1887

Abstract

There is growing evidence that strains of differ in pathogenicity and transmissibility, but little is understood about the contributory factors. We have previously shown that increased expression of the principal sigma factor, SigA, mediates the capacity of strain 210 to grow more rapidly in human monocytes, compared with other strains. Strain 210 is part of the widespread W-Beijing family of strains and includes clinical isolate TB294. To identify genes that respond to changes in SigA levels and that might enhance intracellular growth, we examined RNA and protein expression patterns in TB294-pSigA, a recombinant strain of TB294 that overexpresses from a multicopy plasmid. Lysates from broth-grown cultures of TB294-pSigA contained high levels of Eis, a protein known to modulate host–pathogen interactions. DNA microarray analysis indicated that the gene, Rv2416c, was expressed at levels in TB294-pSigA 40-fold higher than in the vector control strain TB294-pCV, during growth in the human monocyte cell line MonoMac6. Other genes with elevated expression in TB294-pSigA showed much smaller changes from TB294-pCV, and the majority of genes with expression differences between the two strains had reduced expression in TB294-pSigA, including an unexpected number of genes associated with the DNA-damage response. Real-time PCR analyses confirmed that was expressed at very high levels in TB294-pSigA in monocytes as well as in broth culture, and further revealed that, like , was also more highly expressed in wild-type TB294 than in the laboratory strain H37Rv, during growth in monocytes. These findings suggested an association between increased SigA levels and activation, and results of chromatin immunoprecipitation confirmed that SigA binds the promoter in live TB294 cells. Deletion of reduced growth of TB294 in monocytes, and complementation of reversed this effect. We conclude that SigA regulates , that there is a direct correlation between upregulation of SigA and high expression levels of , and that contributes to the enhanced capacity of a clinical isolate of strain 210 to grow in monocytes.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.024638-0
2009-04-01
2020-01-26
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/4/1272.html?itemId=/content/journal/micro/10.1099/mic.0.024638-0&mimeType=html&fmt=ahah

References

  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J.. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res25:3389–3402
    [Google Scholar]
  2. Barnes P. F., Yang Z., Preston-Martin S., Pogoda J. M., Jones B. E., Otaya M., Eisenach K. D., Knowles L., Harvey S., Cave M. D.. 1997; Patterns of tuberculosis transmission in central Los Angeles. JAMA278:1159–1163
    [Google Scholar]
  3. Be N. A., Lamichhane G., Grosset J., Tyagi S., Cheng Q. J., Kim K. S., Bishai W. R., Jain S. K.. 2008; Murine model to study the invasion and survival of Mycobacterium tuberculosis in the central nervous system. J Infect Dis198:1520–1528
    [Google Scholar]
  4. Bernardo L. M., Johansson L. U., Solera D., Skärfstad E., Shingler V.. 2006; The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of σ 54-dependent transcription. Mol Microbiol60:749–764
    [Google Scholar]
  5. Bishai W. R., Graham N. M., Harrington S., Pope D. S., Hooper N., Astemborski J., Sheely L., Vlahov D., Glass G. E., Chaisson R. E.. 1998; Molecular and geographic patterns of tuberculosis transmission after 15 years of directly observed therapy. JAMA280:1679–1684
    [Google Scholar]
  6. Brooks P. C., Dawson L. F., Rand L., Davis E. O.. 2006; The mycobacterium-specific gene Rv2719c is DNA damage inducible independently of RecA. J Bacteriol188:6034–6038
    [Google Scholar]
