1887

Abstract

To identify genes involved in the intracellular survival of we compared the transcriptomes of virulent (H37Rv) and attenuated (H37Ra) strains during their interaction with murine bone-marrow-derived macrophages. Expression profiling was accomplished via the bacterial artificial chromosome fingerprint array (BACFA) technique. Genes identified with BACFA, and confirmed via qPCR to be upregulated in the attenuated H37Ra at 168 h post-infection, were , and . Genes upregulated in the virulent H37Rv were , and . Further qPCR analysis of these genes at 4 and 96 h post-infection revealed that the operon (encoding the fumarate reductase enzyme complex) is expressed at higher levels in the virulent H37Rv at earlier time points while the expression of and is higher in the virulent strain throughout the course of infection. Assessment of transcripts in oxygen-limited cultures of H37Ra and H37Rv showed that the attenuated strain displayed a lag in and expression at the onset of microaerophilic culture, when compared to microaerophilic cultures of H37Rv and aerated cultures of H37Ra. Lastly, treatment of intracellular bacteria with a putative inhibitor of fumarate reductase resulted in a significant reduction of bacterial growth.

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2008-08-01
2019-11-21
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References

  1. Bacon, J., James, B. W., Wernisch, L., Williams, A., Morley, K. A., Hatch, G. J., Mangan, J. A., Hinds, J., Stoker, N. G. & other authors ( 2004; ). The influence of reduced oxygen availability on pathogenicity and gene expression in Mycobacterium tuberculosis. Tuberculosis (Edinb) 84, 205–217.[CrossRef]
    [Google Scholar]
  2. Betts, J. C., Lukey, P. T., Robb, L. C., McAdam, R. A. & Duncan, K. ( 2002; ). Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol Microbiol 43, 717–731.[CrossRef]
    [Google Scholar]
  3. Bhargava, S., Tyagi, A. K. & Tyagi, J. S. ( 1990; ). tRNA genes in mycobacteria: organization and molecular cloning. J Bacteriol 172, 2930–2934.
    [Google Scholar]
  4. Birkholz, S., Knipp, U., Lemma, E., Kroger, A. & Opferkuch, W. ( 1994; ). Fumarate reductase of Helicobacter pylori – an immunogenic protein. J Med Microbiol 41, 56–62.[CrossRef]
    [Google Scholar]
  5. Brosch, R., Gordon, S. V., Billault, A., Garnier, T., Eiglmeier, K., Soravito, C., Barrell, B. G. & Cole, S. T. ( 1998; ). Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics. Infect Immun 66, 2221–2229.
    [Google Scholar]
  6. Brosch, R., Philipp, W. J., Stavropoulos, E., Colston, M. J., Cole, S. T. & Gordon, S. V. ( 1999; ). Genomic analysis reveals variation between Mycobacterium tuberculosis H37Rv and the attenuated M. tuberculosis H37Ra strain. Infect Immun 67, 5768–5774.
    [Google Scholar]
  7. Bryant, C. & Bennet, E. M. ( 1983; ). Observations on the fumarate reductase system in Haemonchus contortus and their relevance to anthelmintic resistance and to strain variations of energy metabolism. Mol Biochem Parasitol 7, 281–292.[CrossRef]
    [Google Scholar]
  8. Chen, M., Zhai, L., Christensen, S. B., Theander, T. G. & Kharazmi, A. ( 2001; ). Inhibition of fumarate reductase in Leishmania major and L. donovani by chalcones. Antimicrob Agents Chemother 45, 2023–2029.[CrossRef]
    [Google Scholar]
  9. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S. & other authors ( 1998; ). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[CrossRef]
    [Google Scholar]
  10. Collins, D. M. & De Lisle, G. W. ( 1984; ). DNA restriction endonuclease analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG. J Gen Microbiol 130, 1019–1021.
    [Google Scholar]
  11. Collins, F. M. & Smith, M. M. ( 1969; ). A comparative study of the virulence of Mycobacterium tuberculosis measured in mice and guinea pigs. Am Rev Respir Dis 100, 631–639.
