Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Apr;65(4):1395–1401. doi: 10.1128/iai.65.4.1395-1401.1997

Effects of overexpression of the alkyl hydroperoxide reductase AhpC on the virulence and isoniazid resistance of Mycobacterium tuberculosis.

B Heym 1, E Stavropoulos 1, N Honoré 1, P Domenech 1, B Saint-Joanis 1, T M Wilson 1, D M Collins 1, M J Colston 1, S T Cole 1
PMCID: PMC175145  PMID: 9119479

Abstract

Mutations to the regulatory region of the ahpC gene, resulting in overproduction of alkyl hydroperoxide reductase, were encountered frequently in a large collection of isoniazid (INH)-resistant clinical isolates of Mycobacterium tuberculosis but not in INH-susceptible strains. Overexpression of ahpC did not seem to be important for INH resistance, however, as most of these strains were already defective for catalase-peroxidase, KatG, the enzyme required for activation of INH. Transformation of the INH-susceptible reference strain, M. tuberculosis H37Rv, with plasmids bearing the ahpC genes of M. tuberculosis or M. leprae did not result in a significant increase in the MIC. Two highly INH-resistant mutants of H37Rv, BH3 and BH8, were isolated in vitro and shown to produce no or little KatG activity and, in the case of BH3, to overproduce alkyl hydroperoxide reductase as the result of an ahpC regulatory mutation that was also found in some clinical isolates. The virulence of H37Rv, BH3, and BH8 was studied intensively in three mouse models: fully immunocompetent BALB/c and Black 6 mice, BALB/c major histocompatibility complex class II-knockout mice with abnormally low levels of CD4 T cells and athymic mice producing no cellular immune response. The results indicated that M. tuberculosis strains producing catalase-peroxidase were considerably more virulent in immunocompetent mice than the isogenic KatG-deficient mutants but that loss of catalase-peroxidase was less important when immunodeficient mice, unable to produce activated macrophages, were infected. Restoration of virulence was not seen in an INH-resistant M. tuberculosis strain that overexpressed ahpC, and this finding was confirmed by experiments performed with appropriate M. bovis strains in guinea pigs. Thus, in contrast to catalase-peroxidase, alkyl hydroperoxide reductase does not appear to act as a virulence factor in rodent infections or to play a direct role in INH resistance, although it may be important in maintaining peroxide homeostasis of the organism when KatG activity is low or absent.

