1887

Abstract

The regulatory mechanisms that control the ESX-1 secretion system, a key player in the pathogenesis of , have not been fully elucidated. However, factors that regulate the ESX-1 substrate EspA usually affect ESX-1 function. Previous studies showed that is directly regulated by the nucleoid-associated protein EspR and the two-component system (TCS) MprAB. The PhoPR TCS also activates , but the direct target of PhoP was unknown. In this report, we reveal that EspR is directly regulated by MprA and PhoP-Rv, but not by PhoP-Ra. PhoP-Rv and MprA binding sites in the promoter were determined by gel-shift and DNase I footprinting assays, which identified a PhoP-protected region centred approximately 205 bp before the start codon and that encompasses MprA Region-1, one of two MprA-protected regions. MprA Region-2 is located approximately 60 bp downstream of MprA Region-1 and overlaps a known EspR binding site. Nucleotides essential for the binding of PhoP and/or MprA were identified through site-directed DNA mutagenesis. Our studies also indicate that MprA Region-2, but not MprA Region-1/PhoP region, is required for the full expression of . Recombinant strains carrying mutations at MprA Region-2 exhibited lower transcription levels for , and , and had reduced EspR and EspA levels in cell lysates. These findings indicate that EspR may mediate the regulatory effect of PhoPR and MprAB, and provide more insight into the mechanisms underlying ESX-1 control.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000023
2015-03-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/161/3/477.html?itemId=/content/journal/micro/10.1099/mic.0.000023&mimeType=html&fmt=ahah

