Skip to main content
Article thumbnail
Location of Repository

Molecular Features and Properties of Mycobacterial Proteins Linked to Tuberculosis Pathogenesis

By Dariush Ilghari


The Mycobacterium tuberculosis genome codes for 11 pairs of CFP-10/ESAT-6 proteins (Esx family) as well as the apparatus required for secretion of these proteins. The core machinery for the secretion of ESAT-6 and CFP-10 is encoded by their surrounding genes. Recent studies also identified a distant region, the Rv3612c-Rv3616c operon, which is essential for the CFP-10/ESAT-6 secretion. Constructs carrying Rv3613c, Rv3614c, Rv3615c and Rv3616c coding regions were produced and used to express the corresponding proteins. However, only Rv3614c and Rv3615c were expressed using an E. coli-based expression system. Analysis using a range of spectroscopic techniques on the purified proteins revealed that both Rv3615c and Rv3614c contain stable secondary structure, but little if any stable tertiary structure and exist in a molten globule-like state. This suggests the proteins probably undergo folding upon binding with possible functional partners. Yeast-two hybrid studies showed no intermolecular interaction between the proteins encoded by the Rv3616c-Rv3612c operon, perhaps suggesting the formation of a higher order multi-protein complex. \ud Together with CFP-10 and ESAT-6, Rv0287 and Rv0288 are the members of the Esx family which are clearly implicated in M. tuberculosis pathogenesis. The expression vectors carrying Rv0287 and Rv0288 coding regions were constructed and used to express the proteins. Analysis using a range of spectroscopic techniques on the purified proteins showed that Rv0288 contains up to 30 % helical secondary structure, but little if any stable tertiary structure and exists in a molten globule-like state. In contrast, Rv0287 has been found to form an unstructured, random coil polypeptide. The work reported here also shows that Rv0287 and Rv0288 form a tight 1:1 complex which is predominantly helical. Furthermore, the Rv0287-Rv0288 complex was found to be significantly more stable to thermal denaturation than CFP-10-ESAT-6. The high resolution solution structure reported here reveals that both proteins, Rv0287 and Rv0288, adopt an elongated helix-turn-helix hairpin structure in which the proteins lie antiparallel to each other, forming a stable four helix bundle. Comparison of the CFP-10-ESAT-6 and Rv0287-Rv0288 complexes also revealed that the overall backbone fold for the complexes is very similar although they display significantly different surface features

Publisher: University of Leicester
Year: 2009
OAI identifier:

Suggested articles


  1. (1989). 3-Dimensional heteronuclear NMR of N-15-labeled proteins
  2. (2007). A genetic screen for Mycobacterium tuberculosis mutants defective for phagosome maturation arrest identifies components of the ESX-1 secretion system.Infect Immun.
  3. (2005). A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor.
  4. (2005). A non-RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis.
  5. (1989). A novel genetic system to detect protein-protein interactions.
  6. (2007). A predicted operon map for Mycobacterium tuberculosis.Nucleic Acids Res.
  7. (1993). Amino-acid type determination in the sequential assignment procedure of uniformly C-13/N-15-enriched proteins.
  8. (2007). analysis of the Mycobacterium tuberculosis Zur (FurB) regulon.
  9. (2001). Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis. Evidence that this lipid is involved in the cell wall permeability barrier.
  10. (2006). Antigen-specific CD8+ T cells and the development of central memory during Mycobacterium tuberculosis infection.
  11. (2006). Antioxidant Dps protein from the thermophilic cyanobacterium Thermosynechococcus elongatus.
  12. B.D.,Young, D.B.(2003) Tuberculosis: a problem with persistence.
  13. (2006). C-terminal signal sequence promotes virulence factor secretion in Mycobacterium tuberculosis.
  14. (2003). CD8+-T-cell responses of Mycobacterium-infected mice to a newly identified major histocompatibility complex class I-restricted epitope shared by proteins of the ESAT-6 family.
  15. (2004). Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility.
  16. (2006). Characterization of the tuberculous granuloma in murine and human lungs: cellular composition and relative tissue oxygen tension.
  17. (1983). Coherence transfer by isotropic mixing application to proton correlation spectroscopy.
  18. (2004). Comparative analysis of different vaccine constructs expressing defined antigens from Mycobacterium tuberculosis.
  19. (2000). Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens. Infect Immun.
  20. (2003). Cutting edge: Mycobacterium tuberculosis blocks Ca 2+ signaling and phagosome maturation in human macrophages via specific inhibition of sphingosine kinase.J Immunol.
  21. (1995). Dali: a network tool for protein structure comparison. Trends Biochem Sci.
  22. (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.
  23. (2003). Deletion of RD1 from Mycobacterium tuberculosis mimics bacille CalmetteGuérin attenuation.
  24. (2002). Distribution of IFN-gamma, IL-4 and TNF-alpha protein and CD8 T cells producing IL-12p40 mRNA in human lung tuberculous granulomas.
  25. (2008). Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis.
  26. (1980). Elucidation of cross relaxation in liquids by twodimensional NMR-spectroscopy.
  27. (2002). Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, which constitute a subfamily of the esat-6 gene family.
  28. (2008). ESAT-6/CFP10 skin test predicts disease in M. tuberculosis-infected guinea pigs.
  29. (2002). Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling.
  30. (2005). Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine: efficient protection and ESAT6-based sensitive monitoring of vaccine efficacy.
  31. (2006). Field evaluation of a novel differential diagnostic reagent for detection of Mycobacterium bovis in cattle. Clin Vaccine Immunol.
  32. (2003). Genes required for mycobacterial growth defined by high density mutagenesis.
  33. (2003). Genes required for mycobacterial growth defined by high density mutagenesis.Mol.
  34. (2006). Global tuberculosis control-surveillance, planning, World Health Organization,
  35. (1992). Gradient-Tailored Excitation for Single-
  36. (1990). H-1-H-1 correlation via isotropic mixing of C-13 magnetization, a new 3-dimensional approach for assigning H-1 and C-13pectra of C-13-enriched proteins.
  37. (2007). Health Protection Agency.
  38. (2006). High frequency of CD4+ T cells specific for the TB10.4 protein correlates with protection against Mycobacterium tuberculosis infection.Infect Immun.
  39. (1993). HNCACB, a High-Sensitivity 3D Nmr Experiment to Correlate Amide-Proton and Nitrogen Resonances with the Alpha-Carbon and Beta-Carbon Resonances in Proteins.
  40. (2005). How to study proteins by circular dichroism.
  41. (2005). Human immune recognition-based multicomponent subunit vaccines against tuberculosis.
  42. (2004). Human tuberculous granulomas induce peripheral lymphoid follicle-like structures to orchestrate local host defence in the lung.
  43. (2003). Identification and macrophage-activating activity of glycolipids released from intracellular Mycobacterium bovis BCGMol.Microbiol.
  44. (2002). ideR, An essential gene in mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response.
  45. (2006). Immunogenicity of Mycobacterium tuberculosis antigens in Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle. Infect Immun.
  46. (1996). Immunopathology of tuberculosis: roles of macrophages and monocytes.
  47. (1991). Improved efficiency of protein structure calculations from NMR data using the program DIANA with redundant dihedral angle constraints.
  48. (2002). In situ detection of Mycobacterium tuberculosis transcripts in human lung granulomas reveals differential gene expression in necrotic lesions.
  49. (2005). In vivo activity of released cell wall lipids of Mycobacterium bovis bacillus Calmette-Guérin is due principally to trehalose mycolates.J.
  50. (2006). Inactivation of Rv2525c, a substrate of the twin arginine translocation (Tat) system of Mycobacterium tuberculosis, increases beta-lactam susceptibility and virulence.
  51. (2007). Induction of CD8 T cells against a novel epitope in TB10.4: correlation with mycobacterial virulence and the presence of a functional region of difference-1.J Immunol.
  52. (2000). Inhibition of Ca (2+) signaling by Mycobacterium tuberculosis is associated with reduced phagosome-lysosome fusion and increased survival within human macrophages.
  53. (2004). Iron-oxo clusters biomineralizing on protein surfaces: structural analysis of Halobacterium salinarum DpsA in its low- and high-iron states.