Full Text

The Full Text of this article is available as a PDF (327.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Banerjee A., Dubnau E., Quemard A., Balasubramanian V., Um K. S., Wilson T., Collins D., de Lisle G., Jacobs W. R., Jr inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science. 1994 Jan 14;263(5144):227–230. doi: 10.1126/science.8284673. [DOI] [PubMed] [Google Scholar]
  2. Barletta R. G., Kim D. D., Snapper S. B., Bloom B. R., Jacobs W. R., Jr Identification of expression signals of the mycobacteriophages Bxb1, L1 and TM4 using the Escherichia-Mycobacterium shuttle plasmids pYUB75 and pYUB76 designed to create translational fusions to the lacZ gene. J Gen Microbiol. 1992 Jan;138(1):23–30. doi: 10.1099/00221287-138-1-23. [DOI] [PubMed] [Google Scholar]
  3. Cole S. T. Mycobacterium tuberculosis: drug-resistance mechanisms. Trends Microbiol. 1994 Oct;2(10):411–415. doi: 10.1016/0966-842x(94)90621-1. [DOI] [PubMed] [Google Scholar]
  4. Cosgrove D., Gray D., Dierich A., Kaufman J., Lemeur M., Benoist C., Mathis D. Mice lacking MHC class II molecules. Cell. 1991 Sep 6;66(5):1051–1066. doi: 10.1016/0092-8674(91)90448-8. [DOI] [PubMed] [Google Scholar]
  5. Deretic V., Philipp W., Dhandayuthapani S., Mudd M. H., Curcic R., Garbe T., Heym B., Via L. E., Cole S. T. Mycobacterium tuberculosis is a natural mutant with an inactivated oxidative-stress regulatory gene: implications for sensitivity to isoniazid. Mol Microbiol. 1995 Sep;17(5):889–900. doi: 10.1111/j.1365-2958.1995.mmi_17050889.x. [DOI] [PubMed] [Google Scholar]
  6. Dessen A., Quémard A., Blanchard J. S., Jacobs W. R., Jr, Sacchettini J. C. Crystal structure and function of the isoniazid target of Mycobacterium tuberculosis. Science. 1995 Mar 17;267(5204):1638–1641. doi: 10.1126/science.7886450. [DOI] [PubMed] [Google Scholar]
  7. Dhandayuthapani S., Zhang Y., Mudd M. H., Deretic V. Oxidative stress response and its role in sensitivity to isoniazid in mycobacteria: characterization and inducibility of ahpC by peroxides in Mycobacterium smegmatis and lack of expression in M. aurum and M. tuberculosis. J Bacteriol. 1996 Jun;178(12):3641–3649. doi: 10.1128/jb.178.12.3641-3649.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dooley S. W., Jarvis W. R., Martone W. J., Snider D. E., Jr Multidrug-resistant tuberculosis. Ann Intern Med. 1992 Aug 1;117(3):257–259. doi: 10.7326/0003-4819-117-3-257. [DOI] [PubMed] [Google Scholar]
  9. Farr S. B., Kogoma T. Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol Rev. 1991 Dec;55(4):561–585. doi: 10.1128/mr.55.4.561-585.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Heym B., Alzari P. M., Honoré N., Cole S. T. Missense mutations in the catalase-peroxidase gene, katG, are associated with isoniazid resistance in Mycobacterium tuberculosis. Mol Microbiol. 1995 Jan;15(2):235–245. doi: 10.1111/j.1365-2958.1995.tb02238.x. [DOI] [PubMed] [Google Scholar]
  11. Heym B., Cole S. T. Isolation and characterization of isoniazid-resistant mutants of Mycobacterium smegmatis and M. aurum. Res Microbiol. 1992 Sep;143(7):721–730. doi: 10.1016/0923-2508(92)90067-x. [DOI] [PubMed] [Google Scholar]
  12. Heym B., Honoré N., Truffot-Pernot C., Banerjee A., Schurra C., Jacobs W. R., Jr, van Embden J. D., Grosset J. H., Cole S. T. Implications of multidrug resistance for the future of short-course chemotherapy of tuberculosis: a molecular study. Lancet. 1994 Jul 30;344(8918):293–298. doi: 10.1016/s0140-6736(94)91338-2. [DOI] [PubMed] [Google Scholar]
  13. Heym B., Zhang Y., Poulet S., Young D., Cole S. T. Characterization of the katG gene encoding a catalase-peroxidase required for the isoniazid susceptibility of Mycobacterium tuberculosis. J Bacteriol. 1993 Jul;175(13):4255–4259. doi: 10.1128/jb.175.13.4255-4259.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kapur V., Li L. L., Hamrick M. R., Plikaytis B. B., Shinnick T. M., Telenti A., Jacobs W. R., Jr, Banerjee A., Cole S., Yuen K. Y. Rapid Mycobacterium species assignment and unambiguous identification of mutations associated with antimicrobial resistance in Mycobacterium tuberculosis by automated DNA sequencing. Arch Pathol Lab Med. 1995 Feb;119(2):131–138. [PubMed] [Google Scholar]
  15. Levin M. E., Hatfull G. F. Mycobacterium smegmatis RNA polymerase: DNA supercoiling, action of rifampicin and mechanism of rifampicin resistance. Mol Microbiol. 1993 Apr;8(2):277–285. doi: 10.1111/j.1365-2958.1993.tb01572.x. [DOI] [PubMed] [Google Scholar]
