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Identification and characterization of a gene encoding a 35-kDa protein from Mycobacterium avium subspecies paratuberculosis
FEMS Microbiology Letters 196 (2001) 195^199
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Identi¢cation and characterization of a gene encoding a 35-kDa protein from Mycobacterium avium subspecies paratuberculosis K.D. Banasure a , S.H. Basagoudanavar a , P. Chaudhury a , V. Tiwari a , N.S. Parihar b , P.P. Goswami a; * a
National Biotechnology Centre, Indian Veterinary Research Institute, Izatnagar 243 122, India b Division of Pathology, Indian Veterinary Research Institute, Izatnagar 243 122, India
Received 7 December 2000; received in revised form 26 January 2001; accepted 29 January 2001
Abstract Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. A gene homologous to that of 35-kDa antigen of Mycobacterium leprae was cloned and sequenced from Mycobacterium paratuberculosis. The database searches revealed 82.79% and 95.67% similarities of its nucleotide sequence, with those of immunodominant 35-kDa protein of M. leprae and M. avium, respectively. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : 35-kDa protein ; Mycobacterium avium paratuberculosis
1. Introduction Mycobacterium avium paratuberculosis (M. a. paratuberculosis), an extremely slow growing, mycobactin-dependent organism, is the causative agent of Johne's disease (paratuberculosis), a chronic enteritis in ruminants [1]. The disease is associated with signi¢cant economic losses all over the world [2,3]. There is no authentic test to detect the early stages of Mycobacterium paratuberculosis infection. The faecal smear examination with Ziehl^Nielsen staining has poor sensitivity while culture-based assays require more than 6^ 8 weeks to identify the organism. The recently developed gene probes and PCR assays for the detection of M. paratuberculosis require specialized equipments and are expensive [4]. Attention is, therefore, paid to the diagnostic tests based on immune responses of the host against the causative M. a. paratuberculosis organism. Utilizing partially puri¢ed, sonicated proteins or recombinant proteins of M. a. paratuberculosis, enzymelinked immunosorbent assays have been reported [5^9] to detect humoral immune response of the host which usually becomes evident in a later stage of infection.
* Corresponding author. Tel. : +91 (0581) 440525; Fax: +91 (0581) 447179; E-mail : [email protected]
Hence emphasis is being put on cell-mediated immunity based tests to identify infection in the early subclinical stage. Therefore tests such as delayed type hypersensitivity (DTH), lymphocyte stimulation assay and Q-interferon assay have been standardized. The available crude and unde¢ned antigen preparations do stimulate lymphocytes but due to commonality of certain antigens with related organisms, it has not been possible to eliminate cross reactions to satisfying levels [1,10]. This factor has sustained the impetus for searching for a speci¢c M. a. paratuberculosis antigen which is easily and speci¢cally recognizable by T-cells [11]. A 35-kDa protein of Mycobacterium leprae has been shown to elicit speci¢c T-cell-mediated immune responses in leprosy patients [12,13]. In this communication, we describe the identi¢cation and characterization of a gene in M. a. paratuberculosis homologous to that of the immunodominant 35-kDa protein of M. leprae. 2. Materials and methods 2.1. Bacterial strains M. paratuberculosis strains 316F and III-V, and M. bovis (AN5) were from the Biological Products Division, IVRI, Izatnagar, India. The other mycobacterial cultures
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 0 6 8 - 4
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used in this study (M. avium, M. intracellulare, M. kansasi, M. scrofulacium, M. smegmatis, M. gordonae, M. microti, M. marinum, M. fortuitum, M. chelonae and M. phlei) were obtained from the Mycobacterial Repository Centre, Jalma Institute for leprosy, Agra, India. The mycobacterial organisms were grown and maintained on MiddleBrook 7H10 agar medium supplemented with 10% OADC (oleic albumin dextrose catalase) and 0.1% glycerol (v/v). For the growth of M. paratuberculosis, the medium had additional supplementation of mycobactin J (0.2%). Escherichia coli JM 109 supplied by Promega (USA) was grown at 37³C in Luria Bertani broth. 2.2. DNA isolation The genomic DNA from the mycobacterial species was isolated by the method of Portillo et al. [14]. Plasmid DNA extraction from E. coli followed the alkaline lysis procedure [15]. M. leprae DNA was obtained from the Jalma Institute for leprosy, Agra, India. 