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. 1996 Aug;178(15):4484–4492. doi: 10.1128/jb.178.15.4484-4492.1996

Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog.

Y Yuan 1, D D Crane 1, C E Barry 3rd 1
PMCID: PMC178214  PMID: 8755875

Abstract

The majority of active tuberculosis cases arise as a result of reactivation of latent organisms which are quiescent within the host. The ability of mycobacteria to survive extended periods without active replication is a complex process whose details await elucidation. We used two-dimensional gel electrophoresis to examine both steady-state protein composition and time-dependent protein synthetic profiles in aging cultures of virulent Mycobacterium tuberculosis. At least seven proteins were maximally synthesized 1 to 2 weeks following the end of log-phase growth. One of these proteins accumulated to become a predominant stationary-phase protein. N-terminal amino acid sequencing and immunoreactivity identified this protein as the 16-kDa alpha-crystallin-like small heat shock protein. The gene for this protein was shown to be limited to the slowly growing M. tuberculosis complex of organisms as assessed by Southern blotting. Overexpression of this protein in wild-type M. tuberculosis resulted in a slower decline in viability following the end of log-phase growth. Accumulation of this protein was observed in log-phase cultures following a shift to oxygen-limiting conditions but not by other external stimuli. The protein was purified to homogeneity from overexpressing M. smegmatis in two steps and shown to have a significant ability to suppress the thermal denaturation of alcohol dehydrogenase. Collectively, these results suggest that the mycobacterial alpha-crystallin protein may play a role in enhancing long-term protein stability and therefore long-term survival of M. tuberculosis.

