part of the fusion protein of β-galactosidase, Map41 is a putative PPE family protein composed of 419 amino acids, and Map39 is also a hypothetical PPE family protein of 391 amino acids produced from a 1,173-bp fragment (Fig. 1).
In order to measure the T-cell response against the recombinant Map proteins, PBMC were obtained from experimentally infected cattle (animal 33) and stimulated with the recombinant proteins for 3 days. The results from the IFN-γ assays of the culture supernatants are depicted in Fig. 2, which shows that all of the recombinant proteins elicited strong IFN-γ responses in a dose-dependent manner. In the comparative study of five experimentally infected cattle and five noninfected controls, the stimulation of PBMC with 5 μg/ml Map10, Map41, and Map39 proteins induced significantly higher levels of IFN-γ production in the infected animals than in the controls (Fig. 3). Furthermore, the MAbs which have been prepared by immunization of BALB/c mice with recombinant Map41 and Map39 strongly reacted with corresponding antigens in immunoblotting with the sonicated extract of M. avium subsp. paratuberculosis ATCC 19698 (Fig. 4), suggesting that these two PPE antigens are actually produced by this organism. On the contrary, MAbs against Map10 protein showed no positive bands in the same immunoblotting. Although IFN-γ was significantly induced in PBMC stimulated with Map10 recombinant protein composed of 88 amino acids, we were unable to identify any open reading frame for this protein on the M. avium subsp. paratuberculosis DNA sequence. Because the complementary sequence coding for Map41 may be expressed as a PPE family protein of M. avium subsp. paratuberculosis, Map10 or a protein containing the same sequence of 88 amino acids may not be encoded on the genome of this organism. However, the cross-reactivity of a portion of the peptide sequences of Map10 with other antigenic proteins may account for this phenomenon.
FIG. 2.
IFN-γ production from the PBMC of infected cattle against recombinant Map proteins.
FIG. 3.
IFN-γ responses in uninfected cattle and cattle experimentally infected with M. avium subsp. paratuberculosis.
FIG. 4.
Immunoblotting profiles of MAbs against Map41 and Map39 antigens. The sonicated extract of M. avium subsp. paratuberculosis ATCC 19698 was electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto a nitrocellulose membrane, and stained with murine immune serum against M. avium subsp. paratuberculosis (lane 1), MAb 5C7 against Map41 (lane 2), or MAb 9B2 against Map39 (lane 3).
Two nucleotide sequences of antigens which share 98% sequence homology with Map41 and Map39 were found by a homology search using the genomic sequence of M. avium subsp. avium strain 104 (The Institute for Genomic Research, http://www.tigr.org); this finding suggests that M. avium subsp. avium may possess almost the same PPE protein family genes as those of M. avium subsp. paratuberculosis. In order to examine whether Map41 and Map39 gene homologs are shared with those of other mycobacterial species, PCR analysis using primer pairs which had previously been employed for the cloning of these genes was performed with 28 strains of nine mycobacterial species (Table 1). Positively amplified bands were obtained from M. avium subsp. paratuberculosis and M. avium subsp. avium only (Table 2).
TABLE 1.
Mycobacterial species tested in this study
| Species | Strain |
|---|---|
| M. avium subsp. avium serovar 1 | ATCC 15769 |
| M. avium subsp. avium serovar 1 | 11907-300 |
| M. avium subsp. avium serovar 2 | 14141-1395 |
| M. avium subsp. avium serovar 3 | 6195 |
| M. avium subsp. avium serovar 3 | 128-Germany |
| M. avium subsp. avium serovar 4 | 13528-1079 |
| M. avium subsp. avium serovar 5 | 4443-1237 |
| M. avium subsp. avium serovar 6 | 34540 |
| M. avium subsp. avium serovar 8 | 14658-1686 |
| M. avium subsp. avium serovar 8 | Kumamoto |
| M. avium subsp. avium serovar 9 | 6450-204 |
| M. avium subsp. avium serovar 10 | 1602-1965 |
| M. avium subsp. avium serovar 11 | 14186-1424 |
| M. avium subsp. avium | P 18 |
| M. avium subsp. paratuberculosis | ATCC 19698 |
| M. intracellulare serovar 7 | Manten 157 |
| M. intracellulare serovar 12 | P 42 |
| M. intracellulare serovar 13 | Chance |
| M. intracellulare serovar 14 | P 39 |
| M. scrofulaceum serovar 41 | Bridge |
| M. scrofulaceum serovar 42 | CDC 1198 |
| M. scrofulaceum serovar 43 | Anderson |
| M. smegmatis | 155 |
| M. bovis | BCG |
| M. bovis | Bovine10 |
| M. tuberculosis | Aoyama B |
| M. kansasii | S-55322 |
| Mycobacterium sp. | 2333 |
TABLE 2.
PCR cross-reactivity among mycobacterial species
| Species | No. of positive samples/totala with target gene:
|
|
|---|---|---|
| map41 | map39 | |
| M. avium subsp. paratuberculosis | 1/1 | 1/1 |
| M. avium subsp. avium | 14/14 | 14/14 |
| M. intracellulare | 0/4 | 0/4 |
| M. scrofulaceum | 0/3 | 0/3 |
| M. smegmatis | 0/1 | 0/1 |
| M. bovis | 0/2 | 0/2 |
| M. kansasii | 0/1 | 0/1 |
| M. tuberculosis | 0/1 | 0/1 |
| Mycobacterium sp. strain 2333 | 0/1 | 0/1 |
DNA were amplified using the specific primers for map41 and map39 genes.