2.3. PCR ampli¢cation and cloning To amplify a 35-kDa protein gene from M. a. paratuberculosis, the primers: (sense) 5P-CCGAGCTCTGACGTCGGCTCAGAATG-3P and (antisense) 5P-CCAAGCTTTCACTTGTACTCATGGAAC-3P, respectively containing SacI and HindIII restriction endonuclease sites, were designed on the basis of DNA sequence information for a homologous protein of M. leprae [12]. DNA ampli¢cation was carried out in 50 Wl containing standard PCR bu¡er, 200 WM of each dNTP, 0.5 WM of each primer, 2 U of Taq DNA polymerase and 50 ng of mycobacterial genomic DNA. Each reaction mix was overlaid with light mineral oil and subjected to 30 cycles of ampli¢cation under the following conditions: denaturation at 94³C for 1 min, primer annealing at 50³C for 1 min and primer extension at 72³C for 1 min 30 s. After 30 cycles, ¢nal extension for 7 min at 72³C was carried out. PCR prod-
ucts were analyzed by agarose (1.0%) gel electrophoresis and also by restriction enzyme analysis. The ampli¢ed MPL35 gene was puri¢ed from agarose gel using the Qiaex II gel extraction kit (Qiagen, USA), digested with SacI and HindIII restriction endonucleases and ligated into the pGEM-3Zf(+) vector. The newly constructed plasmid, designated as pGMPL35, was transformed into E. coli JM 109 competent cells. The transformed E. coli cells grown on LB agar plates containing ampicillin, X-gal and IPTG were screened for the presence of an insert on the basis of the white or blue color of the colonies. Ampicillin resistant transformants were screened by restriction enzyme analysis to identify the presence of the correct insert. 2.4. DNA hybridization The speci¢c distribution of the MPL35 gene in mycobacterial species was con¢rmed by dot blot hybridization. About 2 Wg of chromosomal DNA each from various mycobacterial species was heat-denatured and blotted on Hybond N membrane (Amersham, UK). Hybridization was carried out at 60³C for 18 h. The cloned MPL35 gene fragment was puri¢ed from agarose gel using the Qiaex II gel extraction kit (Qiagen, USA), labelled with [K-32 P]dATP and used as probe. High stringency washing was performed (at 60³C in 0.1USSC) and detection of a hybridization signal was carried out by autoradiography [15]. 2.5. DNA sequence analysis The nucleotide sequence of the cloned MPL35 gene was determined by the dideoxy chain termination method [16]. Nucleotide and deduced amino acid sequences were analyzed using Laser gene software (DNAStar). The nucleotide sequence of the MPL35 gene submitted to the EMBL database has been assigned accession number AJ250887.
Fig. 1. Agarose gel electrophoresis showing the PCR amplicon of 940-bp size from di¡erent mycobacteria. Lanes: M, DNA molecular mass marker pUC18/Sau3AI-pUC18/TaqI digest; 1, M. paratuberculosis 316F ; 2, M. paratuberculosis III-V; 3, M. intracellulare ; 4, M. avium ; 5, M. gordonae ; 6, M. smegmatis ; 7, M. phlei; 8, M. microti; 9, M. kansasi ; 10, M. scrofulacium ; 11, M. bovis (AN5); 12, M. marinum; 13, M. leprae; 14, M. fortuitum ; 15, M. chelonae.
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3. Results and discussion The 35-kDa protein is a major membranous antigenic component of M. leprae and M. avium eliciting T-cell immune response [12,17]. The genes encoding the protein are well conserved among the two species. The present study aimed to isolate and characterize the homologous gene from M. a. paratuberculosis, the causative agent of Johne's disease in ruminants. Hence primers were designed from the nucleotide sequence of 35-kDa protein gene of M. leprae [12]. 3.1. PCR ampli¢cation and construction of recombinant pGMPL35 Upon ampli¢cation, as expected, a PCR product of 940 bp was obtained from M. a. paratuberculosis DNA, and was designated MPL35. Upon restriction enzyme digestion with PvuII and SalI, the expected size of fragments was obtained (data not shown). Only in M. a. paratuberculosis, M. leprae and M. avium an amplicon of the gene encoding a 35-kDa protein was obtained. The designed primers, however, did not amplify a similar gene from M. intracellulare, M. kansasi, M. scrofulacium, M. smegmatis, M. bovis (AN5), M. gordonae, M. microti, M. marinum, M. fortuitum, M. chelonae and M. phlei (Fig. 1). Thus, by utilizing the published nucleotide sequence of a 35-kDa protein of M. leprae [12] speci¢c primers were designed
Fig. 3. Dot blot hybridization of genomic DNA from various mycobacteria, using the cloned MPL35 gene as radiolabelled probe. A1, M. kansasi ; A2, M. scrofulacium ; A3, M. intracellulare ; A4, M. bovis (AN5); B1, PCR-ampli¢ed product of gene encoding 35-kDa protein from M. paratuberculosis ; B2, M. paratuberculosis 316F ; B3, M. paratuberculosis III-V; B4, M. smegmatis; C1, M. gordonae ; C2, M. phlei; C3, M. avium ; C4, M. leprae; D1, M. microti ; D2, M. marinum; D3, M. fortuitum; D4, M. chelonae.