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Selected References

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  1. AUERBACH O., HOBBY G. L., SMALL M. J., LENERT T. F., COMER J. V. The clinicopathologic significance of the demonstration of viable tubercle bacilli in resected lesions. J Thorac Surg. 1955 Feb;29(2):109–135. [PubMed] [Google Scholar]
  2. Adams E., Basten A., Prestidge R., Britton W. J. T cell clones from a non-leprosy exposed subject recognize the Mycobacterium leprae 18-kD protein. Clin Exp Immunol. 1995 Oct;102(1):58–64. doi: 10.1111/j.1365-2249.1995.tb06636.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aldovini A., Husson R. N., Young R. A. The uraA locus and homologous recombination in Mycobacterium bovis BCG. J Bacteriol. 1993 Nov;175(22):7282–7289. doi: 10.1128/jb.175.22.7282-7289.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arrigo A. P., Suhan J. P., Welch W. J. Dynamic changes in the structure and intracellular locale of the mammalian low-molecular-weight heat shock protein. Mol Cell Biol. 1988 Dec;8(12):5059–5071. doi: 10.1128/mcb.8.12.5059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Behlke J., Lutsch G., Gaestel M., Bielka H. Supramolecular structure of the recombinant murine small heat shock protein hsp25. FEBS Lett. 1991 Aug 19;288(1-2):119–122. doi: 10.1016/0014-5793(91)81016-2. [DOI] [PubMed] [Google Scholar]
  6. Caspers G. J., Leunissen J. A., de Jong W. W. The expanding small heat-shock protein family, and structure predictions of the conserved "alpha-crystallin domain". J Mol Evol. 1995 Mar;40(3):238–248. doi: 10.1007/BF00163229. [DOI] [PubMed] [Google Scholar]
  7. Dannenberg A. M., Jr Immunopathogenesis of pulmonary tuberculosis. Hosp Pract (Off Ed) 1993 Jan 15;28(1):51–58. doi: 10.1080/21548331.1993.11442738. [DOI] [PubMed] [Google Scholar]
  8. Das K. P., Surewicz W. K. Temperature-induced exposure of hydrophobic surfaces and its effect on the chaperone activity of alpha-crystallin. FEBS Lett. 1995 Aug 7;369(2-3):321–325. doi: 10.1016/0014-5793(95)00775-5. [DOI] [PubMed] [Google Scholar]
  9. Dellagostin O. A., Esposito G., Eales L. J., Dale J. W., McFadden J. Activity of mycobacterial promoters during intracellular and extracellular growth. Microbiology. 1995 Aug;141(Pt 8):1785–1792. doi: 10.1099/13500872-141-8-1785. [DOI] [PubMed] [Google Scholar]
  10. Dolin P. J., Raviglione M. C., Kochi A. Global tuberculosis incidence and mortality during 1990-2000. Bull World Health Organ. 1994;72(2):213–220. [PMC free article] [PubMed] [Google Scholar]
  11. Friscia G., Vordermeier H. M., Pasvol G., Harris D. P., Moreno C., Ivanyi J. Human T cell responses to peptide epitopes of the 16-kD antigen in tuberculosis. Clin Exp Immunol. 1995 Oct;102(1):53–57. doi: 10.1111/j.1365-2249.1995.tb06635.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. George K. M., Yuan Y., Sherman D. R., Barry C. E., 3rd The biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis. Identification and functional analysis of CMAS-2. J Biol Chem. 1995 Nov 10;270(45):27292–27298. doi: 10.1074/jbc.270.45.27292. [DOI] [PubMed] [Google Scholar]
  13. Groenen P. J., Merck K. B., de Jong W. W., Bloemendal H. Structure and modifications of the junior chaperone alpha-crystallin. From lens transparency to molecular pathology. Eur J Biochem. 1994 Oct 1;225(1):1–19. doi: 10.1111/j.1432-1033.1994.00001.x. [DOI] [PubMed] [Google Scholar]
  14. Horwitz J. Alpha-crystallin can function as a molecular chaperone. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10449–10453. doi: 10.1073/pnas.89.21.10449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jakob U., Gaestel M., Engel K., Buchner J. Small heat shock proteins are molecular chaperones. J Biol Chem. 1993 Jan 25;268(3):1517–1520. [PubMed] [Google Scholar]
  16. Lange R., Hengge-Aronis R. Identification of a central regulator of stationary-phase gene expression in Escherichia coli. Mol Microbiol. 1991 Jan;5(1):49–59. doi: 10.1111/j.1365-2958.1991.tb01825.x. [DOI] [PubMed] [Google Scholar]