  7. Cappelli G., Volpe P., Sanduzzi A., Sacchi A., Colizzi V., Mariani F.. 2001; Human macrophage gamma interferon decreases gene expression but not replication of Mycobacterium tuberculosis : analysis of the host–pathogen reciprocal influence on transcription in a comparison of strains H37Rv and CMT97. Infect Immun69:7262–7270
    [Google Scholar]
  8. Collins D. M., Kawakami R. P., deLisle G. W., Pascopella L., Bloom B. R., Jacobs W. R. Jr. 1995; Mutation of the principal sigma factor causes loss of virulence in a strain of the Mycobacterium tuberculosis complex. Proc Natl Acad Sci U S A92:8036–8040
    [Google Scholar]
  9. Coros A., Callahan B., Battaglioli E., Derbyshire K. M.. 2008; The specialized secretory apparatus ESX-1 is essential for DNA transfer in Mycobacterium smegmatis . Mol Microbiol69:794–808
    [Google Scholar]
  10. Dahl J. L., Wei J., Moulder J. W., Laal S., Friedman R. L.. 2001; Subcellular localization of the intracellular survival-enhancing Eis protein of Mycobacterium tuberculosis . Infect Immun69:4295–4302
    [Google Scholar]
  11. Davis E. O., Springer B., Gopaul K. K., Papavinasasundaram K. G., Sander P., Böttger E. C.. 2002; DNA damage induction of recA in Mycobacterium tuberculosis independently of RecA and LexA. Mol Microbiol46:791–800
    [Google Scholar]
  12. Farewell A., Kvint K., Nyström T.. 1998; Negative regulation by RpoS: a case of sigma factor competition. Mol Microbiol29:1039–1051
    [Google Scholar]
  13. Flint J. L., Kowalski J. C., Karnati P. K., Derbyshire K. M.. 2004; The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis . Proc Natl Acad Sci U S A101:12598–12603
    [Google Scholar]
  14. Fortune S. M., Jaeger A., Sarracino D. A., Chase M. R., Sassetti C. M., Sherman D. R., Bloom B. R., Rubin E. J.. 2005; Mutually dependent secretion of proteins required for mycobacterial virulence. Proc Natl Acad Sci U S A102:10676–10681
    [Google Scholar]
  15. Gamulin V., Cetkovic H., Ahel I.. 2004; Identification of a promoter motif regulating the major DNA damage response mechanism of Mycobacterium tuberculosis . FEMS Microbiol Lett238:57–63
    [Google Scholar]
  16. Gomez M., Doukhan L., Nair G., Smith I.. 1998; SigA is an essential gene in Mycobacterium smegmatis . Mol Microbiol29:617–628
    [Google Scholar]
  17. Gopaul K. K., Brooks P. C., Prost J. F., Davis E. O.. 2003; Characterization of the two Mycobacterium tuberculosis recA promoters. J Bacteriol185:6005–6015
    [Google Scholar]
  18. Grigorova I. L., Phleger N. J., Mutalik V. K., Gross C. A.. 2006; Insights into transcriptional regulation and σ competition from an equilibrium model of RNA polymerase binding to DNA. Proc Natl Acad Sci U S A103:5332–5337
    [Google Scholar]
  19. Hu Y., Coates A. R.. 1999; Transcription of two sigma 70 homologue genes, sigA and sigB , in stationary-phase Mycobacterium tuberculosis . J Bacteriol181:469–476
    [Google Scholar]
  20. Jain S. K., Paul-Satyaseela M., Lamichhane G., Kim K. S., Bishai W. R.. 2006; Mycobacterium tuberculosis invasion and traversal across an in vitro human blood–brain barrier as a pathogenic mechanism for central nervous system tuberculosis. J Infect Dis193:1287–1295
    [Google Scholar]
  21. King T., Ishihama A., Kori A., Ferenci T.. 2004; A regulatory trade-off as a source of strain variation in the species Escherichia coli . J Bacteriol186:5614–5620
    [Google Scholar]
  22. Laemmli U. K.. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685
    [Google Scholar]
  23. Lee J. H., Geiman D. E., Bishai W. R.. 2008; Role of stress response sigma factor SigG in Mycobacterium tuberculosis . J Bacteriol190:1128–1133
    [Google Scholar]
  24. Lella R. K., Sharma C.. 2007; Eis (enhanced intracellular survival) protein of Mycobacterium tuberculosis disturbs the cross regulation of T-cells. J Biol Chem282:18671–18675