that could successfully amplify a similar gene in M. paratuberculosis. This PCR-based strategy simpli¢es the search for analogous genes in related organisms. This gene is reported to be absent in the Mycobacterium tuberculosis complex group and also in some serovars of M. intracellulare [17]. Recombinant plasmid pGMPL35 (4139 bp) generated after cloning of a 940-bp PCR-ampli¢ed fragment in the PGEM-3Zf(+) vector was selected from LB agar plates. A fragment with identical size was released on digestion with HindIII and SacI (Fig. 2). 3.2. Dot blot hybridization Upon dot blot hybridization using the cloned MPL35 gene fragment as probe on genomic DNA from di¡erent mycobacterial species, the hybridization signal was detected also in M. smegmatis, besides M. a. paratuberculosis, M. avium and M. leprae (Fig. 3). These results are in line with earlier reports on the 35-kDa protein [12]. The presence of the 35-kDa protein gene in M. leprae and M. avium was earlier reported [12,17]. Our study extends the list by identifying a homologue of the 35-kDa protein encoding gene in M. paratuberculosis. 3.3. Nucleotide sequencing
Fig. 2. Agarose gel electrophoresis of the cloned fragment of the gene encoding a 35-kDa protein. Lanes: M, DNA molecular mass marker pUC18/Sau3AI-pUC18/TaqI digest; A, PCR-ampli¢ed gene encoding a 35-kDa protein from M. paratuberculosis; B, released insert of 940 bp after SacI and HindIII digestion of recombinant pGMPL35.
To con¢rm the homology with the 35-kDa protein encoding gene of M. leprae and M. avium, the cloned MPL35 gene was sequenced. The PCR fragment of MPL35 gene cloned in pGEM-3Zf(+) vector was sequenced by the dideoxy chain termination method. The DNA sequence contained a 924-bp open reading frame. The potential ATG start codon was followed by 306 codons before a TGA nonsense codon was encountered. The proposed mature protein was 307 amino acids in length with an estimated molecular mass of 33 670.31 Da.
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Fig. 4. Comparison of the deduced amino acid sequence of the M. paratuberculosis 35-kDa protein with sequences of the M. leprae 35-kDa protein and M. avium 35-kDa protein. Amino acids that di¡er from the M. paratuberculosis 35-kDa protein are enclosed in a box.
Alignment of the nucleotide sequence of the MPL35 gene of M. a. paratuberculosis with that of the 35-kDa gene of M. leprae [12] and M. avium [17] showed 82.79% and 95.67% homology, respectively. The deduced amino acid sequence had 90.3% and 95.1% similarities, respectively (Fig. 4). But no signi¢cant similarity of MPL35 was observed with other sequences of M. paratuberculosis. The observed homology of sequences in M. leprae, M.
avium and M. paratuberculosis indicates that the 35-kDa protein is well conserved between these species. The 35-kDa protein of M. leprae is an immunodominant antigen capable of eliciting T-cell and IgG antibody responses. The protein elicited strong DTH in M. leprae or M. avium sensitized animals [13,17]. The identi¢cation of speci¢c antigens recognized by T-cells is required for early diagnosis and vaccine development for M. paratuberculosis
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infections [11]. There are, however, no traceable reports of availability of puri¢ed M. paratuberculosis antigen(s) capable of eliciting T-cell response. The elucidation of this gene is, therefore, likely to pave the way for faster and reliable diagnosis of paratuberculous infections.
