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Molecular Biology and Microbiology, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, FL, USA. Correspondence: Sung ...
RESEARCH ARTICLE

Identi¢cation of seroreactive proteins in the culture ¢ltrate antigen of Mycobacterium avium ssp. paratuberculosis human isolates to sera from Crohn’s disease patients A-Rum Shin1, Hwa-Jung Kim1, Sang Nae Cho2, Michael T. Collins3, Elizabeth J.B. Manning3, Saleh A. Naser4 & Sung Jae Shin1 1

Department of Microbiology, Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon, South Korea; Department of Microbiology and Institute of Immunology and Immunological Diseases, Yonsei University College of Medicine, Seoul, South Korea; 3 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA; and 4Department of Molecular Biology and Microbiology, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, FL, USA

IMMUNOLOGY & MEDICAL MICROBIOLOGY

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Correspondence: Sung Jae Shin, Department of Microbiology, Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747, South Korea. Tel.: 182 42 580 8246; fax: 182 42 585 3686; e-mail: [email protected] Received 26 August 2009; revised 16 September 2009; accepted 21 September 2009. Final version published online 28 October 2009. DOI:10.1111/j.1574-695X.2009.00617.x Editor: Willem van Leeuwen Keywords Mycobacterium paratuberculosis; Crohn’s disease; seroreactivity; diagnosis.

Abstract The etiology of Crohn’s disease (CD) is unresolved, but it is likely that an interplay of host genetic factors and environmental triggers is relevant. Mycobacterium paratuberculosis (MAP) has been focused upon as one of these triggers because it causes a similar chronic inflammatory bowel disease in animals. However, the differences among MAP antigens isolated from humans (H-MAP) and cattle (BMAP) have not been well characterized. In this study, culture filtrate (CF) proteins from MAP isolates were tested with sera from CD patients and healthy controls in enzyme-linked immunosorbent assay (ELISA). Antibody produced by seven CD patients reacted differently according to the antigen source: strong reactivity was seen to H-MAP CF, but not to B-MAP CF. Six proteins, ModD, PepA, transaldolase, EchA9, MAP2120c, and MAP2950c, in H-MAP CF reacting specifically with CD patient sera were identified by liquid chromatography-electrospray ionizationMS. Bioinformatic analysis revealed that ModD and PepA were the same proteins reacting with sera from cattle infected with MAP. The elevated antibody responses of CD patients to rModD and rPepA were confirmed by ELISA (P o 0.001). These results support previous studies showing ModD and PepA as key antigens for the diagnosis of MAP infections. The study also identified additional proteins potentially useful in the design of assays for human MAP infections.

Introduction Crohn’s disease (CD) is an immune-mediated inflammatory bowel disorder of unknown cause, but believed to result from the interplay of host genetics and one or more environmental triggers such as bacteria (Shanahan & O’Mahony, 2005). Mycobacterium avium ssp. paratuberculosis (MAP, also known as Mycobacterium paratuberculosis) infection has been hypothesized as an etiological factor of CD (Engstrand, 1995; Andersen et al., 1997; Greenstein & Collins, 2004; Biet et al., 2005; Alluwaimi, 2007). Infection of ruminants by this organism causes Johne’s disease, a chronic granulomatous enteritis (Cocito et al., 1994; Chacon et al., 2004). Infected animals may be free of symptoms and yet infectious for many years. The organism can be transmitted from cows to 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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calves in unpasteurized milk, and because MAP withstands high heat, the living organism has been found even in pasteurized milk (Lund et al., 2002; Cerf et al., 2007). The contribution that MAP may provide to the pathophysiology of CD is still controversial (Van Kruiningen, 1999; Harris & Lammerding, 2001). There are both similarities and differences in the clinical and histological features among Johne’s disease, human intestinal tuberculosis, and CD (Van Kruiningen, 1999). While some studies have not demonstrated MAP in CD patients, meta-analyses demonstrate a clear association of MAP with CD (Feller et al., 2007; Abubakar et al., 2008). Recent studies show that MAP culture filtrates (CF) contain more proteins reactive with sera from infected cattle than do MAP cellular extracts (CEs) (Cho & Collins, 2006). Fourteen apparently specific proteins FEMS Immunol Med Microbiol 58 (2010) 128–137