  16. Loewen P. C., Switala J., Smolenski M., Triggs-Raine B. L. Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli. Biochem Cell Biol. 1990 Jul-Aug;68(7-8):1037–1044. doi: 10.1139/o90-153. [DOI] [PubMed] [Google Scholar]
  17. MIDDLEBROOK G., COHN M. L. Some observations on the pathogenicity of isoniazid-resistant variants of tubercle bacilli. Science. 1953 Sep 11;118(3063):297–299. doi: 10.1126/science.118.3063.297. [DOI] [PubMed] [Google Scholar]
  18. Morris S., Bai G. H., Suffys P., Portillo-Gomez L., Fairchok M., Rouse D. Molecular mechanisms of multiple drug resistance in clinical isolates of Mycobacterium tuberculosis. J Infect Dis. 1995 Apr;171(4):954–960. doi: 10.1093/infdis/171.4.954. [DOI] [PubMed] [Google Scholar]
  19. Musser J. M., Kapur V., Williams D. L., Kreiswirth B. N., van Soolingen D., van Embden J. D. Characterization of the catalase-peroxidase gene (katG) and inhA locus in isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of mutations associated with drug resistance. J Infect Dis. 1996 Jan;173(1):196–202. doi: 10.1093/infdis/173.1.196. [DOI] [PubMed] [Google Scholar]
  20. Nathan C. F., Root R. K. Hydrogen peroxide release from mouse peritoneal macrophages: dependence on sequential activation and triggering. J Exp Med. 1977 Dec 1;146(6):1648–1662. doi: 10.1084/jem.146.6.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ordway D. J., Sonnenberg M. G., Donahue S. A., Belisle J. T., Orme I. M. Drug-resistant strains of Mycobacterium tuberculosis exhibit a range of virulence for mice. Infect Immun. 1995 Feb;63(2):741–743. doi: 10.1128/iai.63.2.741-743.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Orme I. M. The kinetics of emergence and loss of mediator T lymphocytes acquired in response to infection with Mycobacterium tuberculosis. J Immunol. 1987 Jan 1;138(1):293–298. [PubMed] [Google Scholar]
  23. Rosner J. L., Storz G. Effects of peroxides on susceptibilities of Escherichia coli and Mycobacterium smegmatis to isoniazid. Antimicrob Agents Chemother. 1994 Aug;38(8):1829–1833. doi: 10.1128/aac.38.8.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rosner J. L. Susceptibilities of oxyR regulon mutants of Escherichia coli and Salmonella typhimurium to isoniazid. Antimicrob Agents Chemother. 1993 Oct;37(10):2251–2253. doi: 10.1128/aac.37.10.2251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rouse D. A., Li Z., Bai G. H., Morris S. L. Characterization of the katG and inhA genes of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother. 1995 Nov;39(11):2472–2477. doi: 10.1128/aac.39.11.2472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sherman D. R., Mdluli K., Hickey M. J., Arain T. M., Morris S. L., Barry C. E., 3rd, Stover C. K. Compensatory ahpC gene expression in isoniazid-resistant Mycobacterium tuberculosis. Science. 1996 Jun 14;272(5268):1641–1643. doi: 10.1126/science.272.5268.1641. [DOI] [PubMed] [Google Scholar]
  27. Sherman D. R., Sabo P. J., Hickey M. J., Arain T. M., Mahairas G. G., Yuan Y., Barry C. E., 3rd, Stover C. K. Disparate responses to oxidative stress in saprophytic and pathogenic mycobacteria. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6625–6629. doi: 10.1073/pnas.92.14.6625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Storz G., Tartaglia L. A., Ames B. N. Transcriptional regulator of oxidative stress-inducible genes: direct activation by oxidation. Science. 1990 Apr 13;248(4952):189–194. doi: 10.1126/science.2183352. [DOI] [PubMed] [Google Scholar]
  30. Telenti A., Honoré N., Bernasconi C., March J., Ortega A., Heym B., Takiff H. E., Cole S. T. Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level. J Clin Microbiol. 1997 Mar;35(3):719–723. doi: 10.1128/jcm.35.3.719-723.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wilson T. M., Collins D. M. ahpC, a gene involved in isoniazid resistance of the Mycobacterium tuberculosis complex. Mol Microbiol. 1996 Mar;19(5):1025–1034. doi: 10.1046/j.1365-2958.1996.449980.x. [DOI] [PubMed] [Google Scholar]
  32. Wilson T. M., de Lisle G. W., Collins D. M. Effect of inhA and katG on isoniazid resistance and virulence of Mycobacterium bovis. Mol Microbiol. 1995 Mar;15(6):1009–1015. doi: 10.1111/j.1365-2958.1995.tb02276.x. [DOI] [PubMed] [Google Scholar]
  33. Zhang Y., Heym B., Allen B., Young D., Cole S. The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature. 1992 Aug 13;358(6387):591–593. doi: 10.1038/358591a0. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES