1 National Animal Disease Center, USDA-ARS, 2300 North Dayton Avenue, Ames, IA 50010, USA
2 Iowa State University, Department of Veterinary Pathology, Ames, IA, USA
3 Oregon State University, Department of Biomedical Sciences, Corvallis, OR, USA
Correspondence
John P. Bannantine
jbannant{at}nadc.ars.usda.gov
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ABSTRACT |
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INTRODUCTION |
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Several pathogenic bacteria have exploited M cell function to gain entry into the subepithelium. These include Salmonella enterica subsp. typhimurium (Jepson & Clark, 2001; Jones et al., 1994
), Yersinia pseudotuberculosis (Clark et al., 1998
) and other enteric pathogens (Jepson & Clark, 1998
). M cells are specialized epithelial cells involved in transport of antigens across mucosal epithelia to the underlying lymphoid tissues where protective immune responses are generated. Little is known about the manner in which M. paratuberculosis interacts with the intestinal mucosa in cattle and sheep. One study by Momotani et al. (1988)
showed M. paratuberculosis entry in ligated ileal loops of calves and a second study with similar results was recently performed using ligated distal small intestine in sheep (Sigur-Dardottir et al., 2001
). Both of these studies demonstrated M. paratuberculosis entry through M cells. However, it is not certain whether all mycobacterial bacilli in a given infection use M cells as the sole route of entry. Mycobacterium avium subsp. avium (M. avium), an opportunistic pathogen with greater than 96 % nucleotide identity to M. paratuberculosis, invades the intestine of mice preferentially through enterocytes and not M cells (Sangari et al., 2001
). Specifically, Sangari et al. (2001)
showed that the number of bacteria present in segmented intestinal tissue containing Peyer's patches was 100-fold less than that observed in non-Peyer's-patch regions.
A 35 kDa protein was originally identified in Mycobacterium leprae as an immunodominant antigen (Winter et al., 1995). This antigen was termed the major membrane protein (MMP) because it was purified from a membrane fraction of M. leprae. Sera from patients with leprosy also recognized this MMP, further demonstrating its immunogenicity (Triccas et al., 1996
). This antigen was later found in M. avium (Banasure et al., 2001
; Triccas et al., 1998
) and M. paratuberculosis (Banasure et al., 2001
; Bannantine & Stabel, 2001
). Recent studies with mice have suggested that DNA encoding the M. leprae MMP protects against leprosy infection (Martin et al., 2001
).
In the present study, we characterized the location and immunogenicity of the M. paratuberculosis MMP. In addition, a molecular cloning approach, combined with invasion assays, was used to determine if the MMP plays a role in invasion of bovine epithelial cells.
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METHODS |
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Sera from Johne's disease cattle.
Sera from 13 naturally infected cattle in the clinical stage of Johne's disease and 4 healthy cattle were used in immunoblot assays for detection of antibodies that bind M. paratuberculosis MMP. All 13 infected cattle showed clinical signs of Johne's disease including diarrhoea and shedding of at least 30 M. paratuberculosis bacilli per g faeces. Affinity-purified MBPMMP protein (see below) was used to test these sera for MMP-specific antibody. Cattle sera were diluted 1 : 500 and -bovine immunoglobulinHRP (Pierce Chemical Company) was the secondary antibody in these experiments.
MMP expression in defined conditions.
To determine if MMP expression is increased when mycobacteria are exposed to conditions encountered in the intestinal lumen, transcript levels were measured by RT-PCR and translation was measured by relative protein abundance in defined conditions. An inoculum of 107 M. paratuberculosis was cultured in 7H9 broth and after the culture reached a turbidity equivalent to 5x108 organisms, it was split and placed at 37 °C in several defined environments: hyperosmolarity (complete 7H9 medium supplemented with 0·3 M glucose), anaerobiosis (anaerobic jar), the combination of both conditions, low pH (pH 5·0 was maintained by supplementing 7H9 broth with sulfonate buffers with appropriate pKa values), iso-osmolar and aerobic conditions as control for 24 h.