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of potential diagnostic value for Johne’s disease in cattle were found in the CF of MAP JTC303 (ModD, PepA, ArgJ, CobT, antigen 85c, and nine as yet unnamed proteins) (Cho et al., 2006). Characterization of antigens from MAP strains isolated from humans (H-MAP) and cattle (B-MAP) has not been reported. Nor have there been reports on the reaction of serum antibodies from CD patients with immunogenic proteins from MAP CF. The purpose of the present study was therefore to identify proteins of potential serodiagnostic value found in CF from H-MAP and to establish the antigenic profile of CF proteins differently detected according to the antigen source based on sera from CD patients.

Materials and methods Bacterial strains and culture conditions Two H-MAP (UCF-4 and UCF-5) and two B-MAP (B236 and B238) strains were used in this study. The H-MAP strains were isolated from CD patients, and the two B-MAP strains were obtained from cows diagnosed with Johne’s disease. All strains were initially propagated in 7H9 broth supplemented with 10% v/v oleic acid–albumin–dextrose–catalase (OADC; Becton Dickinson, Sparks, MD) and 2 mg mL1 of mycobactin J (Allied Monitor, Fayette, MO) for 1 month at 37 1C. Singlecell suspensions of each strain were agitated in the presence of glass beads to minimize cell clumping and then quantified by plating on 7H10-OADC agar supplemented with 2 mg mL1 of mycobactin J (Cho & Collins, 2006). Mycobacterium phlei ATCC 11758 and the four strains of MAP were cultivated in modified Watson–Reid (mWR) broth at 37 1C (M. phlei for 2 weeks, and MAP for 8 weeks). The identity of all MAP strains was confirmed by multiplex PCR for insertion elements IS900, IS901, IS1311, and IS1245 as described previously (Uppal et al., 2002).

Preparations of CF and CE antigen CF antigens were prepared from cultures at midlog-phase growth after 8 weeks of incubation at 37 1C as described previously (Sung et al., 2004; Cho et al., 2006). Briefly, the cells grown in mWR were removed by centrifugation at 30 000 g for 30 min. After filtration through a 0.2-mm poresize filter (Nalge Nunc International, Rochester, NY), the filtrate was concentrated 40–50-fold using a Centricon Plus-80 (5-kDa molecular weight cutoff; Amicon, Bevery, MA) and dialyzed five times in 10 mM phosphate-buffered saline (PBS), pH 7.2, using a Slide-A-Lyzer Dialysis Cassette (Pierce, Rockford, IL). To eliminate cross-reacting antibodies due to other Mycobacterium spp., CE antigens of M. phlei ATCC 11758 were prepared as described previously (Cho & Collins, 2006). The CE antigens from all strains of MAP were also produced for FEMS Immunol Med Microbiol 58 (2010) 128–137

enzyme-linked immunosorbent assay (ELISA). The concentration of soluble protein was determined using the BCA protein assay kit (Pierce).

Human sera To assess the antibody responses of patients diagnosed with CD to MAP antigens, a MAP antigen–serum ELISA was developed for use with 94 human sera samples. Forty-eight of these sera were from CD patients randomly selected from a set of sera used in a previous study (Bernstein et al., 2004), and 46 sera were from healthy adult controls. These negative control sera were obtained from Chungnam National University (Daejeon, Korea) (Shin et al., 2008) students with no prior history of clinical CD, ulcerative colitis, or tuberculosis.

Two-dimensional polyacrylamide gel electrophoresis (2-DE) The CF antigens from each MAP strain were prepared using the 2-D Clean-Up Kit (Amersham Biosciences, Uppsala, Sweden). Each sample was separated in the first dimension using 7- or 11-cm immobilized pH gradient (IPG) strips with a pH range of 4–7 (Bio-Rad, Hercules, CA). The samples were then focused as follows using a PROTEAN IEF Cell (Bio-Rad): 250 V for 30 min, from 250 to 4000 V for 2 h, and 4000 V for 20 000 V h for the 7-cm IPG strip, and 250 V for 30 min, from 250 to 8000 V for 2 h, and 8000 V for 35 000 V h for the 11-cm IPG strip. The IPG strips were equilibrated before the second dimension electrophoresis performed as described by Laemmli using 10–20% precast gels (Bio-Rad) (Laemmli, 1970). The gels were stained with 0.25% Coomassie brilliant blue R250 (Bio-Rad).

