Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein

Background The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed. Results MAP0261c is conserved in mycobacteria, showing a 95% amino acid identity in M. avium subspecies avium, 84% in M. intracellulare and 76% in M. bovis and M. tuberculosis. MAP0261c was cloned, expressed, and purified as a fusion protein with the maltose-binding protein (MBP-19 kDa) in Escherichia coli. IFN-γ production was measured from 21 naturally infected and 9 control cattle after peripheral blood mononuclear cells (PBMCs) were stimulated with a whole cell lysate (WCL) of M. avium subsp. paratuberculosis or the recombinant MBP-19 kDa. Overall, the mean response to MBP-19 kDa was not as strong as the mean response to the WCL. By comparison, cells from control, non-infected cattle did not produce IFN-γ after stimulation with either WCL or MBP-19 kDa. To assess the humoral immune response to the 19-kDa protein, sera from cattle with clinical Johne's disease were used in immunoblot analysis. Reactivity to MBP-19 kDa protein, but not MBP alone, was observed in 9 of 14 infected cattle. Antibodies to the 19-kDa protein were not observed in 8 of 9 control cows. Conclusions Collectively, these results demonstrate that while the 19-kDa protein from M. avium subsp. paratuberculosis stimulates a humoral immune response and weak IFN-γ production in infected cattle, the elicited responses are not strong enough to be used in a sensitive diagnostic assay.

dairy herds are infected with M. avium subsp. paratuberculosis [2,3]. Neonatal calves are most susceptible to infection and are likely to become infected after ingestion of contaminated milk or colostrum [4,5]. During the subclinical stage of infection, the host cell-mediated immune response is robust and appears to control the infection. As the disease progresses from the subclinical to the clinical stage, the cell-mediated response diminishes, and a humoral immune response predominates [6]. Vaccines are not completely protective, but have been reported to reduce fecal shedding and delay the onset of clinical disease [7][8][9].
Lipoproteins have long been considered immunomodulators and mycobacteria are especially rich in these posttranslationally modified proteins. There are approximately 100 open reading frames identified in the M. tuberculosis genome that possess a characteristic aminoterminal acylation motif [10]. The 19-kDa lipoprotein from Mycobacterium tuberculosis is immunodominant in both mice [11][12][13] and humans [14,15] and has been shown to stimulate CD4 + T cell proliferation as well as the release of IL-2, IFN-γ, and IL-12 [16,17]. Acylation near the N-terminal portion of the 19-kDa protein is believed to occur at amino acids 19-24 and contributes to its immunogenicity [12]. Furthermore, glycosylation of the M. tuberculosis 19-kDa protein inhibits innate immune responses, such as the release of TNF-α, IL-6, and IL-10 from macrophages, but does not affect antibody binding [18][19][20]. The 19-kDa protein was also shown to induce CD8+ cells to secrete IFN-γ and specifically lyse M. tuberculosis-infected monocytes [21] as well as promote neutrophil priming and activation [22]. Finally, B cell epitopes have been described that localize to the linear sequences of amino acids 11-30, 29-47, 61-80, and 140-159, as well as a conformation-dependent epitope at the amino and carboxy-terminal ends because of intramolecular disulfide bonding of cysteine residues [

Sequence analysis of the mycobacterial 19-kDa coding region
The 19-kDa coding sequence was identified from the M. avium subsp. paratuberculosis genome project as MAP0261c. Comparison of amino acid sequences from other species of mycobacteria show that this gene product is conserved. Sequence alignment shows the N-terminal half is more variable and the region between amino acids 99 and 123 is highly conserved (Figure 1). MAP0261c displays a 95% amino acid identity in M. avium, 84% in M. intracellulare and 76% in M. bovis and M. tuberculosis. MAP0261c has a G+C content of 66.2% and encodes for 161 amino acids with a predicted molecular mass of 15.2 kDa. The first 22 amino acids of the M. tuberculosis 19-kDa protein are hydrophobic and were previously noted to represent a signal peptide that is post-translationally cleaved to expose an N-terminal cysteine [32]. Signal peptidase cleavage analysis of MAP0261c (SignalP3.0; http://www.cbs.dtu.dk/services/SignalP/) detected a signal peptide generated from a putative cleavage site between amino acids 34 and 35, to expose an N-terminal serine ( Figure 1). The SignalP-NN (neural networks) model assigned the highest cleavage probability values to amino acid 35 (C score = 0.324; Y score = 0.439) with the predicted length of the signal peptide being 34 amino acids. This is slightly longer than most signal sequences which range from 18 to about 30 amino acid residues in length [33]. The predicted peptidase cleavage sight for M. tuberculosis is between amino acids 21 and 22 and therefore falls within this range (Figure 1). Despite a predicted signal peptidase cleavage site, PSORTb analysis software http://www.psort.org/psortb/ could not predict if the protein was cytoplasmic or membrane located.