[7]
[8]
Acknowledgements The authors are thankful to the Director, Indian Veterinary Research Institute, Izatnagar, for providing the necessary facilities and also to Dr. V.M. Katoch, Jalma Institute for Leprosy, Agra, U.P. (India) for providing M. leprae DNA.
[9]
[10]
[11]
References
[12]
[1] Chiodini, R.J., Van Kruiningen, H.J. and Merkal, R.S. (1984) Ruminant paratuberculosis (Johne's disease): The current status and future prospects. Cornell Vet. 74, 218^262. [2] Whipple, D.L., Kapke, P.A. and Andersen, P.R. (1992) Comparison of a commercial DNA probe test and three cultivation procedures for the detection of Mycobacterium paratuberculosis in bovine species. J. Vet. Diagn. Invest. 4, 23^27. [3] Whitlock, R.H. (1992) An overview of Johne's disease. In: Proceedings of third International Colloquium on Paratuberculosis (Chiodini, R.J. and Kreeger, J.M., Eds.), pp. 514^522. Providence, RI. [4] Van der Giessen, J.W.B., Haring, R.M., Vauclare, E., Eger, A., Haagsma, J. and Van der Zeist, B.A.M. (1992) Evaluation of the abilities of the three diagnostic tests based on the polymerase chain reaction to detect Mycobacterium paratuberculosis in cattle: application in a controlled programme. J. Clin. Microbiol. 30, 1216^1219. [5] Sugden, E.A., Corner, A.H., Samagh, B.S., Brooks, B.W., Turcotte, C., Nielson, K.H., Stewart, R.B. and Duncan, J.R. (1989) Serodiagnosis of ovine paratuberculosis, using lipoarabinomannan in an enzyme linked immunosorbent assay. Am. J. Vet. Res. 50, 850^854. [6] DeKesel, M., Gilot, P., Misonne, M.C., Cone, M. and Cocito, C. (1993) Cloning and expression of portions of the 34-kilodalton pro-
[13]
[14]
[15]
[16] [17]
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tein gene of Mycobacterium paratuberculosis: Its applications to serological analysis of Johne's disease. J. Clin. Microbiol. 32, 947^954. White, W.B., Whipple, D.L., Stabel, J.R. and Bolin, C.A. (1994) Comparison of cellular and intracellular proteins expressed by various isolates of Mycobacterium paratuberculosis and other mycobacterium species. Am. J. Vet. Res. 55, 1399^1405. Mutharia, L.M., Moreno, K. and Raymond, M. (1997) Analysis of culture ¢ltrate and cell wall associated antigens of Mycobacterium paratuberculosis with monoclonal antibodies. Infect. Immunol. 65, 387^394. El-Zaatari, F.A.K., Naser, S.A. and Grahm, D.Y. (1997) Characterization of a speci¢c Mycobacterium paratuberculosis recombinant clone expressing 35 000-molecular weight antigen and reactivity with sera from animals with clinical and subclinical Johne's disease. J. Clin. Microbiol. 35, 1794^1799. Gilot, P. and Cocito, C. (1993) Comparative analysis of these sensitive used in cutaneous testing for tuberculosis and paratuberculosis in cattle. FEMS Microbiol. Lett. 110, 307^312. Clarke, C.J. (1997) The pathology and pathogenesis of paratuberculosis in ruminants and other species. J. Comp. Pathol. 116, 217^261. Winter, N., Triccas, J.A., Rivoire, B., Pessdani, M.C.V., Eiglmeier, K., Hunter, S.W., Brennan, P.J. and Britton, W.J. (1995) Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae. Mol. Microbiol. 15, 865^876. Triccas, J.A., Roche, P.W., Winter, N., Feng, C.G., Butlin, C.R. and Britton, W.J. (1996) A 35 kilodalton protein is a major target of the human immune response to Mycobacterium leprae. Infect. Immun. 64, 5171^5177. Portillo, P.D., Murillo, L.A. and Patarroyo, M.E. (1991) Ampli¢cation of a species-speci¢c DNA fragment of Mycobacterium paratuberculosis and it's possible use in diagnosis. J. Clin. Microbiol. 29, 2163^ 2168. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: A laboratory manual 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Murphy and Kavanagh (1988) Speeding up the sequencing of double stranded DNA. Nucleic Acid Res. 16, 5198. Triccas, J.A., Winter, N. and Roche, P.W. (1998) Molecular and immunological analysis of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kDa protein. Infect. Immun. 66, 2684^2690.