Immunoblotting Sera from seven CD patients were pooled for immunoblot analysis chosen based on their selectively strong positive H-MAP ELISA results (seropositive to H-MAP, but not to B-MAP). MAP proteins were transferred from polyacrylamide gels to nitrocellulose membranes (0.45 mm pore size; Bio-Rad) as described by Davies et al. (1990) using a Trisglycine buffer containing 0.0375% sodium dodecyl sulfate and 20% methanol. Before the addition of serum, the membranes were incubated for 2 h in blocking buffer (5% skim milk in PBS). Cross-reactive antibodies were removed from each serum sample as described previously (Cho et al., 2006) with slight modifications to fine-tune MAP CF antigen–antibody binding. The sera were exposed to CE antigens collected at the early stationary phase of culture of M. phlei grown in WR medium. The concentration of the absorbent was 2000 mg mL1, and the mixture of sera and the absorbent was incubated at room temperature for 1 h. The MAP proteins were then reacted overnight with the 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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M. phlei-absorbed test sera at 4 1C on a rocking platform (1 : 200 dilutions). This protocol optimized positive results for reactive proteins with sera from CD patients and negative results for sera from healthy controls. Antibody binding was detected using a horseradish peroxidase-conjugated secondary antibody against human immunoglobulin G (IgG) (1 : 3000 dilution; Sigma, St. Louis, MO). All blots were developed using 3,3 0 -diaminobenzidine tetrahydrochloride (Sigma) in 20 mM Tris-buffered saline (pH 7.6) with 30% hydrogen peroxide (H2O2).

software (Nonlinear Dynamics Ltd, Newcastle, UK). The same spots on the different proteome gels were calibrated by outlining the probable spots of H-MAP across the gel image to those of B-MAP (strain B236). The gel of H-MAP was aligned to that of B-MAP by overlapping more than 21 selected areas and the intensity of the protein spots in the gel of B-MAP was allocated 1.0. The backgrounds of the same spots were normalized, and the fold changes in the identified proteins of H-MAP were then quantified by a direct crossgel comparison with the reference spots of B-MAP.

Protein identification and analysis of fold changes in seroreactive proteins differentially expressed

Expression and purification of recombinant ModD (rModD) and PepA (rPepA)

H-MAP (strain UCF-4) CF proteins that reacted with pooled sera from CD patients as shown by the Coomassiestained gel and the immunoblot membrane results were selected. Identification of the protein spots on the stained gels was performed at the Yonsei Proteomics Research Center (Yonsei University, Seoul, Korea) by liquid chromatography-electrospray ionization-MS (LC-ESI-MS). Briefly, nano-LC-MS/MS was performed on an Agilent 1100 Series nano-LC and LTQ-mass spectrometer (Thermo Electron, San Jose, CA). The capillary column used for LC-MS/MS (150 mm  0.075 mm) was obtained from Proxeon (Odense, Denmark) using a slurry packed in house with a 5-mm, 100-A˚ pore size Magic C18 stationary phase (Michrom BioResources, Auburn, CA). For LC, mobile phase A was 0.1% formic acid in deionized water, while mobile phase B was 0.1% formic acid in acetonitrile. The chromatographic gradient was set up to produce a linear increase in B from 5% to 35% over 50 min, from 40% to 60% over 20 min, and from 60% to 80% over 5 min. The flow rate was maintained at 300 nL min1 after splitting, and mass spectra were acquired using data-dependent acquisition with a full mass scan (400–1800 m/z), followed by MS/MS scans. Each MS/MS scan acquired was an average of one microscan on the LTQ. The temperature of the ion transfer tube was maintained at 200 1C and the spray was 1.5–2.0 kV. The normalized collision energy was set at 35% for MS/MS. SEQUEST software was used to identify the peptide sequences. For high-confidence results, dCn = 0.1, Rsp = 4, Xcorr = 1.5 with charge state 11, Xcorr = 2.0 with charge state 21, Xcorr = 2.5 with charge state 31, and peptide probability 4 0.1, were used as cutoffs for protein identification. The methionine residues in the peptides were variably oxidized, while the cysteines were variably carboxyamidomethylated or carboxymethylated.