    [Google Scholar]
  12. Dasgupta, N., Kapur, V., Singh, K. K., Das, T. K., Sachdeva, S., Jyothisri, K. & Tyagi, J. S. ( 2000; ). Characterization of a two-component system, devR-devS, of Mycobacterium tuberculosis. Tuber Lung Dis 80, 141–159.[CrossRef]
    [Google Scholar]
  13. Dolin, P. J., Raviglione, M. C. & Kochi, A. ( 1994; ). Global tuberculosis incidence and mortality during 1990–2000. Bull World Health Organ 72, 213–220.
    [Google Scholar]
  14. Gao, Q., Kripke, K., Arinc, Z., Voskuil, M. & Small, P. ( 2004; ). Comparative expression studies of a complex phenotype: cord formation in Mycobacterium tuberculosis. Tuberculosis (Edinb) 84, 188–196.[CrossRef]
    [Google Scholar]
  15. Ge, Z. ( 2002; ). Potential of fumarate reductase as a novel therapeutic target in Helicobacter pylori infection. Expert Opin Ther Targets 6, 135–146.[CrossRef]
    [Google Scholar]
  16. Ge, Z., Feng, Y., Dangler, C. A., Xu, S., Taylor, N. S. & Fox, J. G. ( 2000; ). Fumarate reductase is essential for Helicobacter pylori colonization of the mouse stomach. Microb Pathog 29, 279–287.[CrossRef]
    [Google Scholar]
  17. Gillespie, J., Barton, L. L. & Rypka, E. W. ( 1988; ). Influence of oxygen tension on the respiratory activity of Mycobacterium phlei. J Gen Microbiol 134, 247–252.
    [Google Scholar]
  18. Goldberg, I., Lonberg-Holm, K., Bagley, E. A. & Stieglitz, B. ( 1983; ). Improved conversion of fumarate to succinate by Escherichia coli strains amplified for fumarate reductase. Appl Environ Microbiol 45, 1838–1847.
    [Google Scholar]
  19. Gordon, S. V., Brosch, R., Billault, A., Garnier, T., Eiglmeier, K. & Cole, S. T. ( 1999a; ). Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol Microbiol 32, 643–655.[CrossRef]
    [Google Scholar]
  20. Gordon, S. V., Heym, B., Parkhill, J., Barrell, B. & Cole, S. T. ( 1999b; ). New insertion sequences and a novel repeated sequence in the genome of Mycobacterium tuberculosis H37Rv. Microbiology 145, 881–892.[CrossRef]
    [Google Scholar]
  21. Graham, J. E. & Clark-Curtiss, J. E. ( 1999; ). Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc Natl Acad Sci U S A 96, 11554–11559.[CrossRef]
    [Google Scholar]
  22. Guest, J. R. ( 1981; ). Partial replacement of succinate dehydrogenase function by phage- and plasmid-specified fumarate reductase in Escherichia coli. J Gen Microbiol 122, 171–179.
    [Google Scholar]
  23. Guinn, K. M., Hickey, M. J., Mathur, S. K., Zakel, K. L., Grotzke, J. E., Lewinsohn, D. M., Smith, S. & Sherman, D. R. ( 2004; ). Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol Microbiol 51, 359–370.[CrossRef]
    [Google Scholar]
  24. Heplar, J. Q., Clifton, C. E., Raffel, S. & Futrelle, C. M. ( 1954; ). Virulence of the tubercle bacillus. I. Effect of oxygen tension upon respiration of virulent and avirulent bacilli. J Infect Dis 94, 90–98.[CrossRef]
    [Google Scholar]
  25. Imaeda, T. ( 1985; ). Deoxyribonucleic acid relatedness among strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium microti and Mycobacterium africanum. Int J Syst Bacteriol 35, 147–150.[CrossRef]
    [Google Scholar]
  26. Jannasch, H. W. ( 1969; ). Estimations of bacterial growth rates in natural waters. J Bacteriol 99, 156–160.
    [Google Scholar]
  27. Jung, Y. J., LaCourse, R., Ryan, L. & North, R. J. ( 2002; ). Virulent but not avirulent Mycobacterium tuberculosis can evade the growth inhibitory action of a T helper 1-dependent, nitric oxide synthase 2-independent defense in mice. J Exp Med 196, 991–998.[CrossRef]
    [Google Scholar]
  28. Kinger, A. K. & Tyagi, J. S. ( 1993; ). Identification and cloning of genes differentially expressed in the virulent strain of Mycobacterium tuberculosis. Gene 131, 113–117.[CrossRef]
    [Google Scholar]
  29. Lari, N., Rindi, L. & Garzelli, C. ( 2001; ). Identification of one insertion site of IS6110 in Mycobacterium tuberculosis H37Ra and analysis of the RvD2 deletion in M. tuberculosis clinical isolates. J Med Microbiol 50, 805–811.