  17. Lee B. Y., Hefta S. A., Brennan P. J. Characterization of the major membrane protein of virulent Mycobacterium tuberculosis. Infect Immun. 1992 May;60(5):2066–2074. doi: 10.1128/iai.60.5.2066-2074.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McCann M. P., Kidwell J. P., Matin A. The putative sigma factor KatF has a central role in development of starvation-mediated general resistance in Escherichia coli. J Bacteriol. 1991 Jul;173(13):4188–4194. doi: 10.1128/jb.173.13.4188-4194.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mehlen P., Briolay J., Smith L., Diaz-latoud C., Fabre N., Pauli D., Arrigo A. P. Analysis of the resistance to heat and hydrogen peroxide stresses in COS cells transiently expressing wild type or deletion mutants of the Drosophila 27-kDa heat-shock protein. Eur J Biochem. 1993 Jul 15;215(2):277–284. doi: 10.1111/j.1432-1033.1993.tb18032.x. [DOI] [PubMed] [Google Scholar]
  20. Molloy A., Laochumroonvorapong P., Kaplan G. Apoptosis, but not necrosis, of infected monocytes is coupled with killing of intracellular bacillus Calmette-Guérin. J Exp Med. 1994 Oct 1;180(4):1499–1509. doi: 10.1084/jem.180.4.1499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Raman B., Ramakrishna T., Rao C. M. Temperature dependent chaperone-like activity of alpha-crystallin. FEBS Lett. 1995 May 29;365(2-3):133–136. doi: 10.1016/0014-5793(95)00440-k. [DOI] [PubMed] [Google Scholar]
  22. SALKIN D., WAYNE L. G. The bacteriology of resected tuberculous pulmonary lesions. I. The effect of interval between reversal of infectiousness and subsequent surgery. Am Rev Tuberc. 1956 Sep;74(3):376–387. doi: 10.1164/artpd.1956.74.3.376. [DOI] [PubMed] [Google Scholar]
  23. Sauer U., Dürre P. Sequence and molecular characterization of a DNA region encoding a small heat shock protein of Clostridium acetobutylicum. J Bacteriol. 1993 Jun;175(11):3394–3400. doi: 10.1128/jb.175.11.3394-3400.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Siegele D. A., Kolter R. Life after log. J Bacteriol. 1992 Jan;174(2):345–348. doi: 10.1128/jb.174.2.345-348.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stover C. K., de la Cruz V. F., Fuerst T. R., Burlein J. E., Benson L. A., Bennett L. T., Bansal G. P., Young J. F., Lee M. H., Hatfull G. F. New use of BCG for recombinant vaccines. Nature. 1991 Jun 6;351(6326):456–460. doi: 10.1038/351456a0. [DOI] [PubMed] [Google Scholar]
  27. Sudre P., ten Dam G., Kochi A. Tuberculosis: a global overview of the situation today. Bull World Health Organ. 1992;70(2):149–159. [PMC free article] [PubMed] [Google Scholar]
  28. Verbon A., Hartskeerl R. A., Schuitema A., Kolk A. H., Young D. B., Lathigra R. The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alpha-crystallin family of low-molecular-weight heat shock proteins. J Bacteriol. 1992 Feb;174(4):1352–1359. doi: 10.1128/jb.174.4.1352-1359.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Voorter C. E., de Haard-Hoekman W., Merck K. B., Bloemendal H., de Jong W. W. Elastase inhibition by the C-terminal domains of alpha-crystallin and small heat-shock protein. Biochim Biophys Acta. 1994 Jan 11;1204(1):43–47. doi: 10.1016/0167-4838(94)90030-2. [DOI] [PubMed] [Google Scholar]
  30. WAYNE L. G. The bacteriology of respected tuberculous pulmonary lesions. 2. Observations on bacilli which are stainable but which cannot be cultured. Am Rev Respir Dis. 1960 Sep;82:370–377. doi: 10.1164/arrd.1960.82.3.370. [DOI] [PubMed] [Google Scholar]
  31. Wayne L. G., Diaz G. A. Autolysis and secondary growth of Mycobacterium tuberculosis in submerged culture. J Bacteriol. 1967 Apr;93(4):1374–1381. doi: 10.1128/jb.93.4.1374-1381.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wayne L. G. Dormancy of Mycobacterium tuberculosis and latency of disease. Eur J Clin Microbiol Infect Dis. 1994 Nov;13(11):908–914. doi: 10.1007/BF02111491. [DOI] [PubMed] [Google Scholar]
  33. Wayne L. G., Sramek H. A. Antigenic differences between extracts of actively replicating and synchronized resting cells of Mycobacterium tuberculosis. Infect Immun. 1979 May;24(2):363–370. doi: 10.1128/iai.24.2.363-370.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wayne L. G. Synchronized replication of Mycobacterium tuberculosis. Infect Immun. 1977 Sep;17(3):528–530. doi: 10.1128/iai.17.3.528-530.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yuan Y., Lee R. E., Besra G. S., Belisle J. T., Barry C. E., 3rd Identification of a gene involved in the biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6630–6634. doi: 10.1073/pnas.92.14.6630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. van den IJssel P. R., Overkamp P., Knauf U., Gaestel M., de Jong W. W. Alpha A-crystallin confers cellular thermoresistance. FEBS Lett. 1994 Nov 21;355(1):54–56. doi: 10.1016/0014-5793(94)01175-3. [DOI] [PubMed] [Google Scholar]

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