Total bacterial RNA was obtained as previously reported (Bermudez et al., 1993). Contaminating DNA was removed from the mycobacterial RNA preparations using DNase digestions performed on RNeasy mini-columns (Qiagen) by adding 82 Kunitz units of enzyme (Qiagen) and incubating the columns at room temperature for 15 min. As a check to ensure all DNA was removed by this method, a control PCR was performed on the RNA preparation. No PCR product was obtained in these controls. Total RNA was initially quantified by A260 and quality determined by A260/A280. Ratios
1·8 were considered acceptable. RNA was then analysed by gel electrophoresis to confirm quality. RNA was submitted to reverse transcriptase treatment to obtain cDNA. Briefly, 6 µl RNA, 6 µl random hexamer (50 µg in 3 µl), 2 µl dNTP mix (10 mM) and distilled water were mixed and incubated at 65 °C for 5 min and subsequently placed on ice for 2 min (RT mix). Then to 18 µl of the RT mix, we added 5 µl 10x RT buffer, 8 µl MgCl2 (25 mM), DTT (0·1 M) and RNase (2 µl). The mix was incubated at 25 °C for 2 min and 2 µl SuperScript RTII (Invitrogen Life Technologies) was added. The sample was incubated for 10 min at 25 °C and then transferred to 42 °C for 50 min. The reaction was then placed on ice, centrifuged briefly and 2 µl RNase H was added followed by incubation at 37 °C for 20 min. The 16S RNA gene was used as the constitutively expressed control. To amplify the cDNA, we used specific primers for the MMP coding sequence and M. paratuberculosis genomic DNA. PCR amplification was carried out at 95 °C for 3 min (1 cycle), 95 °C for 3 s, 62 °C for 30 s, 72 °C for 2 min (35 cycles), and 72 °C for 10 min (1 cycle). Comparison of each amplified cDNA sample with control was carried out using 15 µl of equal amounts of cDNA.
An identical set of M. paratuberculosis cultures was processed as sonicated protein extracts in order to measure relative abundance of MMP in these defined conditions. Mycobacteria were harvested (8000 g for 30 min) and washed twice with cold phosphate-buffered saline. The pellet was resuspended in 0·01 of the culture volume and sonicated on ice with a probe sonicator (Tekmar sonic disruptor). Sonication consisted of three 10 min bursts at 18 W (highest setting) on ice with 10 min chilling periods in between. Debris was centrifuged (12 000 g for 15 min) and supernatants were combined and aliquoted at -20 °C. Protein concentration was determined using the Bio-Rad protein assay.
Cloning and expression of the M. paratuberculosis MMP in E. coli.
A maltose-binding protein (MBP) fusion of MMP (MBPMMP) was constructed using the pMAL-c2 vector (New England Biolabs). The reading frame was amplified using Pwo polymerase (Boehringer Mannheim) and M. paratuberculosis ATCC 19698 genomic DNA as the template. The upstream and downstream oligonucleotide primers for this amplification contain restriction sites for cloning (underlined) and are: 5'-GCGCAGGCCTGATCTGGCTGCCAGGCAACTC-3' and 5'-GCGCAAGCTTCACTTGTACTCATGGAACTG-3'. This primer combination produces the MMP coding sequence minus the initial 12 codons (see GenBank accession number AF232751). The vector was digested sequentially with XmnI and HindIII, and the amplification product was digested with StuI and HindIII. Ligation of these products resulted in an in-frame fusion between the vector-encoded malE gene and a majority of the mmp gene. The resulting recombinant was designated pMAL-mmp. Following ligation, pMAL-mmp was transformed into E. coli DH5. The insert from the construct was confirmed by DNA sequencing. The resulting fusion protein was overexpressed and purified by maltose affinity chromatography using an amylose resin from New England Biolabs. The purified MBPMMP fusion protein was used as antigen in subsequent experiments. An E. coli clone (275-1), producing MMP without the MBP affinity tag, was identified previously from a M. paratuberculosis lambda phage expression library (Bannantine & Stabel, 2001
).
Antibody production and purification.
Two New Zealand White rabbits were immunized with a sonicated protein lysate of M. paratuberculosis using an immunization regimen described previously (Stabel et al., 1996). TiterMax (CytRx Corp.) served as the adjuvant in these experiments. MMP monospecific antibodies were affinity purified from rabbit sera raised against M. paratuberculosis using AminoLink columns (Pierce Chemical Company). Briefly, the MBPMMP protein was coupled by reductive amination to a 4 % agarose support column (Pierce Chemical Company). Antisera from the immunized rabbits (12 ml) were passed over the column followed by three washes and elution according to the instructions of the manufacturer. Eluted fractions were evaluated by spectrophotometry and immunoblot analysis (Bannantine & Stabel, 2001
). Fractions with the highest A280 and the strongest reactivity by immunoblotting were neutralized by addition of 1 M Tris/HCl (pH 9·5) buffer and then dialysed at 4 °C in phosphate-buffered saline. For immunoelectron microscopy, colloidal gold-conjugated goat anti-rabbit IgG was purchased from Ted Pella, Inc.