    [Google Scholar]
  25. MacGurn J. A., Raghavan S., Stanley S. A., Cox J. S.. 2005; A non-RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis . Mol Microbiol57:1653–1663
    [Google Scholar]
  26. Pang X., Vu P., Byrd T. F., Ghanny S., Soteropoulos P., Mukamolova G. V., Wu S., Samten B., Howard S. T.. 2007; Evidence for complex interactions of stress-associated regulons in an mprAB deletion mutant of Mycobacterium tuberculosis . Microbiology153:1229–1242
    [Google Scholar]
  27. Parish T., Stoker N. G.. 2000; Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology146:1969–1975
    [Google Scholar]
  28. Predich M., Doukhan L., Nair G., Smith I.. 1995; Characterization of RNA polymerase and two sigma-factor genes from Mycobacterium smegmatis . Mol Microbiol15:355–366
    [Google Scholar]
  29. Raghavan S., Manzanillo P., Chan K., Dovey C., Cox J. S.. 2008; Secreted transcription factor controls Mycobacterium tuberculosis virulence. Nature454:717–721
    [Google Scholar]
  30. Rand L., Hinds J., Springer B., Sander P., Buxton R. S., Davis E. O.. 2003; The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA. Mol Microbiol50:1031–1042
    [Google Scholar]
  31. Reed M. B., Gagneux S., Deriemer K., Small P. M., Barry C. E. III. 2007; The W/Beijing lineage of Mycobacterium tuberculosis overproduces triglycerides and is constitutively upregulated for the DosR dormancy regulon. J Bacteriol189:2583–2589
    [Google Scholar]
  32. Roberts E. A., Clark A., McBeth S., Friedman R. L.. 2004; Molecular characterization of the eis promoter of Mycobacterium tuberculosis . J Bacteriol186:5410–5417
    [Google Scholar]
  33. Rodrigue S., Brodeur J., Jacques P. E., Gervais A. L., Brzezinski R., Gaudreau L.. 2007; Identification of mycobacterial σ factor binding sites by chromatin immunoprecipitation assays. J Bacteriol189:1505–1513
    [Google Scholar]
  34. Rosas-Magallanes V., Stadthagen-Gomez G., Rauzier J., Barreiro L. B., Tailleux L., Boudou F., Griffin R., Nigou J., Jackson M.. other authors 2007; Signature-tagged transposon mutagenesis identifies novel Mycobacterium tuberculosis genes involved in the parasitism of human macrophages. Infect Immun75:504–507
    [Google Scholar]
  35. Saeed A. I., Sharov V., White J., Li J., Liang W., Bhagabati N., Braisted J., Klapa M., Currier T.. other authors 2003; TM4: a free, open-source system for microarray data management and analysis. Biotechniques34:374–378
    [Google Scholar]
  36. Samuel L. P., Song C. H., Wei J., Roberts E. A., Dahl J. L., Barry C. E. III, Jo E. K., Friedman R. L.. 2007; Expression, production and release of the Eis protein by Mycobacterium tuberculosis during infection of macrophages and its effect on cytokine secretion. Microbiology153:529–540
    [Google Scholar]
  37. Shah I. M., Wolf R. E. J.. 2004; Novel protein–protein interaction between Escherichia coli SoxS and the DNA binding determinant of the RNA polymerase α subunit: SoxS functions as a co-sigma factor and redeploys RNA polymerase from UP-element-containing promoters to SoxS-dependent promoters during oxidative stress. J Mol Biol343:513–532
    [Google Scholar]
  38. Sinsimer D., Huet G., Manca C., Tsenova L., Koo M. S., Kurepina N., Kana B., Mathema B., Marras S. A.. other authors 2008; The phenolic glycolipid of Mycobacterium tuberculosis differentially modulates the early host cytokine response but does not in itself confer hypervirulence. Infect Immun76:3027–3036
    [Google Scholar]
  39. Small P. M., Hopewell P. C., Singh S. P., Paz A., Parsonnet J., Ruston D. C., Schecter G. F., Daley C. L., Schoolnik G. K.. 1994; The epidemiology of tuberculosis in San Francisco. A population-based study using conventional and molecular methods. N Engl J Med330:1703–1709
