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. 1997 Jul;65(7):2537–2541. doi: 10.1128/iai.65.7.2537-2541.1997

Increased intracellular survival of Mycobacterium smegmatis containing the Mycobacterium leprae thioredoxin-thioredoxin reductase gene.

B Wieles 1, T H Ottenhoff 1, T M Steenwijk 1, K L Franken 1, R R de Vries 1, J A Langermans 1
PMCID: PMC175358  PMID: 9199416

Abstract

The thioredoxin (Trx) system of Mycobacterium leprae is expressed as a single hybrid protein containing thioredoxin reductase (TR) at its N terminus and Trx at its C terminus. This hybrid Trx system is unique to M. leprae, since in all other organisms studied to date, including other mycobacteria, both TR and Trx are expressed as two separate proteins. Because Trx has been shown to scavenge reactive oxygen species, we have investigated whether the TR-Trx gene product can inhibit oxygen-dependent killing of mycobacteria by human mononuclear phagocytes and as such could contribute to mycobacterial virulence. The gene encoding M. leprae TR-Trx was cloned into the apathogenic, fast-growing bacterium Mycobacterium smegmatis. Recombinant M. smegmatis containing the gene encoding TR-Trx was killed to a significantly lesser extent than M. smegmatis containing the identical vector with either no insert or a control M. leprae construct unrelated to TR-Trx. Upon phagocytosis, M. smegmatis was shown to be killed predominantly by oxygen-dependent macrophage-killing mechanisms. Coinfection of M. smegmatis expressing the gene encoding TR-Trx together with Staphylococcus aureus, which is known to be killed via oxygen-dependent microbicidal mechanisms, revealed that the TR-Trx gene product interferes with the intracellular killing of this bacterium. A similar coinfection with Streptococcus pyogenes, known to be killed by oxygen-independent mechanisms, showed that the TR-Trx gene product did not influence the oxygen-independent killing pathway. The data obtained in this study suggest that the Trx system of M. leprae can inhibit oxygen-dependent killing of intracellular bacteria and thus may represent one of the mechanisms by which M. leprae can deal with oxidative stress within human mononuclear phagocytes.