Analysis of protein expression The varied expression levels on the protein spots between HMAP and B-MAP were analyzed using PRODIGY SAMESPOTS 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Based on the bioinformatic analysis, ModD and PepA were selected for CD seroanalysis. Preparation of rModD and rPepA was described previously (Cho et al., 2007). Briefly, genomic DNA was isolated from MAP JTC303 strain. The DNA encoding ModD, without its signal sequence, was amplified by PCR. After purification, PCR products were ligated with the pET-22b (1) vector (Novagen, Madison, WI) after digestion with NdeI and XhoI enzymes. Expression of ModD in transformed BL21 (DE3) cells was induced by isopropyl-b-D-thiogalactopyranoside (Promega, Madison, WI). The soluble rModD protein was extracted after cell disruption by sonication as described previously (Cho et al., 2007). rModD containing a C-terminal histidine tag was purified using Ni-nitrilotriacetic acid resin (Qiagen, Chatsworth, CA). The rModD was dialyzed five times in 10 mM PBS, pH 7.2, using a Slide-A-Lyzer Dialysis Cassette (3-kDa molecular weight cutoff; Pierce). After dialysis, the endotoxin contamination of the rModD protein was removed using the Detoxi-Gel Affinity Pak Columns (Pierce). The final concentration of purified rModD protein was determined using a BCA protein assay kit (Pierce).

ELISA procedure details ELISA was performed as described by Voller (1978). Briefly, MAP CF and CE antigens of each strain (2 mg mL1), and recombinant proteins (2 mg mL1) were diluted in coating buffer (10 mM PBS, pH 7.4) and 100 mL was added to each well in 96-well microtiter plates (Maxisorp, Nalge Nunc International). After overnight incubation at 4 1C and washing, the wells were blocked with 10% normal goat serum (Sigma) at room temperature for 2 h. Cross-reactive antibodies were absorbed out of each serum sample by mixing 100 mL of 1 : 25 serum diluted in 10 mM PBS (pH 7.2) with 100 mL of 1000 mg mL1 M. phlei CE antigens (final serum dilution 1 : 50, and final M. phlei CE antigen concentration 500 mg mL1). (For the recombinant protein protocol, the serum preabsorption step was omitted.) The absorbed or nonabsorbed serum (100 mL) was added to each FEMS Immunol Med Microbiol 58 (2010) 128–137

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coated and blocked microtiter plate well and incubated for 30 min at room temperature with shaking at 600 r.p.m. Wells were then washed three times with washing buffer (PBS plus 0.05% of Tween20). After washing five times, 100 mL per well of peroxidase-conjugated goat anti-human IgG (1 : 6000 dilution; Sigma) was added to all wells, and the plates were incubated for an additional 1 h at room temperature. After seven more washes, the reaction was visualized using tetramethylbenzidine (Sigma) and 0.5% v/v H2O2. The reaction was stopped with 1 N sulfuric acid (H2SO4) after 2 min of incubation in the dark. OD450 nm was measured using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA). Because there were no standard positive and negative control sera from H-MAP infections, serum from a confirmed bovine case of Johne’s disease was used as the positive control and pooled sera from two noninfected cows were used as negative controls in each test. All sera were tested in duplicate. ELISA OD values were transformed to sample/positive (S/P) ratios by the following equation: S=P ¼

OD sample  OD negative control Mean OD positive control  Mean OD negative control

The mean S/P values for each serum were used for data analysis.