    [Google Scholar]
  30. Li, M. S., Monahan, I. M., Waddell, S. J., Mangan, J. A., Martin, S. L., Everett, M. J. & Butcher, P. D. ( 2001; ). cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG. Microbiology 147, 2293–2305.
    [Google Scholar]
  31. Madigan, M. T., Martinko, J. M. & Parker, J. ( 1997a; ). Nutrition and metabolism. In Brock Biology of Microorganisms, 8th edn, pp. 111–150. Upper Saddle River, NJ: Prentice-Hall.
  32. Madigan, M. T., Martinko, J. M. & Parker, J. ( 1997b; ). Microbial growth. In Brock Biology of Microorganisms, 8th edn, pp. 151–179. Upper Saddle River, NJ: Prentice-Hall.
  33. Mangan, J. A. & Butcher, P. D. ( 1998; ). Analysis of mycobacterial differential gene expression by RAP-PCR. Methods Mol Biol 101, 307–322.
    [Google Scholar]
  34. Mangan, J. A., Sole, K. M., Mitchison, D. A. & Butcher, P. D. ( 1997; ). An effective method of RNA extraction from bacteria refractory to disruption, including mycobacteria. Nucleic Acids Res 25, 675–676.[CrossRef]
    [Google Scholar]
  35. Menendez Mdel, C., Rebollo, M. J., Nunez Mdel, C., Cox, R. A. & Garcia, M. J. ( 2005; ). Analysis of the precursor rRNA fractions of rapidly growing mycobacteria: quantification by methods that include the use of a promoter (rrnA P1) as a novel standard. J Bacteriol 187, 534–543.[CrossRef]
    [Google Scholar]
  36. Monahan, I. M., Betts, J., Banerjee, D. K. & Butcher, P. D. ( 2001; ). Differential expression of mycobacterial proteins following phagocytosis by macrophages. Microbiology 147, 459–471.
    [Google Scholar]
  37. Monod, J. ( 1949; ). The growth of bacterial cultures. Annu Rev Microbiol 3, 371–394.[CrossRef]
    [Google Scholar]
  38. Mostowy, S., Cleto, C., Sherman, D. R. & Behr, M. A. ( 2004; ). The Mycobacterium tuberculosis complex transcriptome of attenuation. Tuberculosis (Edinb) 84, 197–204.[CrossRef]
    [Google Scholar]
  39. Narain, J. P., Raviglione, M. C. & Kochi, A. ( 1992; ). HIV-associated tuberculosis in developing countries: epidemiology and strategies for prevention. Tuber Lung Dis 73, 311–321.[CrossRef]
    [Google Scholar]
  40. Oatway, W. H. & Steenken, W ( 1936; ). The pathogenesis and fate of tubercle produced by dissociate variants of tubercle bacilli. J Infect Dis 59, 306–325.[CrossRef]
    [Google Scholar]
  41. Omura, S., Miyadera, H., Ui, H., Shiomi, K., Yamaguchi, Y., Masama, R., Nagamitsu, T., Takano, D., Sunazuka, T. & other authors ( 2001; ). An anthelmintic compound, nafuredin, shows selective inhibition of complex I in helminth mitochondria. Proc Natl Acad Sci U S A 98, 60–62.[CrossRef]
    [Google Scholar]
  42. Park, H. D., Guinn, K. M., Harrell, M. I., Liao, R., Voskuil, M. I., Tompa, M., Schoolnik, G. K. & Sherman, D. R. ( 2003; ). Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol Microbiol 48, 833–843.[CrossRef]
    [Google Scholar]
  43. Phillips, M. K., Hederstedt, L., Hasnain, S., Rutberg, L. & Guest, J. R. ( 1987; ). Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex. J Bacteriol 169, 864–873.