For production of monoclonal antibody, 67-week-old BALB/c mice were immunized with MBP (30 µg in 150 µl for each mouse). Splenic lymphocytes from immunized mice were harvested and fused to SP2/0 myeloma cells (Harlow & Lane, 1988). Because of solubility problems associated with the MBP, each well was screened by preparative immunoblotting using MBP as antigen. Positive cell lines were cloned and monoclonal antibodies harvested in hybridoma culture supernatants. Monoclonal antibody (13A4) against a M. paratuberculosis 60 kDa protein was developed using similar methods. Appropriate anti-mouse IgG secondary antibodies were purchased from Pierce Chemical Company.
Electrophoresis and immunoblotting.
E. coli lysates expressing MMP or MBPMMP were prepared as previously described (Rockey & Rosquist, 1994). PAGE was performed using 12 % (w/v) polyacrylamide gels. Electrophoretic transfer of proteins onto nitrocellulose (Schleicher and Schuell) was accomplished with the Bio-Rad Trans Blot Cell (Bio-Rad) in sodium phosphate buffer (25 mM, pH 7·8) at 0·9 A for 90 min. After transfer, filters were blocked with phosphate-buffered saline (PBS; 150 mM NaCl, 10 mM sodium phosphate, pH 7·4) plus 2 % bovine serum albumin (BSA) and 0·1 % Tween 20, referred to hereafter as PBS-BSA. Monospecific MMP antibodies were diluted 1 : 1500 in PBS-BSA and 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 protein Aperoxidase (Pierce Chemical Company) diluted 1 : 20 000 in PBS-BSA. The blots were again washed three times as described above and developed for chemiluminesence using Supersignal detection reagents (Pierce Chemical Company).
Subcellular fractionation of M. paratuberculosis.
In order to test the location of MMP within M. paratuberculosis, cell fractions were obtained from sonicated preparations and analysed by immunoblotting using affinity-purified MMP antibodies. The pellet from a high-speed centrifugation (90 000 g for 60 min at 4 °C) was collected and represented the membrane/cell wall fraction. The supernatant, saved following the high-speed centrifugation and representing the cytosolic proteins, was concentrated in a Savant Speedvac. Both fractions were resuspended in SDS-PAGE loading buffer and an amount of protein equivalent to 450 µl of culture was subjected to SDS-PAGE and immunoblotting. The Middlebrook 7H9 culture supernatant was similarly concentrated and processed to test for secretion of MMP.
Immunoelectron microscopy.
All fixation and staining procedures were conducted at room temperature. M. paratuberculosis ATCC 19698 cells were cultured for 4 weeks in Middlebrook 7H9 medium containing OADC and mycobactin J. Cells were fixed for 24 h in 2·5 % glutaraldehyde in 0·1 M cacodylate buffer, pH 7·4. Fixed cells were washed in the same buffer three times and postfixed in 1 % OsO4 in 0·1 M cacodylate buffer, pH 7·4, for 2 h. After washing in the same buffer, cells were incubated with 30 % ethanol for 10 min. The cells were further dehydrated with a graded series of ethanol and embedded in epoxy resin (Embed 812). Ultrathin sections for immunoelectron microscopy were washed in buffer for 15 min three times and etched with saturated sodium metaperiodate for 15 min. The sections were then blocked with 5 % BSA for 30 min at room temperature, followed by treatment with purified MMP-specific antibodies (diluted 1 : 100) in the blocking solution for 2 h at room temperature. The sections were washed in Tris buffer containing 0·1 % Tween 20 and 0·1 % BSA four times for 10 min each and then incubated with goat anti-rabbit IgG conjugated to colloidal gold (10 nm diameter) in Tris buffer for 2 h. Immunolabelled sections were washed in Tris buffer four times and fixed with 1 % glutaraldehyde in Tris buffer for 10 min. All ultrathin sections were double stained with uranyl acetate and Reynolds lead citrate and then observed under a Philips 410 microscope.