Data analysis The ELISA results for each CF antigen preparation or fraction were subjected to receiver–operator characteristic (ROC) curve analysis. This method estimates the sensitivity and specificity of the ELISA at every possible S/P interpretation cutoff and provides an overall measure of test accuracy as area under the ROC curve (AUC). All statistical analyses were performed using statistical software (GRAPHPAD PRISM version 4.03 for Windows, GraphPad software, San Diego, CA, http://www.graphpad.com). The difference between mean ODs for CE and CF or mean S/P for B-MAP CF and H-MAP CF was evaluated by Wilcoxon’s matched pairs test. The difference in the mean S/P for human sera from CF and healthy controls was also evaluated using the Mann–Whitney test. Differences were considered significant at P o 0.05.

Results Comparison of CF vs. CE antigens by ELISA Sera from 48 CD patients and 46 healthy controls were tested by ELISA using CE and CF antigens of MAP UCF-4 strain as the solid-phase antigen. Elevated antibody responses to both antigens were observed in CD patients (P o 0.001) (data not shown). Interestingly, the mean ELISA OD of CE and CF antigens was significantly different FEMS Immunol Med Microbiol 58 (2010) 128–137

Fig. 1. Comparison of mean OD in ELISA results using the CE and CF antigens prepared from MAP UCF-4 strain for 48 CD patients and 46 healthy controls. A significant difference was reported when P o 0.05.

(P o 0.05) for CD patients with a low background for healthy subjects (Fig. 1). Although similar in sensitivity and specificity, the ELISA based on CF antigens yielded a higher signal to noise ratio (S/N = mean OD of the sera from the CD patients/mean OD of the sera from the healthy controls) for CD patients compared with the ELISA based on CE antigens (mean S/N: 4.71 vs. 2.73, respectively; P o 0.05). These results indicated that CF antigens were serologically superior to CE antigens and thus only CF antigens from each MAP strain were used for subsequent experiments.

Comparison of CF antigens from different MAP isolates by ELISA CD patient sera responded with higher ELISA S/P values than controls when CF antigens from H-MAP and B-MAP was used as the solid-phase antigen (P o 0.05) (Fig. 2). However, not all CD patient sera showed elevated ELISA results. From an overall comparison in ELISA results, the antibody responses for CD patients differed according to the antigen source: strong reactivity was seen to H-MAP CF, but not to B-MAP CF. No reactivity to either type of CF was seen with 46 control sera (P o 0.05). Of four strains compared, CF antigens from MAP UCF-4 strain showed the highest ROC AUC (Table 1) (Fig. 2). The S/N ratio was higher for strain UCF-4 and UCF-5 than for B-MAP (P o 0.05). ELISA results using CF antigens from H-MAP were highly correlated (r = 0.98); however, a relatively low correlation was observed (r = 0.57) of ELISA results between CF antigens from B-MAP and H-MAP. Interestingly, seven sera (15.2%) from CD patients had little or no antibody to CF antigens from B-MAP isolates, but were strongly immunoreactive with the CF antigen of UCF-4. The reverse case was not observed. 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Fig. 2. Level of antibody responses of patients to the CF from H-MAP and B-MAP isolates. Serum samples were from 48 patients with CD and 46 healthy subjects. All results are expressed as individual S/P ratios (a), and were compared by ROC analysis (b). The horizontal bars indicate geometric means. Significantly higher antibody responses of CD patient to CF antigen from H-MAP isolates were found than from the B-MAP isolates (P o 0.05).

Table 1. Overall comparison of ELISA results for CF antigens prepared from B-MAP, H-MAP, and recombinant proteins to sera from patients diagnosed with CD and those from healthy controls B-MAP

H-MAP

Recombinant protein

Strain parameters

B236

B244

UCF-4

UCF-5

rModD

rPepA

Cutoff

0.079 43.75 (29.48–58.82) 0.78 (0.69–0.87)

0.081 41.67 (27.61–56.79) 0.77 (0.65–0.89)

0.083 66.67 (51.59–79.60) 0.89 (0.82–0.96)

0.083 60.42 (45.27–74.23) 0.86 (0.78–0.94)

0.095 53.85 (33.37–73.41) 0.85 (0.74–0.97)

0.086 62.50 (43.69–78.90) 0.86 (0.76–0.96)

2.94

2.87

4.73

4.21

2.87

3.71

Sensitivity (95% confidence intervals) AUC (95% confidence intervals) Average of S/Nw

Cutoff values for each ELISA were determined when the specificity was 100%. w S/N was considered as a standard for the overall comparison of antibody response by the following formula: (mean OD of sera from CD patients/mean OD of sera from healthy donors).