    [Google Scholar]
  44. Prichard, R. K. ( 1973; ). The fumarate reductase reaction of Haemonchus contortus and the mode of action of some anthelmintics. Int J Parasitol 3, 409–417.[CrossRef]
    [Google Scholar]
  45. Rahn, O. ( 1930; ). The non-logarithmic order of death of some bacteria. J Gen Physiol 13, 395–407.[CrossRef]
    [Google Scholar]
  46. Ramakrishnan, T., Indira, M. & Maller, R. K. ( 1962; ). Evaluation of the routes of glucose utilization in virulent and avirulent strains of Mycobacterium tuberculosis. Biochim Biophys Acta 59, 529–532.[CrossRef]
    [Google Scholar]
  47. Raman, S., Song, T., Puyang, X., Bardarov, S., Jacobs, W. R., Jr & Husson, R. N. ( 2001; ). The alternative sigma factor SigH regulates major components of oxidative and heat stress responses in Mycobacterium tuberculosis. J Bacteriol 183, 6119–6125.[CrossRef]
    [Google Scholar]
  48. Rengarajan, J., Bloom, B. R. & Rubin, E. J. ( 2005; ). Genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages. Proc Natl Acad Sci U S A 102, 8327–8332.[CrossRef]
    [Google Scholar]
  49. Rindi, L., Lari, N. & Garzelli, C. ( 1999; ). Search for genes potentially involved in Mycobacterium tuberculosis virulence by mRNA differential display. Biochem Biophys Res Commun 258, 94–101.[CrossRef]
    [Google Scholar]
  50. Rindi, L., Lari, N. & Garzelli, C. ( 2001; ). Genes of Mycobacterium tuberculosis H37Rv downregulated in the attenuated strain H37Ra are restricted to M. tuberculosis complex species. New Microbiol 24, 289–294.
    [Google Scholar]
  51. Rivera-Marrero, C. A., Burroughs, M. A., Masse, R. A., Vannberg, F. O., Leimbach, D. L., Roman, J. & Murtagh, J. J., Jr ( 1998; ). Identification of genes differentially expressed in Mycobacterium tuberculosis by differential display PCR. Microb Pathog 25, 307–316.[CrossRef]
    [Google Scholar]
  52. Rooyakkers, A. W. & Stokes, R. W. ( 2005; ). Absence of complement receptor 3 results in reduced binding and ingestion of Mycobacterium tuberculosis but has no significant effect on the induction of reactive oxygen and nitrogen intermediates or on the survival of the bacteria in resident and interferon-gamma activated macrophages. Microb Pathog 39, 57–67.[CrossRef]
    [Google Scholar]
  53. Saini, D. K., Malhotra, V., Dey, D., Pant, N., Das, T. K. & Tyagi, J. S. ( 2004; ). DevR-DevS is a bona fide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR. Microbiology 150, 865–875.[CrossRef]
    [Google Scholar]
  54. Sassetti, C. M. & Rubin, E. J. ( 2003; ). Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci U S A 100, 12989–12994.[CrossRef]
    [Google Scholar]
  55. Schmittgen, T. D., Zakrajsek, B. A., Mills, A. G., Gorn, V., Singer, M. J. & Reed, M. W. ( 2000; ). Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal Biochem 285, 194–204.[CrossRef]
    [Google Scholar]
  56. Schnappinger, D., Ehrt, S., Voskuil, M. I., Liu, Y., Mangan, J. A., Monahan, I. M., Dolganov, G., Efron, B., Butcher, P. D. & other authors ( 2003; ). Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment. J Exp Med 198, 693–704.[CrossRef]
    [Google Scholar]
  57. Selwyn, P. A., Hartel, D., Lewis, V. A., Schoenbaum, E. E., Vermund, S. H., Klein, R. S., Walker, A. T. & Friedland, G. H. ( 1989; ). A prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection. N Engl J Med 320, 545–550.[CrossRef]
    [Google Scholar]
  58. Sirakova, T. D., Thirumala, A. K., Dubey, V. S., Sprecher, H. & Kolattukudy, P. E. ( 2001; ). The Mycobacterium tuberculosis pks2 gene encodes the synthase for the hepta- and octamethyl-branched fatty acids required for sulfolipid synthesis. J Biol Chem 276, 16833–16839.[CrossRef]
    [Google Scholar]