Invasion assay.
To test bovine epithelial cell invasion in vitro, MadinDarby bovine kidney (MDBK) cells were used. MDBK cells were cultured to 80 % confluence in RPMI 1640 plus Dulbecco's Minimal Essential Medium supplemented with 10 % heat-inactivated fetal bovine serum. MDBK cells in culture for approximately 4 days were incubated with specific antibody or purified protein for 30 min prior to addition of 106 c.f.u. of M. paratuberculosis in Hanks' Balanced Salts Solution (HBSS; bacteria : cell ratio 1 : 1). To avoid clumping, the bacterial suspension was passed through a 16 gauge needle 10 times and then placed in a 15 ml tube and allowed to rest on the bench for 10 min. Only the top half of the suspension was used in the assays. An aliquot of the bacterial suspension was stained using the LIVE and DEAD assay (Molecular Probes) and only disperse inocula with more than 85 % of viable bacteria were used in the assays. After 2 h at 37 °C in 5 % CO2, monolayers were washed twice with HBSS to remove extracellular bacteria and then treated with amikacin (200 µg ml-1) for 2 h at 37 °C. Amikacin at this concentration either kills or detaches the extracellular bacteria without any effect on the viability of the intracellular bacteria (Bermudez et al., 1997). Supernatant was removed and monolayers lysed with 0·1 % Triton X-100 for 15 min. Lysates were then added to 0·025 % SDS for 10 min, serially diluted and plated onto Middlebrook 7H10 agar slants containing mycobactin J.
Statistical analysis.
The results of experimental groups obtained from at least three invasion assays were compared with the controls at the same time point. A statistical analysis of these comparisons was done by using the Student's t test. P<0·05 was considered significant.
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RESULTS |
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MBPMMP was analysed by SDS-PAGE and immunoblotting to assess purity and further characterize the fusion protein (Fig. 2AC). Lane 2 of Fig. 2(A)
shows a single band at 75 kDa indicating the purity of the fusion protein. The calculated size of MMP alone is 34 kDa and that of MBP is 42 kDa. MBPLacZ
peptide was purified and used as a control in these experiments (lane 1). This control protein migrated at 50 kDa (42 kDa MBP plus the 8 kDa LacZ
peptide). An E. coli clone (275-1) expressing MMP without the MBP affinity tag was previously identified from a M. paratuberculosis phage expression library (Bannantine & Stabel, 2001
). This recombinant E. coli lysate was loaded in lane 3 of Fig. 2(AC). One immunoblot was probed with a monoclonal antibody that detects only the MBP tag of each fusion protein in lanes 1 and 2 (Fig. 2B
). A second identical immunoblot (Fig. 2C
), probed with affinity purified MMP-specific antibodies, detects only the MMP portion of the MBPMMP fusion (lane 2) as well as MMP in the recombinant E. coli 275-1 lysate (lane 3).
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MMP protein is present in higher relative abundance in selected conditions
With specific antibodies to MMP obtained, we again addressed the question of MMP expression in low-oxygen and hyperosmolarity conditions. M. paratuberculosis was cultured in defined conditions for 24 h as described in Methods. Immunoblot analyses using the specific MMP antibodies were conducted in duplicate to assay for MMP abundance in M. paratuberculosis protein lysates produced from each condition. The results of these experiments show that a higher relative abundance of MMP is produced by M. paratuberculosis in low oxygen as compared to bacilli grown in aerobic conditions (Fig. 3). The relative abundance of MMP appears to decrease slightly in hyperosmolarity conditions but is still higher than that observed in aerobic cultures (Fig. 3
). A 60 kDa protein served as an internal control to normalize MMP abundance because its expression is unaffected by oxygen and osmolarity (data not shown). Collectively, these experiments show that both transcription and translation of MMP is increased when exposed to these conditions.
