Chapter 17 : Expression of Foreign Genes in Mycobacteria

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This chapter reviews the development of genetic systems for the expression of foreign genes in mycobacteria. Mycobacteriophage have been used for many years to type mycobacterial isolates and have more recently been modified as vectors for efficient delivery of foreign DNA into mycobacteria. Plasmid-based expression systems extend the capabilities of phage-based systems by providing increased cloning capacity, increased copy number in the mycobacterial host, and ease of manipulation. Various derivatives of the pAL5000 plasmid replicon have been combined with plasmid replication origins, such as ColEl and pl5A, by several groups to fashion -mycobacterium shuttle vectors. Expression of foreign proteins in recombinant organisms is influenced by many factors. Much of the increased interest in mycobacteria is a result of the emergence of multidrug-resistant . Complex surface macromolecules, such as oligosaccharides and glycolipids, require the products of many genes and the correct cell envelope structure for proper assembly. BCG, the current vaccine against tuberculosis, has provided variable protective efficacy against tuberculosis in different vaccine trials. Immunization of chickens with recombinant expressing an surface antigen elicits partial protection against coccidiosis.

Citation: Burlein J, Offutt S, Hanson M, Stover C. 1994. Expression of Foreign Genes in Mycobacteria, p 239-252. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch17
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Figure 1

Mycobacterial extrachromosomal (pMV261) and integrative (pMV361) expression vectors (modified from ). Common elements in these two vectors include an expression cassette, the Tn905-derived gene (kanamycin resistance), and an origin of replication functional in derived from pUC19. The expression cassette contains 0.4 kb of the 5ʹ end of the BCG gene, including the promoter region, ribosome-binding site, and first six codons (MAKTIA) as well as a multiple cloning site and the transcriptional terminator. The vectors differ by inclusion of either a mycobacterial plasmid replication origin or the and genes of mycobacteriophage L5.

Citation: Burlein J, Offutt S, Hanson M, Stover C. 1994. Expression of Foreign Genes in Mycobacteria, p 239-252. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch17
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Figure 2

Expression of foreign antigens in different BCG substrains. BCG culture lysates equivalent to approximately 5 x 106 bacteria were analyzed by SDS-PAGE and Coomassie blue staining (A) or by immunoblotting with antibodies specific for foreign antigens OspA (B, C) or HIV-1 gpl20 (D). Panel A shows the total proteins expressed by nonrecombinant BCG. Panels B through D show relevant portions of immunoblots of different BCG recombinants expressing the indicated foreign antigens.

Citation: Burlein J, Offutt S, Hanson M, Stover C. 1994. Expression of Foreign Genes in Mycobacteria, p 239-252. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch17
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1. Aldovini, A.,, R. N. Husson,, and R. A. Young. 1993. The uraA locus and homologous recombination in Mycobacterium bovis BCG. J. Bacterial. 175:72827289.
2. Aldovini, A.,, and R. A. Young. 1991. Humoral and cell-mediated responses to live recombinant BCG-HIV vaccines. Nature (London) 351:479482.
3. Andersen, A. B.,, and E. B. Hansen. 1989. Structure and mapping of antigenic domains of protein antigen b, a 38,000-molecular weight protein of Mycobacterium tuberculosis. Infect. Immun. 57:24812488.
4. Ashbridge, K. R.,, R. J. Booth,, J. D. Watson,, and R. Lathigra. 1989. Nucleotide sequence of the 19 kDa antigen gene from Mycobacterium tuberculosis. Nucleic Acids Res. 17:1249.
5. Banerjee, A.,, E. Dubnau,, A. Quemard,, V. Balasubra-manian,, K. S. Um,, T. Wilson,, G. de Lisle,, and W. R. JacobsJr.. 1994. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 263:227230.
6. Barletta, R. G.,, D. D. Kim,, S. B. Snapper,, B. R. Bloom,, and W. R. JacobsJr.. 1992. Identification of expression signals of the mycobacteriophages Bxbl, LI, and TM4 using the Escherichia-mycobacterium shuttle plasmids pYUB75, and pYUB76 designed to create translational fusions to the lacZ gene. J. Gen. Microbiol. 138:2330.