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Identi¢cation and characterization of a gene encoding a 35-kDa protein from Mycobacterium avium subspecies paratuberculosis K.D. Banasure a , S.H. Basagoudanavar a , P. Chaudhury a , V. Tiwari a , N.S. Parihar b , P.P. Goswami a; * a
National Biotechnology Centre, Indian Veterinary Research Institute, Izatnagar 243 122, India b Division of Pathology, Indian Veterinary Research Institute, Izatnagar 243 122, India
Received 7 December 2000; received in revised form 26 January 2001; accepted 29 January 2001
Abstract Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. A gene homologous to that of 35-kDa antigen of Mycobacterium leprae was cloned and sequenced from Mycobacterium paratuberculosis. The database searches revealed 82.79% and 95.67% similarities of its nucleotide sequence, with those of immunodominant 35-kDa protein of M. leprae and M. avium, respectively. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : 35-kDa protein ; Mycobacterium avium paratuberculosis
1. Introduction Mycobacterium avium paratuberculosis (M. a. paratuberculosis), an extremely slow growing, mycobactin-dependent organism, is the causative agent of Johne's disease (paratuberculosis), a chronic enteritis in ruminants [1]. The disease is associated with signi¢cant economic losses all over the world [2,3]. There is no authentic test to detect the early stages of Mycobacterium paratuberculosis infection. The faecal smear examination with Ziehl^Nielsen staining has poor sensitivity while culture-based assays require more than 6^ 8 weeks to identify the organism. The recently developed gene probes and PCR assays for the detection of M. paratuberculosis require specialized equipments and are expensive [4]. Attention is, therefore, paid to the diagnostic tests based on immune responses of the host against the causative M. a. paratuberculosis organism. Utilizing partially puri¢ed, sonicated proteins or recombinant proteins of M. a. paratuberculosis, enzymelinked immunosorbent assays have been reported [5^9] to detect humoral immune response of the host which usually becomes evident in a later stage of infection.
* Corresponding author. Tel. : +91 (0581) 440525; Fax: +91 (0581) 447179; E-mail : [email protected]
Hence emphasis is being put on cell-mediated immunity based tests to identify infection in the early subclinical stage. Therefore tests such as delayed type hypersensitivity (DTH), lymphocyte stimulation assay and Q-interferon assay have been standardized. The available crude and unde¢ned antigen preparations do stimulate lymphocytes but due to commonality of certain antigens with related organisms, it has not been possible to eliminate cross reactions to satisfying levels [1,10]. This factor has sustained the impetus for searching for a speci¢c M. a. paratuberculosis antigen which is easily and speci¢cally recognizable by T-cells [11]. A 35-kDa protein of Mycobacterium leprae has been shown to elicit speci¢c T-cell-mediated immune responses in leprosy patients [12,13]. In this communication, we describe the identi¢cation and characterization of a gene in M. a. paratuberculosis homologous to that of the immunodominant 35-kDa protein of M. leprae. 2. Materials and methods 2.1. Bacterial strains M. paratuberculosis strains 316F and III-V, and M. bovis (AN5) were from the Biological Products Division, IVRI, Izatnagar, India. The other mycobacterial cultures
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 0 6 8 - 4
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used in this study (M. avium, M. intracellulare, M. kansasi, M. scrofulacium, M. smegmatis, M. gordonae, M. microti, M. marinum, M. fortuitum, M. chelonae and M. phlei) were obtained from the Mycobacterial Repository Centre, Jalma Institute for leprosy, Agra, India. The mycobacterial organisms were grown and maintained on MiddleBrook 7H10 agar medium supplemented with 10% OADC (oleic albumin dextrose catalase) and 0.1% glycerol (v/v). For the growth of M. paratuberculosis, the medium had additional supplementation of mycobactin J (0.2%). Escherichia coli JM 109 supplied by Promega (USA) was grown at 37³C in Luria Bertani broth. 2.2. DNA isolation The genomic DNA from the mycobacterial species was isolated by the method of Portillo et al. [14]. Plasmid DNA extraction from E. coli followed the alkaline lysis procedure [15]. M. leprae DNA was obtained from the Jalma Institute for leprosy, Agra, India. 2.3. PCR ampli¢cation and cloning To amplify a 35-kDa protein gene from M. a. paratuberculosis, the primers: (sense) 5P-CCGAGCTCTGACGTCGGCTCAGAATG-3P and (antisense) 5P-CCAAGCTTTCACTTGTACTCATGGAAC-3P, respectively containing SacI and HindIII restriction endonuclease sites, were designed on the basis of DNA sequence information for a homologous protein of M. leprae [12]. DNA ampli¢cation was carried out in 50 Wl containing standard PCR bu¡er, 200 WM of each dNTP, 0.5 WM of each primer, 2 U of Taq DNA polymerase and 50 ng of mycobacterial genomic DNA. Each reaction mix was overlaid with light mineral oil and subjected to 30 cycles of ampli¢cation under the following conditions: denaturation at 94³C for 1 min, primer annealing at 50³C for 1 min and primer extension at 72³C for 1 min 30 s. After 30 cycles, ¢nal extension for 7 min at 72³C was carried out. PCR prod-