Comparative proteome analysis of CFs prepared from different MAP isolates CF proteins were compared between H-MAP (UCF-4 and UCF-5) and B-MAP (B236 and B238) isolates by 2-DE (Fig. 3). H-MAP and B-MAP CF proteins produced similar patterns, except for a few spots. Their string of major spots around 25 kDa resembled those of mycolyl transferase seen in 2-DE of Mycobacterium tuberculosis (Jungblut et al., 1999). This pattern, seen in all four strains tested, suggests various forms of the same protein differing slightly in mass and pI in 2-D gels. The presence of proteins modified by post-translational changes is in agreement with previous reports (Cho et al., 2006). Most CF proteins from H-MAP isolates were o 50 kDa and located between pH 4.5 and 6. Most CF proteins from B-MAP isolates, however, were

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evenly distributed between pH 4 and 7 on the gel. The protein spots of UCF-4 (Fig. 3a) were similar to those of UCF-5 (Fig. 3b), but less numerous. The protein spot patterns of B236 (Fig. 3c) and B238 (Fig. 3d) resembled each other while differing from H-MAP patterns.

Identification of differentially expressed proteins in the H-MAP isolate Ten protein spots reacting strongly with a pool of seven CD patient sera in H-MAP (UCF-4) CF were identified using LC-ESI-MS (Fig. 4). Four proteins were identified as ModD, PepA, EchA9, and transaldolase, and two additional spots were designated as hypothetical proteins (Table 2). Three of the identified proteins were detected in more than one spot. In particular, one of the identified proteins, ModD, was FEMS Immunol Med Microbiol 58 (2010) 128–137

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Fig. 3. The 2-DE patterns of CF antigens from human and bovine isolates of MAP. CFs were harvested at the midlog phase. After concentration, 200 mg of CF antigens of (a) UCF-4, (b) UCF-5 from H-MAP isolates, and (c) B236, (d) B238 from B-MAP isolates were applied to a first dimension of 7 cm, pH 4–7 nonlinear IPG strips and a second dimension of 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were stained with 0.25% Coomassie brilliant blue R250.

found in three distinct neighboring spots. With the help of Clusters of Orthologous Groups website (http://www.ncbi. nlm.nih.gov/COG/) and the TubercuList website (http:// genolist.pasteur.fr/TubercuList/), the proteins identified in this study were placed into the function categories of M. tuberculosis genes (Table 2). Finally, the precise intensities of identified spots between H-MAP and B-MAP were analyzed using the software program as mentioned above (Fig. 5). H-MAP-identified protein spots were expressed much more strongly than those of B-MAP. Among 10 protein spots identified in UCF4 CF, ModD, PepA and transaldolase showed expression levels 20-fold higher than those of B-MAP CF. Thus, ModD and PepA were targeted to evaluate the serological application for CD patients.

Antibody responses to recombinant proteins Because ModD and PepA showed the highest expression level with sera from CD patients, rModD and rPepA were produced for further evaluation. CD patient sera contained significant levels of antibodies against both proteins (Mann–Whitney rank test, P o 0.001) (Table 1). However, no recombinant protein provided more sensitivity based on ROC AUC than the original CF antigens from UFC-4 (Table 1).