7. Belisle, J. T.,, L. Pascopella,, J. M. Inamine,, P. J. Brennan,, and W. R. JacobsJr.. 1991. Isolation and expression of a gene cluster responsible for biosynthesis of the glycopeptidolipid antigens of Mycobacterium avium. J. Bacteriol. 173:69916997.
8. Bergstrom, S.,, V. G. Bundoc,, and A. G. Barbour. 1989. Molecular analysis of linear* plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi. Mol. Microbiol. 3:479486.
9. Borremans, M.,, L. de Wit,, G. Volckaert,, J. Ooms,, J. de Bruyn,, K. Huygen,, J.-P. van Vooren,, M. Stelandre,, R. Verhofstadt,, and J. Content. 1989. Cloning, sequence determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis. Infect. Immun. 57:31233130.
10. Buchmeier, N. A.,, and F. Heffron. 1990. Induction of Salmonella stress proteins upon infection of macrophages. Science 248:730732.
11. Buell, G.,, and N. Panayotatos,. 1986. Mechanism and practice, p. 345363. In W. Reznikoff, and L. Gold (ed.), Maximizing Gene Expression. Butterworths, Boston.
12. Cassatt, D. R.,, V. F. de la Cruz,, J. E. Burlein,, J. Young,, S. Koenig,, and C. Kendall Stover,. 1993. Protection of mice against tetanus challenge using an experimental tetanus vaccine based on recombinant BCG, p. 385389. In H. S. Ginsberg,, F. Brown,, R. M. Channock,, and R. A. Lerner (ed.), Vaccines 93. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
13. Clark-Curtiss, J. E.,, W. R. Jacobs,, M. A. Docherty,, L. R. Ritchie,, and R. Curtiss. 1985. Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. J. Bacteriol. 161:10931102.
14. Connell, N. D.,, E. Medina-Acosta,, W. R. McMaster,, B. R. Bloom,, and D. G. Russell. 1993. Effective immunization against cutaneous leishmaniasis with recombinant BCG expressing the Leishmania surface proteinase (gp63). Proc Natl Acad. Sci. USA 90:1147311477.
15. Content, J.,, A. de la Cuvellerie,, L. de Wit,, V. Vincent-Levy-Frebault,, J. Ooms,, and J. de Bruyn. 1991. The gene coding for the antigen 85 complexes of Mycobacterium tuberculosis and Mycobacterium bovis BCG are members of a gene family: cloning, sequence determination, and genomic organization of the gene coding for antigen 85-C of M. tuberculosis. Infect. Immun. 59:32053212.
16. Convit, J.,, N. Aranzazu,, M. Ulrich,, M. E. Pinardi,, O. Reyes,, and J. Alvarado. 1982. Immunotherapy with a mixture of Mycobacterium leprae and BCG in different forms of leprosy and Matsuda negative contacts. Int. J. Lepr. 50:415.
17. Cooksey, R. C, J. T. Crawford, W. R. Jacobs, and T. M. Shinnick. 1993. A rapid method for screening antimicrobial agents for activity against a strain of Mycobacterium tuberculosis expressing firefly luciferase. Antimicrob. Agents Chemother. 37:13481352.
18. Dale, J. W.,, and A. Patki. 1990. Mycobacterial gene expression and regulation, p. 173198. InJ. McFadden (ed.), Molecular Biology of the Mycobacteria. Surrey University Press, London.
19. de Boer, H. A.,, and R. A. Kastelein,. 1986. Biased codon usage: an exploration of its role in optimization of translation, p. 225286. In W. Reznikoff, and L. Gold (ed.), Maximizing Gene Expression. Butterworths, Boston.
20. Donnelly-Wu, M. K.,, W. R. JacobsJr.,, and G. F. Hatfull. 1993. Superinfection immunity of mycobacteriophage LS: applications for genetic transformation of mycobacteria. Mol. Microbiol. 7:407417.
21. Finlay, B. B.,, and S. Falkow. 1989. Common themes in microbial pathogenicity. Microbiol. Rev. 53:210230.
22. Fortier, A. H.,, B. A. Mock,, M. S. Meitzer,, and C. A. Nacy. 1987. Mycobacterium bovis BCG-induced protection against cutaneous and systemic Leishmania major infections of mice. Infect. Immun. 55: 17071714.