ucts were analyzed by agarose (1.0%) gel electrophoresis and also by restriction enzyme analysis. The ampli¢ed MPL35 gene was puri¢ed from agarose gel using the Qiaex II gel extraction kit (Qiagen, USA), digested with SacI and HindIII restriction endonucleases and ligated into the pGEM-3Zf(+) vector. The newly constructed plasmid, designated as pGMPL35, was transformed into E. coli JM 109 competent cells. The transformed E. coli cells grown on LB agar plates containing ampicillin, X-gal and IPTG were screened for the presence of an insert on the basis of the white or blue color of the colonies. Ampicillin resistant transformants were screened by restriction enzyme analysis to identify the presence of the correct insert. 2.4. DNA hybridization The speci¢c distribution of the MPL35 gene in mycobacterial species was con¢rmed by dot blot hybridization. About 2 Wg of chromosomal DNA each from various mycobacterial species was heat-denatured and blotted on Hybond N membrane (Amersham, UK). Hybridization was carried out at 60³C for 18 h. The cloned MPL35 gene fragment was puri¢ed from agarose gel using the Qiaex II gel extraction kit (Qiagen, USA), labelled with [K-32 P]dATP and used as probe. High stringency washing was performed (at 60³C in 0.1USSC) and detection of a hybridization signal was carried out by autoradiography [15]. 2.5. DNA sequence analysis The nucleotide sequence of the cloned MPL35 gene was determined by the dideoxy chain termination method [16]. Nucleotide and deduced amino acid sequences were analyzed using Laser gene software (DNAStar). The nucleotide sequence of the MPL35 gene submitted to the EMBL database has been assigned accession number AJ250887.
Fig. 1. Agarose gel electrophoresis showing the PCR amplicon of 940-bp size from di¡erent mycobacteria. Lanes: M, DNA molecular mass marker pUC18/Sau3AI-pUC18/TaqI digest; 1, M. paratuberculosis 316F ; 2, M. paratuberculosis III-V; 3, M. intracellulare ; 4, M. avium ; 5, M. gordonae ; 6, M. smegmatis ; 7, M. phlei; 8, M. microti; 9, M. kansasi ; 10, M. scrofulacium ; 11, M. bovis (AN5); 12, M. marinum; 13, M. leprae; 14, M. fortuitum ; 15, M. chelonae.
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3. Results and discussion The 35-kDa protein is a major membranous antigenic component of M. leprae and M. avium eliciting T-cell immune response [12,17]. The genes encoding the protein are well conserved among the two species. The present study aimed to isolate and characterize the homologous gene from M. a. paratuberculosis, the causative agent of Johne's disease in ruminants. Hence primers were designed from the nucleotide sequence of 35-kDa protein gene of M. leprae [12]. 3.1. PCR ampli¢cation and construction of recombinant pGMPL35 Upon ampli¢cation, as expected, a PCR product of 940 bp was obtained from M. a. paratuberculosis DNA, and was designated MPL35. Upon restriction enzyme digestion with PvuII and SalI, the expected size of fragments was obtained (data not shown). Only in M. a. paratuberculosis, M. leprae and M. avium an amplicon of the gene encoding a 35-kDa protein was obtained. The designed primers, however, did not amplify a similar gene from M. intracellulare, M. kansasi, M. scrofulacium, M. smegmatis, M. bovis (AN5), M. gordonae, M. microti, M. marinum, M. fortuitum, M. chelonae and M. phlei (Fig. 1). Thus, by utilizing the published nucleotide sequence of a 35-kDa protein of M. leprae [12] speci¢c primers were designed
Fig. 3. Dot blot hybridization of genomic DNA from various mycobacteria, using the cloned MPL35 gene as radiolabelled probe. A1, M. kansasi ; A2, M. scrofulacium ; A3, M. intracellulare ; A4, M. bovis (AN5); B1, PCR-ampli¢ed product of gene encoding 35-kDa protein from M. paratuberculosis ; B2, M. paratuberculosis 316F ; B3, M. paratuberculosis III-V; B4, M. smegmatis; C1, M. gordonae ; C2, M. phlei; C3, M. avium ; C4, M. leprae; D1, M. microti ; D2, M. marinum; D3, M. fortuitum; D4, M. chelonae.