Both Map41 and Map39 are characterized as belonging to the same subgroup of the PPE protein family, whereby there are at least three groups in M. tuberculosis. All of the 68 members of the PPE protein family of M. tuberculosis have a conserved N-terminal domain that consists of 180 to 200 amino acid residues. The first subgroup constitutes the major polymorphic tandem repeat characterized by the multiple repetition of the motif N-X-G-X-G-N-X-G in the C-terminal segment. The second subgroup shares the characteristic conserved motif G-X-X-S-V-P-X-X-W around position 350, and the third subgroup contains unrelated proteins, except for the above-mentioned N-terminal conserved domain (4). Map41 and Map39 proteins are characterized by conserved N-terminal amino acid sequences and the motif G-X-X-S-V-P-X-X-W around positions 320 to 330, suggesting that these proteins are members of the second subgroup of the PPE protein family. In addition to these two PPE protein genes which have been listed as locus tags, MAP1518 (Map41) and MAP3184 (Map39), we were able to identify 12 additional PPE protein genes which belonged to the second subgroup of the PPE family on the genome of this organism, according to an analysis of the genomic sequence of M. avium subsp. paratuberculosis (GenBank accession no. AE016958). Interestingly, 8 of 14 PPE protein family genes categorized into the second subgroup were situated in succession in a short DNA segment of about 23 kbp (positions 1,651,644 to 1,674,579) (Fig. 5A). The gene for Map39 was also followed by another PPE family protein gene (Fig. 5B). Although the reason for this characteristic clustering of the PPE genes of the second subgroup remains unclear, some type of functional relevance may be indicated.
FIG. 5.
PE and PPE family proteins adjacent to Map41 and Map39. The hypothetical PE (open squares) and PPE (closed rectangles) protein genes have been listed as locus tags, i.e., MAP1505, -1506, -1507, -1514, -1515, -1516, -1518, -1519, -1521, -1522, -3184, and -3185, on the genome of M. avium subsp. paratuberculosis K-10.
After the completion of the genome sequencing of M. tuberculosis in which the nomenclature for this antigenic protein family was introduced (4), the PE and PPE protein family has been widely assumed to represent immunologically important antigens of the mycobacterial species (10). Recently, some of the PPE proteins of M. tuberculosis have been reported to be potent T-cell and B-cell antigens (3, 7, 19, 22), and it has also been suggested that these proteins might be responsible for generating antigenic variation (4). Thus, the PE and PPE protein family has gained much interest as a promising target for systematic molecular and immunological characterization. The findings of the present study demonstrated for the first time that the PPE family proteins of M. avium subsp. paratuberculosis are potent IFN-γ-inducing antigens that are recognized by the immune systems of experimentally infected cattle.
A number of previous studies have focused on identifying the immunogenic antigens and the virulent factors of M. avium subsp. paratuberculosis (1, 6, 8, 9). In particular, the identification of antigens that stimulate the T-cell response, as well as the induction of IFN-γ production, seems to be an initial requirement for the development of effective vaccines and diagnostic tests based on CMI. Several types of M. avium subsp. paratuberculosis antigen, the secreted 14-kDa protein MPP14 (21), alkyl hydroperoxidase reductase C (AhpC) and AhpD (20), the 30-kDa antigen P30 (2), a superoxide dismutase (16), the 85B antigen (17), and a thiol peroxidase (15), have been reported as potent IFN-γ-inducing antigens in studies employing immunized mice or the experimental infection of cattle and sheep. In addition to these antigens, we have identified three recombinant proteins which induce IFN-γ production only from PBMC of cattle infected with M. avium subsp. paratuberculosis.
The present database search and the cross-reaction test using PCR indicated that M. avium subsp. avium also has PPE family proteins that are similar to Map41 and Map39, but no cross-reactions were observed among other mycobacterial species tested, thus suggesting that these PPE proteins are specific to M. avium. It seems preferable to use specific antigens which do not cross-react, especially with M. avium subsp. avium, for the diagnosis of paratuberculosis; however, such specific diagnostic tests are not yet available. Olsen and coworkers have reported that the AhpC and -D proteins of M. avium subsp. paratuberculosis revealed high specificity without cross-reacting against M. avium subsp. avium antigens, and these enzymes were constitutively expressed in large amounts in M. avium subsp. paratuberculosis without exposure to oxidative stress (20). These findings appear to be important because the ahpC gene of M. avium subsp. avium that shows 99% nucleotide sequence homology with M. avium subsp. paratuberculosis may not be equally regulated under the same culture conditions as those used for M. avium subsp. paratuberculosis (GenBank accession no. AX094821 for ahpC of M. avium subsp. paratuberculosis and U18263 for M. avium). This finding suggests that these genetically and antigenically closely related organisms share a number of similar genes but that their gene products are not equally regulated in these organisms. In the same context, the PPE protein family genes may be highly homologous between these two organisms, but it still appears worthwhile to evaluate the specificity and reactivity of the PPE proteins of M. avium subsp. paratuberculosis as diagnostic antigens.
Acknowledgments
We thank Goran Bolske, National Veterinary Institute, Uppsala, Sweden, for kindly supplying Mycobacterium species strain 2333. We are also grateful to Kouichi Kojima and Naoyuki Nishimura, Shimadzu Corporation, for providing the lysis buffer for DNA extraction, to Shigeki Inumaru for kindly supplying of purified recombinant bovine IFN-γ, and to The Institute for Genomic Research (website at http: //www.tigr.org) for providing the sequence data for M. avium 104.
This work was supported by research funds from the Ministry of Agriculture, Forestry and Fisheries of Japan.
Editor: J. L. Flynn
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