References

  1. Baker J. J., Johnson B. K., Abramovitch R. B.(2014). Slow growth of Mycobacterium tuberculosis at acidic pH is regulated by phoPR and host-associated carbon sources. Mol Microbiol 94, 5669. [View Article][PubMed] [Google Scholar]
  2. Blasco B., Stenta M., Alonso-Sarduy L., Dietler G., Peraro M. D., Cole S. T., Pojer F.(2011). Atypical DNA recognition mechanism used by the EspR virulence regulator of Mycobacterium tuberculosis. Mol Microbiol 82, 251264. [View Article][PubMed] [Google Scholar]
  3. Blasco B., Chen J. M., Hartkoorn R., Sala C., Uplekar S., Rougemont J., Pojer F., Cole S. T.(2012). Virulence regulator EspR of Mycobacterium tuberculosis is a nucleoid-associated protein. PLoS Pathog 8, e1002621. [View Article][PubMed] [Google Scholar]
  4. Boritsch E. C., Supply P., Honoré N., Seeman T., Stinear T. P., Brosch R.(2014). A glimpse into the past and predictions for the future: the molecular evolution of the tuberculosis agent. Mol Microbiol 93, 835852. [View Article][PubMed] [Google Scholar]
  5. Bretl D. J., He H., Demetriadou C., White M. J., Penoske R. M., Salzman N. H., Zahrt T. C.(2012). MprA and DosR coregulate a Mycobacterium tuberculosis virulence operon encoding Rv1813c and Rv1812c. Infect Immun 80, 30183033. [View Article][PubMed] [Google Scholar]
  6. Cao G., Howard S. T., Zhang P., Hou G., Pang X.(2013). Functional analysis of the EspR binding sites upstream of espR in Mycobacterium tuberculosis. Curr Microbiol 67, 572579. [View Article][PubMed] [Google Scholar]
  7. Chesne-Seck M. L., Barilone N., Boudou F., Gonzalo Asensio J., Kolattukudy P. E., Martín C., Cole S. T., Gicquel B., Gopaul D. N., Jackson M.(2008). A point mutation in the two-component regulator PhoP-PhoR accounts for the absence of polyketide-derived acyltrehaloses but not that of phthiocerol dimycocerosates in Mycobacterium tuberculosis H37Ra. J Bacteriol 190, 13291334. [View Article][PubMed] [Google Scholar]
  8. Cimino M., Thomas C., Namouchi A., Dubrac S., Gicquel B., Gopaul D. N.(2012). Identification of DNA binding motifs of the Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction system. PLoS ONE 7, e42876. [View Article][PubMed] [Google Scholar]
  9. 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 A 102, 1067610681. [View Article][PubMed] [Google Scholar]
  10. Frigui W., Bottai D., Majlessi L., Monot M., Josselin E., Brodin P., Garnier T., Gicquel B., Martin C.& other authors (2008). Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP. PLoS Pathog 4, e33. [View Article][PubMed] [Google Scholar]
  11. Garces A., Atmakuri K., Chase M. R., Woodworth J. S., Krastins B., Rothchild A. C., Ramsdell T. L., Lopez M. F., Behar S. M.& other authors (2010). EspA acts as a critical mediator of ESX1-dependent virulence in Mycobacterium tuberculosis by affecting bacterial cell wall integrity. PLoS Pathog 6, e1000957. [View Article][PubMed] [Google Scholar]
  12. Gonzalo Asensio J., Maia C., Ferrer N. L., Barilone N., Laval F., Soto C. Y., Winter N., Daffé M., Gicquel B.& other authors (2006). The virulence-associated two-component PhoP-PhoR system controls the biosynthesis of polyketide-derived lipids in Mycobacterium tuberculosis. J Biol Chem 281, 13131316. [View Article][PubMed] [Google Scholar]
  13. Gonzalo-Asensio J., Soto C. Y., Arbués A., Sancho J., del Carmen Menéndez M., García M. J., Gicquel B., Martín C.(2008). The Mycobacterium tuberculosis phoPR operon is positively autoregulated in the virulent strain H37Rv. J Bacteriol 190, 70687078. [View Article][PubMed] [Google Scholar]
  14. Gonzalo-Asensio J., Malaga W., Pawlik A., Astarie-Dequeker C., Passemar C., Moreau F., Laval F., Daffé M., Martin C.& other authors (2014). Evolutionary history of tuberculosis shaped by conserved mutations in the PhoPR virulence regulator. Proc Natl Acad Sci U S A 111, 1149111496. [View Article][PubMed] [Google Scholar]
  15. Gordon S. V., Brosch R., Billault A., Garnier T., Eiglmeier K., Cole S. T.(1999). Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol Microbiol 32, 643655. [View Article][PubMed] [Google Scholar]