  54. (2004). Isolation of Mycobacterium tuberculosis mutants defective in the arrest of phagosome maturation.
  55. (2008). Long term efficacy of DOTS regimens for tuberculosis: systematic review 336(7642):485-487.
  56. (2002). Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti.
  57. (2007). M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells.
  58. M.(2002) Microarray analysis of the Mycobacterium tuberculosis transcriptional response to the acidic conditions found in phagosomes.
  59. (2001). Massive gene decay in the leprosy bacillus.
  60. Molecular cloning a laboratory manual, Third Edition,
  61. (2008). Molecular features governing the stability and specificity of functional complex formation by Mycobacterium tuberculosis CFP10/ESAT-6 family proteins.
  62. (1996). MOLMOL: A program for display and analysis of macromolecular structures.
  63. (2005). Mutually dependent secretion of proteins required for mycobacterial virulence.
  64. (1996). Mycobacterium avium- and Mycobacterium tuberculosis-containing vacuoles are dynamic, fusion-competent vesicles that are accessible to glycosphingolipids from the host cell plasmalemma
  65. (2005). Mycobacterium tuberculosis Four-minute Phagosome.
  66. (2006). Mycobacterium tuberculosis inhibition of phagolysosome biogenesis and autophagy as a host defence mechanismCell.
  67. (2003). Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence.
  68. (1980). Natural abundance N-15 NMR by enhanced heteronuclear spectroscopy.
  69. (2006). New insights into the function of granulomas in human tuberculosis.
  70. (2001). of Mycobacterium tuberculosis H37Rv downregulated in the attenuated strain H37Ra are restricted to M. tuberculosis complex species.New Microbiol.
  71. (1989). Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional hetero nuclear quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta.
  72. (2005). Peripheral blood and pleural fluid mononuclear cell responses to low-molecularmass secretory polypeptides of Mycobacterium tuberculosis in human models of immunity to tuberculosis. Infect Immun.
  73. (2000). Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase.
  74. (1985). Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria.
  75. (2006). Prospects for a novel vaccine against tuberculosis.
  76. (2007). Protective Immune Responses to a Recombinant Adenovirus Type 35 Tuberculosis Vaccine
  77. (1999). Protein backbone angle restraints from searching a database for chemical shift and sequence homology.
  78. (2002). Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA.
  79. (2007). Protein secretion systems in Mycobacteria.
  80. (2004). Protein-protein interactions of proteins from the ESAT-6 family of Mycobacterium tuberculosis.
  81. (2003). Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis.
  82. (2005). Recombinant BCG expressing Mycobacterium tuberculosis major extracellular proteins.
  83. (2005). Rv2358 and FurB: two transcriptional regulators from Mycobacterium tuberculosis which respond to zinc.
  84. (2008). Screening highly expressed mycobacterial genes identifies Rv3615c as a useful differential diagnostic antigen for the Mycobacterium tuberculosis complex.
  85. (1999). Search for genes potentially involved in Mycobacterium tuberculosis virulence by mRNA differential display.Biochem Biophys Res Commun.
  86. (2003). SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis.
  87. (2008). Secreted transcription factor controls Mycobacterium tuberculosis virulence.
  88. (1998). Sequential unfolding of papain in molten globule state.Biochem.
  89. (2008). Structural studies on the second Mycobacterium smegmatis Dps: invariant and variable features of structure, assembly and function.
  90. (2005). Structure and function of the complex formed by the tuberculosis virulence factors
  91. (2004). T7 lysozyme represses T7 RNA polymerase transcription by destabilizing the open complex during initiation.
  92. (1992). The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy.
  93. (1997). The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
  94. (2003). The complete genome sequence of Mycobacterium bovis.
  95. (2006). The crystal structure of the E. coli stress protein YciF. Protein Sci.
  96. (2001). The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria .Genome Biol..;2(10):RESEARCH0044.
  97. (2002). The ESAT-6/WXG100 superfamily - and a new Gram-positive secretion system? Trends Microbiol.
  98. (2001). The evolution of mycobacterial pathogenicity: clues from comparative genomics.
  99. (2003). The nature of the di-iron site in the bacterioferritin from Desulfovibrio desulfuricans.
  100. (2003). The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue.
  101. (2004). The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis.
  102. (2005). The twin-arginine translocation pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases.
  103. (1997). Torsion angle dynamics for NMR structure calculation with the new program DYANA.
  104. (2000). Towards the proteome of Mycobacterium tuberculosis.
  105. (2002). Transformation of yeast by lithium acetate/singlestranded carrier DNA/polyethylene glycol mwthod.
  106. (1994). Treatment of tuberculosis and tuberculosis infection in adults and children. American Thoracic Society and The Centers for Disease Control and Prevention.
  107. (2007). Tuberculosis, from basic science to patient care.
  108. (2008). Tuberculosis: Shrewd survival strategy
  109. (2004). Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant.
  110. (2007). Type VII secretion--mycobacteria show the way. Nat Rev Microbiol.
  111. (2006). Using circular dichroism spectra to estimate protein secondary structure.
  112. (2005). What's good for the host is good for the bug.
  113. (2007). Who puts the tubercle in tuberculosis?
  114. (2001). Yeast Two-Hybrid Screening for Proteins that Interact with Nuclear Hormone Receptors. Methods in Molecular Biology.
  115. Z-buffer/X-gal/β-mercaptoethanol solution: 5 ml Z buffer, 18 μl β-mercaptoethanol, 50 μl

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.