23. Galen, J.. Unpublished data.
24. Garbe, T.,, D. Harris,, M. Vordermeier,, R. Lathigra,, J. Ivanyi,, and D. Young. 1993. Expression of the Mycobacterium tuberculosis 19-kilodalton antigen in Mycobacterium smegmatis: immunological analysis and evidence of glycosylation. Infect. Immun. 61:260267.
25. Goldberg, A., L-, and S. A. Goff. 1986. The selective degradation of abnormal proteins in bacteria, p. 287314. In W. Reznikoff, and L. Gold (ed.), Maximizing Gene Expression. Butterworths, Boston.
26. Goldberg, M. B.,, S. A. Boyko,, and S. B. Calderwood. 1990. Transcriptional regulation by iron of a Vibrio cholerae virulence gene and homology of the gene to the Escherichia coli Fur system. J. Bacteriol. 172: 68636870.
27. Haeseleer, F.,, J.-F. Pollet,, M. Haumont,, C. Dubeaux,, A. Bollen,, P. Jacobs,, M. Modesti,, and J. Cohen,. 1993. Stable integration and expression of Plasmodium falciparum antigen coding sequences in mycobacteria, p. 413417. In H. S. Ginsberg,, F. Brown,, R. M. Channock,, and R. A. Lerner (ed.), Vaccines 93. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
28. Hanson, M. S.,, C. V. Lapcevich,, and S. L. Haun. Progress on development of the live BCG recombinant (rBCG) vaccine vehicle for combined delivery. Ann. N.Y. Acad. Sci., in press.
29. Hatful), G. F.,, and G. J. Sarkis. 1993. DNA sequence, structure, and gene expression of mycobacterio-phage L5: a phage system for mycobacterial genetics. Mol. Microbiol. 7:395405.
30. Hermans, J.,, C. Martin,, G. N. M. Huijberts,, T. Goosen,, and J. A. M. de Bont. 1991. Transformation of Mycobacterium aurum and Mycobacterium smegmatis with the broad host-range gram-negative cosmid vector pJRD215. Mol. Microbiol. 5:15611566.
31. Hull, S. I.,, R. J. WaUaceJr.,, D. G. Bobey,, K. E. Price,, R. A. Goodhines,, J. M. Swenon,, and V. A. Silcox. 1984. Presence of aminoglycoside acetyltransferase and plasmids in Mycobacterium fortuitum. Am. Rev. Respir. Dis. 129:614618.
32. Jacobs, W. R.Jr.,, R. G. Barletta,, R. Udani,, J. Chan,, G. Kalkut,, G. Sosne,, T. Keiser,, G. J. Sartas,, G. F. Hatfull,, and B. R. Bloom. 1993. Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science 260:819822.
33. Jacobs, W. R.Jr.,, G. V. Kalpana,, J. D. Cirillo,, L. Pascopella,, S. B. Snapper,, R. A. Udani,, W. Jones,, R. G. Barletta,, and B. R. Bloom. 1991. Genetic systems for mycobacteria. Methods Enzymol. 204: 537555.
34. Jacobs, W. R.Jr.,, M. Tuckman,, and B. R. Bloom. 1987. Introduction of foreign DNA into mycobacteria using a shuttle phasmid. Nature (London) 327: 532535.
35. Jenkins, M. C.,, and H. S. Lillehoj. Personal communication.
36. Kalpana, G. V.,, B. R. Bloom,, and W. R. Jacobs Jr.. 1991. Insertional mutagenesis and illegitimate recombination in mycobacteria. Proc. NatL Acad. Sci. USA 88:54335437.
37. Labidi, A.,, H. L. David,, and D. Roulland-Dussoix. 1985. Cloning and expression of mycobacterial plasmid DNA in Escherichia coli. FEMS Microbiol. Lett. 30:221225.
38. Lanar, D.. Unpublished data.
39. Lathigra, R., et al. Unpublished data.
40. Lazraq, R.,, S. Clavel-Seres,, and H. L. David. 1991. Transformation of distinct mycobacterial species by shuttle vectors derived from the Mycobacterium fortuitum pAL5000 plasmid. Curr. Microbiol. 22:913.
41. Lazraq, R.,, S. Clavel-Seres,, H. L. David,, and D. Roulland-Dussoix. 1990. Conjugative transfer of a shuttle plasmid for Escherichia coli to Mycobacterium smegmatis. FEMS Microbiol. Lett. 69:135138.