Cloning and expression of the M. avium subsp. paratuberculosis 19-kDa protein
In order to perform immunoassays with purified 19-kDa protein, MAP0261c was amplified from M. avium subsp. paratuberculosis genomic DNA and cloned into the pMal-c2 expression vector and transformed in E. coli. Induced expression resulted in production of a maltose binding protein (MBP)-19 kDa fusion protein that was affinitypurified from E. coli lysates. MBP-19 kDa was analyzed by SDS-PAGE ( Figure 2) to assess yield, purity and size. The predicted mass of MBP alone is 42 kDa, while the predicted mass of the MBP-19 kDa fusion protein is 56 kDa. The purified MBP-19 kDa protein migrated to a position around 50 kDa in SDS-PAGE ( Figure 2A). Approximately 5 mg of purified protein was easily obtained from a 500ml broth culture at O.D. 600 nm = 0.9. The purified protein was further characterized by immunoblot analysis using a monoclonal antibody (mAb) that detects the MBP affinity tag ( Figure 2B). Both the fusion protein and MBP alone are detected by the mAb. In addition, the fusion protein is expressed at higher levels under inducing conditions.

Immunoblot analysis of the 19-kDa protein
The M. tuberculosis 19-kDa protein is known to be immunodominant, therefore immunoblot analysis was performed to determine if cattle naturally infected with M. avium subsp. paratuberculosis produce antibodies against the 19-kDa protein. Immunoblots were probed with sera from 9 non-infected and 14 clinically infected cattle. Sera from 8 of 9 non-infected cattle did not react with either MBP or MBP-19 kDa, while 1 non-infected cattle weakly recognized both MBP and MBP-19 kDa protein (data not shown). By comparison, sera from 9 of 14 infected cattle reacted specifically with the 19-kDa protein, but not MBP alone. Antibody reactivity to the 19-kDa protein from 3 clinical cows is shown in Figure 3. Sera from the remaining five infected cattle detected both MBP and MBP-19 kDa proteins. This result made it difficult to distinguish if Amino acid sequence comparison of the mycobacterial 19 kDa protein sera from the animal recognized the MAP0261c gene product or if it simply recognized the MBP affinity tag. Collectively, these data suggest that the 19-kDa protein is detectable and immunogenic in cattle with Johne's disease.

IFN-γ responses of infected cattle
As an indicator of the cell-mediated responses of infected cattle to the 19-kDa protein, whole blood containing PBMCs from 9 control and 21 infected cows was stimulated with M. avium subsp. paratuberculosis sonicated whole cell lysate (WCL), MBP, or MBP-19 kDa and IFN-γ production was assayed by ELISA. These cattle were selected from a larger group because they showed no IFNγ stimulation in response to MBP alone. IFN-γ production in response to WCL stimulation allowed for the segregation of infected cattle into three groups: suspect, positive, and high positive. After subtracting the IFN-γ responses of non-stimulated cells, suspect animals had less than 0.1 absorbance units of IFN-γ production, positive animals had 0.1 -0.3 absorbance units of IFN-γ production, and high positive animals had more than 0.3 absorbance units of IFN-γ production. IFN-γ responses by blood mononuclear cells from infected cattle exceeded responses from control cattle for both the WCL and MBP-19 kDa (Table 1. Significant differences (P < 0.05) were found between control and high positive groups for both WCL and MBP-19 kDa protein stimulation. However, direct comparisons of the two antigen preps using mononuclear cells from the same animal clearly showed the WCL was a stronger stimulator of IFN-γ production ( Table  1).