that could successfully amplify a similar gene in M. paratuberculosis. This PCR-based strategy simpli¢es the search for analogous genes in related organisms. This gene is reported to be absent in the Mycobacterium tuberculosis complex group and also in some serovars of M. intracellulare [17]. Recombinant plasmid pGMPL35 (4139 bp) generated after cloning of a 940-bp PCR-ampli¢ed fragment in the PGEM-3Zf(+) vector was selected from LB agar plates. A fragment with identical size was released on digestion with HindIII and SacI (Fig. 2). 3.2. Dot blot hybridization Upon dot blot hybridization using the cloned MPL35 gene fragment as probe on genomic DNA from di¡erent mycobacterial species, the hybridization signal was detected also in M. smegmatis, besides M. a. paratuberculosis, M. avium and M. leprae (Fig. 3). These results are in line with earlier reports on the 35-kDa protein [12]. The presence of the 35-kDa protein gene in M. leprae and M. avium was earlier reported [12,17]. Our study extends the list by identifying a homologue of the 35-kDa protein encoding gene in M. paratuberculosis. 3.3. Nucleotide sequencing
Fig. 2. Agarose gel electrophoresis of the cloned fragment of the gene encoding a 35-kDa protein. Lanes: M, DNA molecular mass marker pUC18/Sau3AI-pUC18/TaqI digest; A, PCR-ampli¢ed gene encoding a 35-kDa protein from M. paratuberculosis; B, released insert of 940 bp after SacI and HindIII digestion of recombinant pGMPL35.
To con¢rm the homology with the 35-kDa protein encoding gene of M. leprae and M. avium, the cloned MPL35 gene was sequenced. The PCR fragment of MPL35 gene cloned in pGEM-3Zf(+) vector was sequenced by the dideoxy chain termination method. The DNA sequence contained a 924-bp open reading frame. The potential ATG start codon was followed by 306 codons before a TGA nonsense codon was encountered. The proposed mature protein was 307 amino acids in length with an estimated molecular mass of 33 670.31 Da.
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Fig. 4. Comparison of the deduced amino acid sequence of the M. paratuberculosis 35-kDa protein with sequences of the M. leprae 35-kDa protein and M. avium 35-kDa protein. Amino acids that di¡er from the M. paratuberculosis 35-kDa protein are enclosed in a box.
Alignment of the nucleotide sequence of the MPL35 gene of M. a. paratuberculosis with that of the 35-kDa gene of M. leprae [12] and M. avium [17] showed 82.79% and 95.67% homology, respectively. The deduced amino acid sequence had 90.3% and 95.1% similarities, respectively (Fig. 4). But no signi¢cant similarity of MPL35 was observed with other sequences of M. paratuberculosis. The observed homology of sequences in M. leprae, M.
avium and M. paratuberculosis indicates that the 35-kDa protein is well conserved between these species. The 35-kDa protein of M. leprae is an immunodominant antigen capable of eliciting T-cell and IgG antibody responses. The protein elicited strong DTH in M. leprae or M. avium sensitized animals [13,17]. The identi¢cation of speci¢c antigens recognized by T-cells is required for early diagnosis and vaccine development for M. paratuberculosis
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infections [11]. There are, however, no traceable reports of availability of puri¢ed M. paratuberculosis antigen(s) capable of eliciting T-cell response. The elucidation of this gene is, therefore, likely to pave the way for faster and reliable diagnosis of paratuberculous infections.