Discussion Serologic tests are commonly used for the diagnosis of Johne’s disease, although they have a low diagnostic sensiFEMS Immunol Med Microbiol 58 (2010) 128–137

tivity for animals in all but late stages of infection. Improved assay sensitivity may be achieved using MAP CF antigens (Cho & Collins, 2006). Previous studies reported that 14 proteins of serodiagnostic value for bovine paratuberculosis were identified in the CF of MAP (Cho et al., 2006). Comparative proteome analysis between strains has revealed differences in expression level, although encoded ORFs are highly homologous or identical. Several studies of the differently expressed proteins have been reported including Rv0927c from the analysis of proteome between WBeijing and non-Beijing strains, the analysis of membrane and cytosolic proteins from MAP strains K10 and 186 (Jungblut et al., 1999; Jiang et al., 2007; Radosevich et al., 2007). In particular, Rv0927c, a probable short-chain alcohol dehydrogenase, was also found to be present in the proteome of CDC1551, but not H37Rv; however, the Rv0927c gene was present in the H37Rv genome as well as CDC1551 (Jungblut et al., 1999). In addition, HisA, an isomerase that catalyzes the fourth step in the histidine biosynthetic pathway, was present in the proteome of CDC1551, but not H37Rv, although the corresponding gene is present in the genome of H37Rv strain (Jungblut et al., 1999; Betts et al., 2000). This study revealed that protein expression differed between strains isolated from cattle vs. humans. The CF proteins identified as ModD, EchA9, PepA, transaldolase, and two hypothetical proteins were expressed at higher levels in H-MAP than in B-MAP isolates, with the greatest expression seen in UCF-4 (data not shown). In addition, 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Fig. 4. Identification of the protein spots reacting with sera from patients with CD. The strain UCF-4 of MAP was separated on 11 cm, pH 4–7, nonlinear IPG strips and electrophoresed on a 10–20% sodium dodecyl sulfate polyacrylamide gel electrophoresis precasting gel. Then, one strip of the proteins was stained with 0.25% Coomassie brilliant blue R250 (a); the other strip was transferred to a nitrocellulose membrane. After the proteins were incubated with patient sera, it was detected with peroxidase-conjugated anti-human IgG and 3, 3 0 -diaminobenzidine tetrahydrochloride (b). Protein spots corresponding to those on the immunoblot gel were identified using LC-ESI-MS. Identified spots are ringed. Their identities are listed in Table 2.

there were proteins that were abundantly expressed in the UCF-5, but not in UCF-4. Three proteins were identified as sodA and two hypothetical proteins (MAP1420, MAP4056c) (data not shown). Overall, CD patient sera were found to contain antibodies to ModD and PepA. Interestingly, these two proteins are also highly reactive with sera from cattle with paratuberculosis and are known to be secreted or exported proteins of M. tuberculosis (Braunstein et al., 2003). Native ModD and PepA had a significantly higher diagnostic sensitivity than the recombinant forms of these proteins (Cameron et al., 1994; Cho et al., 2007). PepA is a serine protease and has a putative signal sequence at the N-terminus. Native PepA was significantly more antigenic in infected sheep, goats, and deer (Cameron et al., 1994), and PepA is conserved among Mycobacterium 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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spp. for example a virulent H37Ra strain, virulent Erdman strain, H37Rv strain, and clinical isolate CSU93 with 100% identity (Ratliff et al., 1993). Mycobacterial ModD is an alanine and proline-rich secreted 45–47-kDa immunogenic glycoprotein belonging to a fibronectin attachment protein (FAP) (Ratliff et al., 1993; Romain et al., 1993). FAPs are a family of fibronectin-binding glycoproteins that are conserved in several species of mycobacteria: Mycobacterium bovis, M. tuberculosis, Mycobacterium vaccae, M. avium, and Mycobacterium leprae (Laqueyrerie et al., 1995; Schorey et al., 1996). FAPs are necessary for internalization and invasion of epithelial cells by MAP, and deletion of this gene leads to enhanced aggregation in Mycobacterium smegmatis (Miyamoto et al., 2004). Other H-MAP CF proteins reactive with CD patient sera included transaldolase, EchA9 (enoyl-CoA hydratase), and two hypothetical proteins. Transaldolase takes part in the pentose (5-carbon) phosphate (phosphogluconate) cytosolic pathway to generate NADPH and synthesize pentose sugar (Klutts et al., 2002). As a critical enzymatic component of carbohydrate metabolism, transaldolase is highly conserved in several species of mycobacteria (http:// www.biohealthbase.org). EchA9 belongs to the enoyl-CoA hydratase/isomerase family of proteins that catalyzes the reversible hydration of unsaturated fatty acyl-CoA to bhydroxyacyl-CoA (Fujita et al., 1980). EchA9 is found in several species of mycobacteria: M. tuberculosis H37Rv, H37Ra, CDC1551, M. smegmatis, M. bovis, M. bovis BCG, M. leprae, M. avium, and Mycobacterium ulcerans (http:// www.biohealthbase.org). MAP2950c is a hypothetical protein that corresponds to the 16-kDa immunoprotective secreted extracellular MPT63 protein from M. bovis and M. tuberculosis H37Rv (Manca et al., 1997; Kobayashi et al., 2003). MPT63 is found only in M. tuberculosis complex species, and the polyclonal antibody against MPT63 does not cross-react with environmental mycobacteria (Manca et al., 1997). It has been reported previously that a multiantigen complex including MPT63 is useful for detecting tuberculosis patients (Wang et al., 2005). The epitopes of MAP2950c may also be useful for detection of MAP infections in CD patients. MAP2120c is a hypothetical protein that has 61% amino acid identity to Rv1464 encoding the cysteine desulfurase (csd) gene. It belongs to the CSD subfamily participating in cysteine metabolism. However, the contribution of MAP2120c and Rv1464 proteins to the serodiagnosis for mycobacterial infections has not been completely evaluated. Conclusively, this is the first report on the analysis of CD patient serum antibody responses to H-MAP by 2-DE immunoblot. The six H-MAP CF antigens identified may be useful as diagnostic tools to detect MAP infection of CD patients. The data suggest that four proteins including FEMS Immunol Med Microbiol 58 (2010) 128–137