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Selected References

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  1. Buchanan B. B., Schürmann P., Decottignies P., Lozano R. M. Thioredoxin: a multifunctional regulatory protein with a bright future in technology and medicine. Arch Biochem Biophys. 1994 Nov 1;314(2):257–260. doi: 10.1006/abbi.1994.1439. [DOI] [PubMed] [Google Scholar]
  2. Drevets D. A., Campbell P. A. Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria. J Immunol Methods. 1991 Aug 28;142(1):31–38. doi: 10.1016/0022-1759(91)90289-r. [DOI] [PubMed] [Google Scholar]
  3. Fernando M. R., Nanri H., Yoshitake S., Nagata-Kuno K., Minakami S. Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells. Eur J Biochem. 1992 Nov 1;209(3):917–922. doi: 10.1111/j.1432-1033.1992.tb17363.x. [DOI] [PubMed] [Google Scholar]
  4. Garbe T. R., Barathi J., Barnini S., Zhang Y., Abou-Zeid C., Tang D., Mukherjee R., Young D. B. Transformation of mycobacterial species using hygromycin resistance as selectable marker. Microbiology. 1994 Jan;140(Pt 1):133–138. doi: 10.1099/13500872-140-1-133. [DOI] [PubMed] [Google Scholar]
  5. Geertsma M. F., Broos H. R., van den Barselaar M. T., Nibbering P. H., van Furth R. Lung surfactant suppresses oxygen-dependent bactericidal functions of human blood monocytes by inhibiting the assembly of the NADPH oxidase. J Immunol. 1993 Mar 15;150(6):2391–2400. [PubMed] [Google Scholar]
  6. Geertsma M. F., Nibbering P. H., Pos O., Van Furth R. Interferon-gamma-activated human granulocytes kill ingested Mycobacterium fortuitum more efficiently than normal granulocytes. Eur J Immunol. 1990 Apr;20(4):869–873. doi: 10.1002/eji.1830200423. [DOI] [PubMed] [Google Scholar]
  7. Gleason F. K., Holmgren A. Thioredoxin and related proteins in procaryotes. FEMS Microbiol Rev. 1988 Dec;4(4):271–297. doi: 10.1111/j.1574-6968.1988.tb02747.x. [DOI] [PubMed] [Google Scholar]
  8. Hancock J. T., Jones O. T. The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages. Biochem J. 1987 Feb 15;242(1):103–107. doi: 10.1042/bj2420103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Harboe M., Closs O., Bjorvatn B., Kronvall G., Axelsen N. H. Antibody response in rabbits to immunization with Mycobacterium leprae. Infect Immun. 1977 Dec;18(3):792–805. doi: 10.1128/iai.18.3.792-805.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Klebanoff S. J., Shepard C. C. Toxic effect of the peroxidase-hydrogen peroxide-halide antimicrobial system on Mycobacterium leprae. Infect Immun. 1984 May;44(2):534–536. doi: 10.1128/iai.44.2.534-536.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Leijh P. C., Nathan C. F., van den Barselaar M. T., van Furth R. Relationship between extracellular stimulation of intracellular killing and oxygen-dependent microbicidal systems of monocytes. Infect Immun. 1985 Feb;47(2):502–507. doi: 10.1128/iai.47.2.502-507.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lygren S. T., Closs O., Bercouvier H., Wayne L. G. Catalases, peroxidases, and superoxide dismutases in Mycobacterium leprae and other mycobacteria studied by crossed immunoelectrophoresis and polyacrylamide gel electrophoresis. Infect Immun. 1986 Dec;54(3):666–672. doi: 10.1128/iai.54.3.666-672.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mitsui A., Hirakawa T., Yodoi J. Reactive oxygen-reducing and protein-refolding activities of adult T cell leukemia-derived factor/human thioredoxin. Biochem Biophys Res Commun. 1992 Aug 14;186(3):1220–1226. doi: 10.1016/s0006-291x(05)81536-0. [DOI] [PubMed] [Google Scholar]
  15. Mundayoor S., Shinnick T. M. Identification of genes involved in the resistance of mycobacteria to killing by macrophages. Ann N Y Acad Sci. 1994 Aug 15;730:26–36. doi: 10.1111/j.1749-6632.1994.tb44236.x. [DOI] [PubMed] [Google Scholar]
  16. Nathan C., Xie Q. W. Nitric oxide synthases: roles, tolls, and controls. Cell. 1994 Sep 23;78(6):915–918. doi: 10.1016/0092-8674(94)90266-6. [DOI] [PubMed] [Google Scholar]
  17. Neill M. A., Klebanoff S. J. The effect of phenolic glycolipid-1 from Mycobacterium leprae on the antimicrobial activity of human macrophages. J Exp Med. 1988 Jan 1;167(1):30–42. doi: 10.1084/jem.167.1.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Shinnick T. M., King C. H., Quinn F. D. Molecular biology, virulence, and pathogenicity of mycobacteria. Am J Med Sci. 1995 Feb;309(2):92–98. doi: 10.1097/00000441-199502000-00007. [DOI] [PubMed] [Google Scholar]
  19. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R., Jr Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol. 1990 Nov;4(11):1911–1919. doi: 10.1111/j.1365-2958.1990.tb02040.x. [DOI] [PubMed] [Google Scholar]
  20. Stuehr D. J., Fasehun O. A., Kwon N. S., Gross S. S., Gonzalez J. A., Levi R., Nathan C. F. Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs. FASEB J. 1991 Jan;5(1):98–103. doi: 10.1096/fasebj.5.1.1703974. [DOI] [PubMed] [Google Scholar]
  21. Wieles B., Nagai S., Wiker H. G., Harboe M., Ottenhoff T. H. Identification and functional characterization of thioredoxin of Mycobacterium tuberculosis. Infect Immun. 1995 Dec;63(12):4946–4948. doi: 10.1128/iai.63.12.4946-4948.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wieles B., van Agterveld M., Janson A., Clark-Curtiss J., Rinke de Wit T., Harboe M., Thole J. Characterization of a Mycobacterium leprae antigen related to the secreted Mycobacterium tuberculosis protein MPT32. Infect Immun. 1994 Jan;62(1):252–258. doi: 10.1128/iai.62.1.252-258.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wieles B., van Noort J., Drijfhout J. W., Offringa R., Holmgren A., Ottenhoff T. H. Purification and functional analysis of the Mycobacterium leprae thioredoxin/thioredoxin reductase hybrid protein. J Biol Chem. 1995 Oct 27;270(43):25604–25606. doi: 10.1074/jbc.270.43.25604. [DOI] [PubMed] [Google Scholar]
  24. Wieles B., van Soolingen D., Holmgren A., Offringa R., Ottenhoff T., Thole J. Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae. Mol Microbiol. 1995 Jun;16(5):921–929. doi: 10.1111/j.1365-2958.1995.tb02318.x. [DOI] [PubMed] [Google Scholar]
  25. Zhang Y., Lathigra R., Garbe T., Catty D., Young D. Genetic analysis of superoxide dismutase, the 23 kilodalton antigen of Mycobacterium tuberculosis. Mol Microbiol. 1991 Feb;5(2):381–391. doi: 10.1111/j.1365-2958.1991.tb02120.x. [DOI] [PubMed] [Google Scholar]

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