  16. Gordon B. R., Li Y., Wang L., Sintsova A., van Bakel H., Tian S., Navarre W. W., Xia B., Liu J.(2010). Lsr2 is a nucleoid-associated protein that targets AT-rich sequences and virulence genes in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 107, 51545159. [View Article][PubMed] [Google Scholar]
  17. Goyal R., Das A. K., Singh R., Singh P. K., Korpole S., Sarkar D.(2011). Phosphorylation of PhoP protein plays direct regulatory role in lipid biosynthesis of Mycobacterium tuberculosis. J Biol Chem 286, 4519745208. [View Article][PubMed] [Google Scholar]
  18. Gupta S., Sinha A., Sarkar D.(2006). Transcriptional autoregulation by Mycobacterium tuberculosis PhoP involves recognition of novel direct repeat sequences in the regulatory region of the promoter. FEBS Lett 580, 53285338. [View Article][PubMed] [Google Scholar]
  19. Gupta S., Pathak A., Sinha A., Sarkar D.(2009). Mycobacterium tuberculosis PhoP recognizes two adjacent direct-repeat sequences to form head-to-head dimers. J Bacteriol 191, 74667476. [View Article][PubMed] [Google Scholar]
  20. Haydel S. E., Clark-Curtiss J. E.(2006). The Mycobacterium tuberculosis TrcR response regulator represses transcription of the intracellularly expressed Rv1057 gene, encoding a seven-bladed β-propeller. J Bacteriol 188, 150159. [View Article][PubMed] [Google Scholar]
  21. He H., Zahrt T. C.(2005). Identification and characterization of a regulatory sequence recognized by Mycobacterium tuberculosis persistence regulator MprA. J Bacteriol 187, 202212. [View Article][PubMed] [Google Scholar]
  22. He H., Hovey R., Kane J., Singh V., Zahrt T. C.(2006). MprAB is a stress-responsive two-component system that directly regulates expression of sigma factors SigB and SigE in Mycobacterium tuberculosis. J Bacteriol 188, 21342143. [View Article][PubMed] [Google Scholar]
  23. He H., Bretl D. J., Penoske R. M., Anderson D. M., Zahrt T. C.(2011). Components of the Rv0081-Rv0088 locus, which encodes a predicted formate hydrogenlyase complex, are coregulated by Rv0081, MprA, and DosR in Mycobacterium tuberculosis. J Bacteriol 193, 51055118. [View Article][PubMed] [Google Scholar]
  24. Hunt D. M., Sweeney N. P., Mori L., Whalan R. H., Comas I., Norman L., Cortes T., Arnvig K. B., Davis E. O.& other authors (2012). Long-range transcriptional control of an operon necessary for virulence-critical ESX-1 secretion in Mycobacterium tuberculosis. J Bacteriol 194, 23072320. [View Article][PubMed] [Google Scholar]
  25. Lee J. S., Krause R., Schreiber J., Mollenkopf H. J., Kowall J., Stein R., Jeon B. Y., Kwak J. Y., Song M. K.& other authors (2008). Mutation in the transcriptional regulator PhoP contributes to avirulence of Mycobacterium tuberculosis H37Ra strain. Cell Host Microbe 3, 97103. [View Article][PubMed] [Google Scholar]
  26. Li A. H., Waddell S. J., Hinds J., Malloff C. A., Bains M., Hancock R. E., Lam W. L., Butcher P. D., Stokes R. W.(2010). Contrasting transcriptional responses of a virulent and an attenuated strain of Mycobacterium tuberculosis infecting macrophages. PLoS ONE 5, e11066. [View Article][PubMed] [Google Scholar]
  27. Mahairas G. G., Sabo P. J., Hickey M. J., Singh D. C., Stover C. K.(1996). Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 178, 12741282.[PubMed] [Google Scholar]
  28. McGillivray A., Golden N. A., Gautam U. S., Mehra S., Kaushal D.(2014). The Mycobacterium tuberculosis Rv2745c plays an important role in responding to redox stress. PLoS ONE 9, e93604. [View Article][PubMed] [Google Scholar]
  29. Pang X., Howard S. T.(2007). Regulation of the α-crystallin gene acr2 by the MprAB two-component system of Mycobacterium tuberculosis. J Bacteriol 189, 62136221. [View Article][PubMed] [Google Scholar]
  30. 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. Microbiology 153, 12291242. [View Article][PubMed] [Google Scholar]
  31. Pang X., Cao G., Neuenschwander P. F., Haydel S. E., Hou G., Howard S. T.(2011). The β-propeller gene Rv1057 of Mycobacterium tuberculosis has a complex promoter directly regulated by both the MprAB and TrcRS two-component systems. Tuberculosis (Edinb) 91 (Suppl 1), S142S149. [View Article][PubMed] [Google Scholar]