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Table 2. List of protein spots identified in UCF-4 from MAP Spot

Identified proteinw

ORFw

Locus

Molecular mass (kDa)

1, 2, 3

ModD

MAP1569

modD

45–47

4

Hypothetical protein

MAP2120c



69.9

5

Transaldolase

MAP1177c

tal

40.3

6, 7

EchA9

MAP1018c

echA9

36.8

8, 9

PepA

MAP3527

pepA

32.0

10

Hypothetical protein

MAP2950c



20.5

Putative functionz Alanine- and proline-rich 45/47-kDa glycoprotein Fibronectinattachment protein (FAP) Belongs to class V of pyridoxal-phosphate-dependent aminotransferases and csd (cysteine desulfurase) subfamily Catalyzes the reversible formation of D-erythrose 4-phosphate and Dfructose 6-phosphate from sedoheptulose 7-phosphate and Dglyceraldehyde 3-phosphate Enoyl-CoA hydratase/isomerase family Catalyzes the reversible hydration of unsaturated fatty acyl-CoA to b-hydroxyacyl-CoA Belongs to the serine protease family and has a putative signal sequence at the N-terminus 16-kDa immunoprotective extracellular protein and immunogenic protein mpt63 (containing the N-terminal signal sequence) Identical to MPT63 from Mycobacterium bovis

Spot numbers indicated in Fig. 3. w

The nomenclature from the MAP K10 genome was used (http://www.ncbi.nlm.nih.gov). The list putative functions were obtained from BLAST (http://www.ncbi.nlm.nih.gov/COG/).

z

Fig. 5. Expression level of the identified proteins originating from H-MAP UCF-4 strain. The spots identified between B-MAP (B236) and H-MAP (UCF-4) were analyzed using PRODIGY SAMESPOTS software (Nonlinear Dynamics Ltd). The backgrounds of the same spots were normalized to those in MAP B236 and the fold changes of the identified proteins in MAP UCF-4 were then quantified by a direct cross-gel comparison with the reference spots of MAP B236. The conformation changes of the proteins reacting to sera from CD patients in UCF-4 CF proteins were expressed in the right-top portion.

transaldolase, enoyl-CoA hydratase (EchA9), and two hypothetical proteins (MAP2120c and MAP2950c) may be antigens uniquely important for detection of human antiFEMS Immunol Med Microbiol 58 (2010) 128–137

body responses to MAP infection. Further characterization of these proteins and their epitopes should clarify their potential diagnostic value. 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Acknowledgement This study was supported by a grant from the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Korea (A090184).

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