  59. Steenken, W. & Gardner, L. U. ( 1946; ). History of H37 strain of tubercle bacillus. Am Rev Respir Dis 54, 62–66.
    [Google Scholar]
  60. Steenken, W., Jr, Oatway, W. H. & Petroff, S. A. ( 1934; ). Biological studies of the tubercle bacillus. III. Dissociation and pathogenicity of the R and S variants of the human tubercle bacillus (H37). J Exp Med 60, 515–540.[CrossRef]
    [Google Scholar]
  61. Stewart, G. R., Wernisch, L., Stabler, R., Mangan, J. A., Hinds, J., Laing, K. G., Young, D. B. & Butcher, P. D. ( 2002; ). Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148, 3129–3138.
    [Google Scholar]
  62. Stokes, R. W., Haidl, I. D., Jefferies, W. A. & Speert, D. P. ( 1993; ). Mycobacteria–macrophage interactions. Macrophage phenotype determines the nonopsonic binding of Mycobacterium tuberculosis to murine macrophages. J Immunol 151, 7067–7076.
    [Google Scholar]
  63. Talaat, A. M., Hunter, P. & Johnston, S. A. ( 2000; ). Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis. Nat Biotechnol 18, 679–682.[CrossRef]
    [Google Scholar]
  64. Turrens, J. F., Watts, B. P., Jr, Zhong, L. & Docampo, R. ( 1996; ). Inhibition of Trypanosoma cruzi and T. brucei NADH fumarate reductase by benznidazole and anthelmintic imidazole derivatives. Mol Biochem Parasitol 82, 125–129.[CrossRef]
    [Google Scholar]
  65. Turrens, J. F., Newton, C. L., Zhong, L., Hernandez, F. R., Whitfield, J. & Docampo, R. ( 1999; ). Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts. FEMS Microbiol Lett 175, 217–221.[CrossRef]
    [Google Scholar]
  66. Van Hellemond, J. J. & Tielens, A. G. ( 1994; ). Expression and functional properties of fumarate reductase. Biochem J 304, 321–331.
    [Google Scholar]
  67. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. & Speleman, F. ( 2002; ). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3, RESEARCH0034
    [Google Scholar]
  68. Waddell, S. J., Chung, G. A., Gibson, K. J., Everett, M. J., Minnikin, D. E., Besra, G. S. & Butcher, P. D. ( 2005; ). Inactivation of polyketide synthase and related genes results in the loss of complex lipids in Mycobacterium tuberculosis H37Rv. Lett Appl Microbiol 40, 201–206.[CrossRef]
    [Google Scholar]
  69. Wang, S., Liu, F. & Zhao, B. ( 2000; ). Study on mRNA differential display in Mycobacterium tuberculosis H37Rv and H37Ra. Zhonghua Jie He He Hu Xi Za Zhi 23, 669–671.
    [Google Scholar]
  70. Wang, S. Y., Zheng, H. J., Wang, B. F., Zhang, X. L., Pu, S. Y., Zhu, G. F. & Zhao, G. P. ( 2007; ). Complete genomic sequence of Mycobacterium tuberculosis strain H37Ra, a non-pathogenic variant closely related to the well-characterized pathogenic strain H37Rv. GenBank Accession Number: CP000611, http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=148503909.
  71. Wayne, L. G. ( 1976; ). Dynamics of submerged growth of Mycobacterium tuberculosis under aerobic and microaerophilic conditions. Am Rev Respir Dis 114, 807–811.
    [Google Scholar]
  72. Wei, J., Dahl, J. L., Moulder, J. W., Roberts, E. A., O'Gaora, P., Young, D. B. & Friedman, R. L. ( 2000; ). Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages. J Bacteriol 182, 377–384.[CrossRef]
    [Google Scholar]
  73. Wernisch, L., Kendall, S. L., Soneji, S., Wietzorrek, A., Parish, T., Hinds, J., Butcher, P. D. & Stoker, N. G. ( 2003; ). Analysis of whole-genome microarray replicates using mixed models. Bioinformatics 19, 53–61.[CrossRef]
    [Google Scholar]
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