  32. Pang X., Samten B., Cao G., Wang X., Tvinnereim A. R., Chen X. L., Howard S. T.(2013). MprAB regulates the espA operon in Mycobacterium tuberculosis and modulates ESX-1 function and host cytokine response. J Bacteriol 195, 6675. [View Article][PubMed] [Google Scholar]
  33. Pathak A., Goyal R., Sinha A., Sarkar D.(2010). Domain structure of virulence-associated response regulator PhoP of Mycobacterium tuberculosis: role of the linker region in regulator-promoter interaction(s). J Biol Chem 285, 3430934318. [View Article][PubMed] [Google Scholar]
  34. Raghavan S., Manzanillo P., Chan K., Dovey C., Cox J. S.(2008). Secreted transcription factor controls Mycobacterium tuberculosis virulence. Nature 454, 717721. [View Article][PubMed] [Google Scholar]
  35. Rickman L., Scott C., Hunt D. M., Hutchinson T., Menéndez M. C., Whalan R., Hinds J., Colston M. J., Green J., Buxton R. S.(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. Mol Microbiol 56, 12741286. [View Article][PubMed] [Google Scholar]
  36. Rosenberg O. S., Dovey C., Tempesta M., Robbins R. A., Finer-Moore J. S., Stroud R. M., Cox J. S.(2011). EspR, a key regulator of Mycobacterium tuberculosis virulence, adopts a unique dimeric structure among helix-turn-helix proteins. Proc Natl Acad Sci U S A 108, 1345013455. [View Article][PubMed] [Google Scholar]
  37. Ryndak M., Wang S., Smith I.(2008). PhoP, a key player in Mycobacterium tuberculosis virulence. Trends Microbiol 16, 528534. [View Article][PubMed] [Google Scholar]
  38. Sherrid A. M., Rustad T. R., Cangelosi G. A., Sherman D. R.(2010). Characterization of a Clp protease gene regulator and the reaeration response in Mycobacterium tuberculosis. PLoS ONE 5, e11622. [View Article][PubMed] [Google Scholar]
  39. Simeone R., Bottai D., Brosch R.(2009). ESX/type VII secretion systems and their role in host–pathogen interaction. Curr Opin Microbiol 12, 410. [View Article][PubMed] [Google Scholar]
  40. Singh R., Anil Kumar V., Das A. K., Bansal R., Sarkar D.(2014). A transcriptional co-repressor regulatory circuit controlling the heat-shock response of Mycobacterium tuberculosis. Mol Microbiol 94, 450465. [View Article][PubMed] [Google Scholar]
  41. Solans L., Aguiló N., Samper S., Pawlik A., Frigui W., Martín C., Brosch R., Gonzalo-Asensio J.(2014a). A specific polymorphism in Mycobacterium tuberculosis H37Rv causes differential ESAT-6 expression and identifies WhiB6 as a novel ESX-1 component. Infect Immun 82, 34463456. [View Article][PubMed] [Google Scholar]
  42. Solans L., Gonzalo-Asensio J., Sala C., Benjak A., Uplekar S., Rougemont J., Guilhot C., Malaga W., Martín C., Cole S. T.(2014b). The PhoP-dependent ncRNA Mcr7 modulates the TAT secretion system in Mycobacterium tuberculosis. PLoS Pathog 10, e1004183. [View Article][PubMed] [Google Scholar]
  43. 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. Nature 351, 456460. [View Article][PubMed] [Google Scholar]
  44. Walters S. B., Dubnau E., Kolesnikova I., Laval F., Daffe M., Smith I.(2006). The Mycobacterium tuberculosis PhoPR two-component system regulates genes essential for virulence and complex lipid biosynthesis. Mol Microbiol 60, 312330. [View Article][PubMed] [Google Scholar]
  45. Wang Y., Huang Y., Xue C., He Y., He Z. G.(2011). ClpR protein-like regulator specifically recognizes RecA protein-independent promoter motif and broadly regulates expression of DNA damage-inducible genes in mycobacteria. J Biol Chem 286, 3115931167. [View Article][PubMed] [Google Scholar]
  46. Wang Y., Cen X. F., Zhao G. P., Wang J.(2012). Characterization of a new GlnR binding box in the promoter of amtB in Streptomyces coelicolor inferred a PhoP/GlnR competitive binding mechanism for transcriptional regulation of amtB. J Bacteriol 194, 52375244. [View Article][PubMed] [Google Scholar]
  47. Zhang P., Wu H., Chen X. L., Deng Z., Bai L., Pang X.(2014). Regulation of the biosynthesis of thiopeptide antibiotic cyclothiazomycin by the transcriptional regulator SHJG8833 in Streptomyces hygroscopicus 5008. Microbiology 160, 13791392. [View Article][PubMed] [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000023
Loading
/content/journal/micro/10.1099/mic.0.000023
Loading

Data & Media loading...

Supplements

Supplementary Data



PDF
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