Discussion
A majority of the research on individual mycobacterial proteins has been performed in M. tuberculosis, whereas little is known about the M. avium subsp. paratuberculosis proteome. Indeed, all currently available antigen-based diagnostic tests for Johne's disease use an undefined mixture of proteins, such as purified protein derivative (PPD) or WCL, which may not be specific for M. avium subsp. paratuberculosis. Recent completion of the M. avium subsp. paratuberculosis genome has already advanced efforts to identify novel antigens [26,34]. Furthermore, the genome will be a critical resource in proteomic studies directed at defining the proteins present in mixtures such as johnin PPD. The present study was performed in order to characterize the M. avium subsp. paratuberculosis 19-kDa protein, as well as assess its immunostimulatory capabilities in cattle.
In this study, we show that the 19-kDa protein of M. avium subsp. paratuberculosis can be readily overexpressed as a fusion protein in E. coli. This is not true for many other proteins encoded by M. avium subsp. paratuberculosis Although not demonstrated directly by our studies, it is possible that the 19-kDa was not posttranslationally modified by the heterologous E. coli host. It is unclear whether posttranslational modification of this protein would affect its immunological activity. The recombinant antigen was detected by sera from cattle with Johne's disease; however, it was not as strong a stimulator of proliferative T-cell responses as has been reported for its counterpart in M. tuberculosis [36]. Furthermore, the 19-kDa protein from M. tuberculosis was shown to induce both cellular and humoral immune responses from mice and humans [11,14]. These studies, combined with the present study, may suggest that acylation is more important in cell-mediated immune responses than in the humoral immune response.
It is generally accepted that cellular and humoral immune responses of M. avium subsp. paratuberculosis-infected cattle are biphasic, with IFN-γ responses detected early and antibody responses detected late in infection. However, evidence suggests that an unknown M. avium subsp. paratuberculosis protein can be detected by antibodies from cattle just 3 weeks after infection [37]. As a measure of cellular immune responses, blood mononuclear cells from infected cattle were stimulated with both the recombinant 19-kDa protein and a whole-cell sonicated lysate of M. avium subsp. paratuberculosis (WCL), and IFN-γ production was measured. Results from this study suggest that while the 19-kDa protein is a stimulator of IFN-γ production, it is not as potent when compared to WCL. Additionally, we found that a majority (9 of 14) of infected cattle produced antibodies to the 19-kDa protein, as determined by immunoblot analysis. By comparison, sera from the majority of control, non-infected cattle (8 of 9) did not react to the 19-kDa protein. The single noninfected cow that did show reactivity to the MAP0261c gene product may be attributed to exposure of environmental mycobacteria such as M. avium subsp. avium, which has a similar protein (Figure 1). Sera from four clinical cows reacted to the MBP protein, but this reactivity was extremely weak. MBP is found in environmental E. coli and likely accounts for reactivity seen in some cattle. In order to avoid potential cross-reactivity with MBP, we attempted to cleave the MBP portion from the MBP-19 kDa fusion protein, but cleavage was not 100% efficient (data not shown).
The M. tuberculosis 19-kDa protein was reported to contain a 21 amino acid N-terminal signal peptide [22], however our SignalP analysis for M. avium subsp. paratuberculosis identified a putative 34 amino acid N-terminal signal sequence. Furthermore, the computer algorithm PSORTb predicts a putative signal sequence, but it cannot determine if the protein is actually secreted. Antibodies will be produced against the recombinant MBP-19 kDa protein to determine if the protein is secreted by M. avium subsp. paratuberculosis.

Conclusions
The results from this study show that the recombinant 19-kDa protein stimulates a weak host immune response in infected cattle. The 19-kDa protein may be used in conjunction with other antigens from M. avium subsp. paratuberculosis to identify infected cattle but should not be used as a "stand alone antigen" in new diagnostic assays.

Bacterial strains and culture conditions
M. avium subsp. paratuberculosis strain 19698-1974 (originally isolated in 1974 from a clinical cow housed at the National Animal Disease Center, Ames, IA) was grown in Middlebrook 7H9 liquid media (pH 6.0) supplemented

Cattle
The Johne's Disease Research Project at the National Animal Disease Center has a repository of sera from cattle that were euthanized with clinical signs of Johne's disease, which included shedding, weight loss and diarrhea. Fecal samples from each of these clinical cattle were found to contain more than 100 CFU per gram of feces as deter- The noninfected control cows were characterized by repeated negative fecal cultures performed quarterly over a 3-to 5-year period. In addition, these animals were negative on all immunological assays (i.e. ELISA and IFN-γ production) performed during that period. Subclinical cattle (suspect, positive, and high-positive) were characterized by shedding less than 10 CFU/g of feces and were intermittently positive by IFN-γ assays (response > 0.1) performed quarterly over a 3-to 5-year period. The institutional Animal Care and Use Committee approved all animal procedures described in this study.

Comparison of the mycobacterial 19 kDa coding region
The nucleotide sequences for the 19  Tween 20, blots were incubated for 1.5 hours in anti-goat peroxidase-conjugated secondary antibody diluted 1:20,000 in PBS-BSA (Pierce Biotechnology Inc.). After secondary antibody incubation, the blots were washed 3 times as described above and were developed for chemiluminescent detection using SuperSignal detection reagents (Pierce Biotechnology Inc.).

IFN-γ assays
Blood was collected from the jugular vein of subclinicallyinfected cattle into sodium heparin vacutainer blood collection tubes. One ml aliquots of whole blood from each animal were plated into 4 wells of 24-well culture plates and cultured alone (non-stimulated) or with 10 µg/ml of M. avium subsp. paratuberculosis sonicate (WCL), 10 µg/ml of MBP, or 10 µg/ml of MBP-19 kDa. The WCL was prepared by sonication of bacilli and centrifugation exactly as described previously [37]. Blood-antigen mixtures were incubated for 18 hours at 39°C in a 5% CO 2 humidified atmosphere. Plates containing blood-antigen samples were centrifuged at 500 × g for 15 minutes and the plasma was harvested from each well. Plasma samples were frozen at -20°C until being analyzed for IFN-γ concentration by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Bovigam, BioCor, Omaha, NE) as recommended by the manufacturer. Samples were analyzed in duplicate and were determined to be positive for IFN-γ production if the absorbance of the stimulated sample (WCL, MBP, MBP-19 kDa) was 0.1 units greater than the absorbance of the nonstimulated well for that animal. This classification of IFN-γ positive samples has been previously reported by our laboratory as well as others [41,42].

Statistical analysis
ANOVA and unpaired t tests were performed to analyze the IFN-γ stimulation data. Analyses were performed to compare average stimulation of control, non-infected cattle to the infected cattle groups (suspect, positive, high positive). Differences were considered significant when P < 0.05.