42. Lee, C.,, P. Li,, H. Inouye,, E. Brickman,, and J. Beckwith. 1989. Genetic studies on the inability of β-ga-lactosidase to be translocated across the Escherichia coli cytoplasmic membrane. J. Bacterial. 171:46094616.
43. Lee, M. H.,, L. Pascopella,, W. R. JacobsJr.,, and G. F. Hatfull. 1991. Site-specific integration of mycobac-teriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis and bacille Calmette-Guerin. Proc. Natl. Acad. Sei. USA 88:31113115.
44. Martin, C.,, J. Timm,, J. Rauzier,, R. Gomez-Lus,, J. Davies,, and B. Gicquel. 1990. Transposition of an antibiotic resistance element in mycobacteria. Nature (London) 345:739743.
45. Matsuo, K.,, R. Yamaguchi,, A. Yamazata,, H. Tasaka,, K. Terasaka,, M. Totsuka,, K. Kobayashi,, H. Yukitake,, and T. Yamada. 1990a. Establishment of a foreign secretion system in mycobacteria. Infect. Immun. 58:40494054.
46. Matsuo, K.,, R. Yamaguchi,, A. Yamazaki,, H. Tasaka,, K. Terasaka,, and T. Yamada. 1990b. Cloning and expression of the gene for the cross-reactive a antigen of Mycobacterium kansasii. Infect. Immun. 58:550556.
47. Mehra, V.,, D. Sweetser,, and R. A. Young. 1986. Efficient mapping of protein antigen determinants. Proc. Natl. Acad. Sci. USA 83:70137017.
48. Meissner, P. S.,, and J. O. Falkingham. 1984. Plasmid-encoded mercuric reductase in Mycobacterium scrofulaceum. J. Bacterial. 157:669672.
49. Miller, J. F.,, C. R. Roy,, and S. Falkow. 1989. Analysis of Bordetella pertussis virulence gene regulation by use of transcriptional fusions in Escherichia coli. J. Bacteriol 171:63456348.
50. Murray, A.,, N. Winter,, M. Lagranderie,, D. F. Hill,, J. Rauzier,, J. Timm,, C. Leclerc,, K. M. Moriarty,, M. Gheorghiu,, and B. Gicquel. 1992. Expression of Escherichia coli β-galactosidase in Mycobacterium bovis BCG using an expression system from Mycobacterium paratuberculosis which induced humoral and cellular immune responses. Mol. Microbiol. 6:33313342.
51. Nair, J.,, D. A. Rouse,, and S. L. Morris. 1992. Nucleotide sequence analysis and serologic characterization of the Mycobacterium intracelhilare homologue of the Mycobacterium tuberculosis 19 kDa antigen. Mol Microbiol. 6:14311439.
52. Nakayama, K.,, S. M. Kelly,, and R. Curtiss IH. 1988. Construction of an Asd+ expression-cloning vector: stable maintenance and high level expression of cloned genes in a Salmonella vaccine vector strain. Bio/Technology 6:693697.
53. Osborne, T. W. 1983. Changes in BCG strains. Tubercle 64:1132.
54. Park, S. F.,, G. S. A. B. Stewart,, and R. G. Kroll. 1992. The use of bacterial luciferase for monitoring the environmental regulation of expression of genes encoding virulence factors in Listeria monocytogenes. /. Gen. Microbiol. 138:26192627.
55. Pearce, E. J.,, S. L. James,, S. Hierny,, D. E. Lanar,, and A. Sher. 1988. Induction of protective immunity against Schistosoma mansoni by vaccination with schistosome paramyosin (Sm97), a nonsurface parasite antigen. Proc. Natl. Acad. Sei. USA 85:56795682.
56. Radford, A. J.,, and A. L. M. Hodgson. 1991. Construction and characterization of a mycobacterium-Escherichia coli shuttle vector. Plasmid 25:149153.
57. Ranes, M. G.,, J. Rauzier,, M. Lagranderie,, M. Gheorghiu,, and B. Gicquel. 1990. Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a "mini" mycobacterium-Escherichia coli shuttle vector. J. Bacteriol 172:27932797.