[7]
[8]
Acknowledgements The authors are thankful to the Director, Indian Veterinary Research Institute, Izatnagar, for providing the necessary facilities and also to Dr. V.M. Katoch, Jalma Institute for Leprosy, Agra, U.P. (India) for providing M. leprae DNA.
[9]
[10]
[11]
References
[12]
[1] Chiodini, R.J., Van Kruiningen, H.J. and Merkal, R.S. (1984) Ruminant paratuberculosis (Johne's disease): The current status and future prospects. Cornell Vet. 74, 218^262. [2] Whipple, D.L., Kapke, P.A. and Andersen, P.R. (1992) Comparison of a commercial DNA probe test and three cultivation procedures for the detection of Mycobacterium paratuberculosis in bovine species. J. Vet. Diagn. Invest. 4, 23^27. [3] Whitlock, R.H. (1992) An overview of Johne's disease. In: Proceedings of third International Colloquium on Paratuberculosis (Chiodini, R.J. and Kreeger, J.M., Eds.), pp. 514^522. Providence, RI. [4] Van der Giessen, J.W.B., Haring, R.M., Vauclare, E., Eger, A., Haagsma, J. and Van der Zeist, B.A.M. (1992) Evaluation of the abilities of the three diagnostic tests based on the polymerase chain reaction to detect Mycobacterium paratuberculosis in cattle: application in a controlled programme. J. Clin. Microbiol. 30, 1216^1219. [5] Sugden, E.A., Corner, A.H., Samagh, B.S., Brooks, B.W., Turcotte, C., Nielson, K.H., Stewart, R.B. and Duncan, J.R. (1989) Serodiagnosis of ovine paratuberculosis, using lipoarabinomannan in an enzyme linked immunosorbent assay. Am. J. Vet. Res. 50, 850^854. [6] DeKesel, M., Gilot, P., Misonne, M.C., Cone, M. and Cocito, C. (1993) Cloning and expression of portions of the 34-kilodalton pro-
[13]
[14]
[15]
[16] [17]
199
tein gene of Mycobacterium paratuberculosis: Its applications to serological analysis of Johne's disease. J. Clin. Microbiol. 32, 947^954. White, W.B., Whipple, D.L., Stabel, J.R. and Bolin, C.A. (1994) Comparison of cellular and intracellular proteins expressed by various isolates of Mycobacterium paratuberculosis and other mycobacterium species. Am. J. Vet. Res. 55, 1399^1405. Mutharia, L.M., Moreno, K. and Raymond, M. (1997) Analysis of culture ¢ltrate and cell wall associated antigens of Mycobacterium paratuberculosis with monoclonal antibodies. Infect. Immunol. 65, 387^394. El-Zaatari, F.A.K., Naser, S.A. and Grahm, D.Y. (1997) Characterization of a speci¢c Mycobacterium paratuberculosis recombinant clone expressing 35 000-molecular weight antigen and reactivity with sera from animals with clinical and subclinical Johne's disease. J. Clin. Microbiol. 35, 1794^1799. Gilot, P. and Cocito, C. (1993) Comparative analysis of these sensitive used in cutaneous testing for tuberculosis and paratuberculosis in cattle. FEMS Microbiol. Lett. 110, 307^312. Clarke, C.J. (1997) The pathology and pathogenesis of paratuberculosis in ruminants and other species. J. Comp. Pathol. 116, 217^261. Winter, N., Triccas, J.A., Rivoire, B., Pessdani, M.C.V., Eiglmeier, K., Hunter, S.W., Brennan, P.J. and Britton, W.J. (1995) Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae. Mol. Microbiol. 15, 865^876. Triccas, J.A., Roche, P.W., Winter, N., Feng, C.G., Butlin, C.R. and Britton, W.J. (1996) A 35 kilodalton protein is a major target of the human immune response to Mycobacterium leprae. Infect. Immun. 64, 5171^5177. Portillo, P.D., Murillo, L.A. and Patarroyo, M.E. (1991) Ampli¢cation of a species-speci¢c DNA fragment of Mycobacterium paratuberculosis and it's possible use in diagnosis. J. Clin. Microbiol. 29, 2163^ 2168. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: A laboratory manual 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Murphy and Kavanagh (1988) Speeding up the sequencing of double stranded DNA. Nucleic Acid Res. 16, 5198. Triccas, J.A., Winter, N. and Roche, P.W. (1998) Molecular and immunological analysis of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kDa protein. Infect. Immun. 66, 2684^2690.
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