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DISCUSSION |
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M. paratuberculosis invades the bovine intestinal mucosa as a first step in the pathogenesis of Johne's disease. The interaction between M. paratuberculosis and the bovine intestinal mucosa has been described to occur through M cells (Momotani et al., 1988), although it is plausible that the bacterium crosses the intestinal wall by invading intestinal epithelial cells as well. M. paratuberculosis surface proteins probably play the dominant role in the initial interactions with bovine or ovine intestinal cells. Until very recently, no M. paratuberculosis surface proteins had been described. MMP is currently one of only two surface proteins identified for M. paratuberculosis (Secott et al., 2002
). We have demonstrated that MMP is surface located using two independent methods. Despite the location of this protein, no predicted signal peptide was observed using the method of Krogh et al. (2001)
and SignalP analysis (http://www.cbs.dtu.dk/services/SignalP/). This situation is not unprecedented, as other exported mycobacterial proteins do not posses a classical signal sequence (Pethe et al., 2000
; Thole et al., 1995
).
The majority of intestinal pathogens are able to regulate the expression of virulence factors once in the intestinal environment. For example, Salmonella HilA, a global regulator of invasion of the intestinal mucosa, and Yersinia ail are both regulated in conditions of oxygen tension (Lee et al., 1992; Pederson & Pierson, 1995
). Similarly, osmolarity has been shown to be an environmental signal controlling the expression of genes associated with virulence in several other pathogens, among them the toxR gene of Vibrio cholerae (Miller & Mekalanos, 1988
) and ompR genes of Salmonella and Shigella species (Bernardini et al., 1990
; Chatfield et al., 1991
). M. avium, a pathogen that is mainly acquired through the gastrointestinal tract (Bermudez et al., 1997
), has been demonstrated to have its uptake by human intestinal epithelial cells increased by several-fold when it is pre-incubated in low oxygen tension or hyperosmolarity, two conditions encountered in the intestinal tract.
In M. paratuberculosis, RNA transcription and protein expression data show that MMP expression is increased in both low-oxygen and hyperosmolarity conditions. It is plausible to assume that M. paratuberculosis is subject to the influence of the intestinal environment and therefore regulates proteins associated with the interaction of the mucosa accordingly. The present study also shows that the role of MMP in invasion is increased when M. paratuberculosis is exposed to low-oxygen conditions. A host immune response is produced against MMP, giving further evidence of its in vivo expression. We show that MMP elicits antibody production in infected cattle, which is consistent with observations that show MMP is recognized in sera from leprosy patients (Triccas et al., 1996).
Entry into the mucosal surface is a complex phenomenon that in many pathogens involves the participation of a number of virulence genes. We used both the purified MMP protein and antibody to show a specific but small diminution in invasion. It is clear from these data that MMP is a player probably among several moieties and it is not surprising that the differences in invasion are not greater, though they are statistically significant. Regarding the level of invasion of M. paratuberculosis, it is similar to that observed with other mycobacteria (Bermudez et al., 1997; Sangari et al., 2001
).
MMP is not the first mycobacterial invasion protein described. A recent report by Secott et al. (2002) showed that decreased expression of the fibronectin attachment protein, FAP-P, reduced attachment and ingestion of M. paratuberculosis in Caco-2 cells. In addition, a 21 kDa M. leprae protein has been shown to mediate invasion of Schwann cells of the peripheral nerves (Shimoji et al., 1999
). In that study, the 21 kDa protein was coated onto polystyrene beads and shown to invade in a concentration-dependent manner after 12 h using fluorescence and electron microscopy. Another M. leprae protein, which binds fibronectin, has been found to play a role in invasion using a strategy similar to that described here (Schorey et al., 1995
). M. tuberculosis also produces a protein shown to promote entry into mammalian cells (Arruda et al., 1993
; Flesselles et al., 1999
). This protein, termed Mce, conferred invasiveness upon a non-pathogenic E. coli strain (Arruda et al., 1993
). The M. paratuberculosis 35 kDa protein is present in M. leprae (Triccas et al., 1996
; Winter et al., 1995
) and M. avium (Triccas et al., 1998
), but is absent in M. tuberculosis and M. bovis (Banasure et al., 2001
; Triccas et al., 1998
). M. avium has been shown previously to invade intestinal epithelial cells (Sangari et al., 2001
) and, although not demonstrated by experiments reported in this study, MMP may play a role in these interactions as well (Miltner et al., 2001
).
In summary, we have identified a novel M. paratuberculosis protein associated with the invasion of epithelial cells. Future studies involving monoclonal antibody production and protein interaction studies between MMP and MDBK cells are under way.
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ACKNOWLEDGEMENTS |
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Received 27 February 2003;
revised 24 April 2003;
accepted 28 April 2003.