58. Schein, C. 1989. Production of soluble recombinant proteins in bacteria. Bio/Technology 7:11411149.
59. Shinnick, T. M. 1987. The 65-kilodalton antigen of Mycobacterium tuberculosis. J. Bacteriol. 169: 10801088.
60. Snapper, S. B.,, L. Lugosi,, A. Jekkel,, R. Melton,, T. Kieser,, B. R. Bloom,, and W. R. JacobsJr.. 1988. Lysogeny and transformation of mycobacteria: stable expression of foreign genes. Proc. Natl. Acad. Sci. USA 85:69876991.
61. Stover, C. K.,, G. P. Bansal,, M. S. Hanson,, J. E. Burlein,, S. R. Palaszynski,, J. F. Young,, S. Koenig,, D. B. Young,, A. Sadziene,, and A. Barbour. 1993a. Protective immunity elicited by a recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease vaccine. J. Exp. Med. 178:197209.
62. Stover, C. K.,, V. F. de la Cruz,, T. R. Fuerst,, J. E. Burlein,, L. A. Benson,, L. T. Bennett,, G. P. Bansal,, J. F. Young,, M. H. Lee,, G. F. Hatfull,, S. B. Snapper,, R. G. Barletta,, W. R. JacobsJr.,, and B. R. Bloom. 1991. New use of BCG for recombinant vaccines. Nature (London) 351:458460.
63. Stover, C. K.,, S. Langermann,, M. S. Hanson,, J. E. Burlein,, S. R. Palaszynski,, S. Koenig,, L. S. McDaniel,, and D. E. Briles. 1993b. Candidate pneumococcal vaccines based on the recombinant BCG vaccine vehicle, abstr. E-79. Abstr. 93rd Gen. Meet. Am. Soc. Microbiol. 1993.
64. Thole, J. E.,, H. G. Dauwerse,, P. K. Das,, D. G. Groothuis,, L. M. Schouls,, and J. D. A. van Embden. 1985. Cloning of Mycobacterium bovis DNA and expression of antigens in Escherichia coli. Infect. Immun. 50:800806.
65. Thole, J. E. R.,, R. Schoningh,, A. A. M. Janson,, T. Garbe,, Y. E. Cornelisse,, J. E. Clark-Curtiss,, A. H. J. Kolk,, T. H. M. Ottenhoff,, R. R. R. De Vries,, and C. Abou-Zeid. 1992. Molecular and immunological analysis of a fibronectin-binding protein antigen secreted by Mycobacterium leprae. Mol. Microbiol. 6:153163.
66. Wada, K.,, Y. Wada,, F. Ishibashi,, T. Gojobori,, and T. Ikemura. 1992. Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Res. 20:S2111S2118.
67. Winter, N.,, M. Lagranderie,, J. Rauzier,, J. Timm,, C. Leclerc,, B. Guy,, M. P. Kieny,, M. Gheorghiu,, and B. Gicquel. 1991. Expression of heterologous genes in Mycobacterium bovis BCG: induction of a cellular response against HIV-1 Nef protein. Gene 109:4754.
68. Yasutomi, Y.,, S. Koenig,, S. S. Haun,, C. K. Stover,, R. K. Jackson,, P. Conrad,, A. J. Conley,, E. A. Emini,, T. R. Fuerst,, and N. L. Letvin. 1993. Immunization with recombinant BCG-SIV elicits SIV-specific cytotoxic T lymphocytes in rhesus monkeys. J. Immunol. 150:31013107.
69. Young, D. B.,, and T. R. Garbe. 1991. Lipoprotein antigens of Mycobacterium tuberculosis. Res. Microbiol. 142:5565.
70. Young, R. A.,, B. R. Bloom,, C. M. Grosskinsky,, J. Ivanyi,, D. Thomas,, and R. W. Davis. 1985a. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA. Proc. Natl. Acad. Sci. USA 82:25832587.
71. Young, R. A.,, V. Mehra,, D. Sweetser,, T. Buchanan,, J. Clark-Curtiss,, R. W. Davis,, and B. R. Bloom. 1985b. Genes for the major protein antigens of the leprosy parasite, Mycobacterium leprae. Nature (London) 316:450452.
72. Zhang, Y.,, B. Heym,, B. Allen,, D. Young,, and S. Cole. 1992. The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature (London) 358:591593.

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