    [Google Scholar]
  40. Steyn A. J., Collins D. M., Hondalus M. K., Jacobs W. R. Jr, Kawakami R. P., Bloom B. R.. 2002; Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth. Proc Natl Acad Sci U S A99:3147–3152
    [Google Scholar]
  41. 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.. other authors 1991; New use of BCG for recombinant vaccines. Nature351:456–460
    [Google Scholar]
  42. Strickland M. S., Thompson N. E., Burgess R. R.. 1988; Structure and function of the σ -70 subunit of Escherichia coli RNA polymerase. Monoclonal antibodies: localization of epitopes by peptide mapping and effects on transcription. Biochemistry27:5755–5762
    [Google Scholar]
  43. Talaat A. M., Hunter P., Johnston S. A.. 2000; Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis. Nat Biotechnol18:679–682
    [Google Scholar]
  44. Talaat A. M., Lyons R., Howard S. T., Johnston S. A.. 2004; The temporal expression profile of Mycobacterium tuberculosis infection in mice. Proc Natl Acad Sci U S A101:4602–4607
    [Google Scholar]
  45. Theus S. A., Cave M. D., Eisenach K. D.. 2005; Intracellular macrophage growth rates and cytokine profiles of Mycobacterium tuberculosis strains with different transmission dynamics. J Infect Dis191:453–460
    [Google Scholar]
  46. Tsenova L., Ellison E., Harbacheuski R., Moreira A. L., Kurepina N., Reed M. B., Mathema B., Barry C. E. III, Kaplan G.. 2005; Virulence of selected Mycobacterium tuberculosis clinical isolates in the rabbit model of meningitis is dependent on phenolic glycolipid produced by the bacilli. J Infect Dis192:98–106
    [Google Scholar]
  47. Typas A., Barembruch C., Possling A., Hengge R.. 2007; Stationary phase reorganisation of the Escherichia coli transcription machinery by Crl protein, a fine-tuner of σ S activity and levels. EMBO J26:1569–1578
    [Google Scholar]
  48. Velmurugan K., Chen B., Miller J. L., Azogue S., Gurses S., Hsu T., Glickman M., Jacobs W. R. Jr, Porcelli S. A., Briken V.. 2007; Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells. PLoS Pathog3:e110
    [Google Scholar]
  49. Vetting M. W., de Carvalho L. P. S., Yu M., Hegde S. S., Magnet S., Roderick S. L., Blanchard J. S.. 2005; Structure and functions of the GNAT superfamily of acetyltransferases. Arch Biochem Biophys433:212–226
    [Google Scholar]
  50. 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 Bacteriol182:377–384
    [Google Scholar]
  51. Weis S. E., Pogoda J. M., Yang Z., Cave M. D., Wallace C., Kelley M., Barnes P. F.. 2002; Transmission dynamics of tuberculosis in Tarrant county, Texas. Am J Respir Crit Care Med166:36–42
    [Google Scholar]
  52. Wu S., Howard S. T., Lakey D. L., Kipnis A., Samten B., Safi H., Gruppo V., Wizel B., Shams H.. other authors 2004; The principal sigma factor sigA mediates enhanced growth of Mycobacterium tuberculosis strains in vivo . Mol Microbiol51:1551–1562
    [Google Scholar]
  53. Yang Z., Barnes P. F., Chaves F., Eisenach K. D., Weis S. E., Bates J. H., Cave M. D.. 1998; Diversity of DNA fingerprints of Mycobacterium tuberculosis isolates in the United States. J Clin Microbiol36:1003–1007
    [Google Scholar]
  54. Zhang M., Gong J., Yang Z., Samten B., Cave M. D., Barnes P. F.. 1999; Enhanced capacity of a widespread strain of Mycobacterium tuberculosis to grow in human macrophages. J Infect Dis179:1213–1217
    [Google Scholar]
  55. Ziegler-Heitbrock H. W., Thiel E., Futterer A., Herzog V., Wirtz A., Riethmuller G.. 1988; Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. Int J Cancer41:456–461
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.024638-0
Loading
/content/journal/micro/10.1099/mic.0.024638-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error