1887

Abstract

Chaperone and protease systems play essential roles in cellular homeostasis and have vital functions in controlling the abundance of specific cellular proteins involved in processes such as transcription, replication, metabolism and virulence. Bacteria have evolved accurate regulatory systems to control the expression and function of chaperones and potentially destructive proteases. Here, we have used a combination of transcriptomics, proteomics and targeted mutagenesis to reveal that the gene regulator (ClgR) of activates the transcription of at least ten genes, including four that encode protease systems (ClpP1/C, ClpP2/C, PtrB and HtrA-like protease Rv1043c) and three that encode chaperones (Acr2, ClpB and the chaperonin Rv3269). Thus, ClgR controls a larger network of protein homeostatic and regulatory systems than ClgR in any other bacterium studied to date. We demonstrate that ClgR-regulated transcriptional activation of these systems is essential for to replicate in macrophages. Furthermore, we observe that this defect is manifest early in infection, as lacking ClgR is deficient in the ability to control phagosome pH 1 h post-phagocytosis.

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2010-11-01
2020-01-22
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References

  1. Arsène, F., Tomoyasu, T. & Bukau, B. ( 2000; ). The heat shock response of Escherichia coli. Int J Food Microbiol 55, 3–9.[CrossRef]
    [Google Scholar]
  2. Bailey, T. L. & Elkan, C. ( 1994; ). Fitting a mixture model by expectation maximization to discover motifs in biopolymers. In Second International Conference on Intelligent Systems for Molecular Biology, pp. 28–36. Menlo Park, CA: AAAI Press.
  3. Bailey, T. L. & Gribskov, M. ( 1998; ). Combining evidence using p-values: application to sequence homology searches. Bioinformatics 14, 48–54.[CrossRef]
    [Google Scholar]
  4. Barik, S., Sureka, K., Mukherjee, P., Basu, J. & Kundu, M. ( 2010; ). RseA, the SigE specific anti-sigma factor of Mycobacterium tuberculosis, is inactivated by phosphorylation-dependent ClpC1P2 proteolysis. Mol Microbiol 75, 592–606.
    [Google Scholar]
  5. Bellier, A. & Mazodier, P. ( 2004; ). ClgR, a novel regulator of clp and lon expression in Streptomyces. J Bacteriol 186, 3238–3248.[CrossRef]
    [Google Scholar]
  6. Bellier, A., Gominet, M. & Mazodier, P. ( 2006; ). Post-translational control of the Streptomyces lividans ClgR regulon by ClpP. Microbiology 152, 1021–1027.[CrossRef]
    [Google Scholar]
  7. Breitling, R., Armengaud, P., Amtmann, A. & Herzyk, P. ( 2004; ). Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments. FEBS Lett 573, 83–92.[CrossRef]
    [Google Scholar]
  8. Brötz-Oesterhelt, H., Beyer, D., Kroll, H. P., Endermann, R., Ladel, C., Schroeder, W., Hinzen, B., Raddatz, S., Paulsen, H. & other authors ( 2005; ). Dysregulation of bacterial proteolytic machinery by a new class of antibiotics. Nat Med 11, 1082–1087.[CrossRef]
    [Google Scholar]
  9. Butler, S. M., Festa, R. A., Pearce, M. J. & Darwin, K. H. ( 2006; ). Self-compartmentalized bacterial proteases and pathogenesis. Mol Microbiol 60, 553–562.[CrossRef]
    [Google Scholar]
  10. Darwin, K. H. ( 2009; ). Prokaryotic ubiquitin-like protein (Pup), proteasomes and pathogenesis. Nat Rev Microbiol 7, 485–491.[CrossRef]
    [Google Scholar]
  11. Darwin, K. H., Ehrt, S., Gutierrez-Ramos, J. C., Weich, N. & Nathan, C. F. ( 2003; ). The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science 302, 1963–1966.[CrossRef]
    [Google Scholar]
  12. Dussurget, O., Stewart, G., Neyrolles, O., Pescher, P., Young, D. & Marchal, G. ( 2001; ). Role of Mycobacterium tuberculosis copper-zinc superoxide dismutase. Infect Immun 69, 529–533.[CrossRef]
    [Google Scholar]
  13. Engels, S., Schweitzer, J. E., Ludwig, C., Bott, M. & Schaffer, S. ( 2004; ). clpC and clpP1P2 gene expression in Corynebacterium glutamicum is controlled by a regulatory network involving the transcriptional regulators ClgR and HspR as well as the ECF sigma factor σ H. Mol Microbiol 52, 285–302.[CrossRef]
    [Google Scholar]
  14. Engels, S., Ludwig, C., Schweitzer, J. E., Mack, C., Bott, M. & Schaffer, S. ( 2005; ). The transcriptional activator ClgR controls transcription of genes involved in proteolysis and DNA repair in Corynebacterium glutamicum. Mol Microbiol 57, 576–591.[CrossRef]
    [Google Scholar]
  15. Flynn, J. M., Neher, S. B., Kim, Y. I., Sauer, R. T. & Baker, T. A. ( 2003; ). Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. Mol Cell 11, 671–683.[CrossRef]
    [Google Scholar]
  16. Frees, D., Chastanet, A., Qazi, S., Sorensen, K., Hill, P., Msadek, T. & Ingmer, H. ( 2004; ). Clp ATPases are required for stress tolerance, intracellular replication and biofilm formation in Staphylococcus aureus. Mol Microbiol 54, 1445–1462.[CrossRef]
    [Google Scholar]
  17. Frees, D., Savijoki, K., Varmanen, P. & Ingmer, H. ( 2007; ). Clp ATPases and ClpP proteolytic complexes regulate vital biological processes in low GC, Gram-positive bacteria. Mol Microbiol 63, 1285–1295.[CrossRef]
    [Google Scholar]
  18. Gaillot, O., Pellegrini, E., Bregenholt, S., Nair, S. & Berche, P. ( 2000; ). The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes. Mol Microbiol 35, 1286–1294.
    [Google Scholar]
  19. Gerth, U., Kock, H., Kusters, I., Michalik, S., Switzer, R. L. & Hecker, M. ( 2008; ). Clp-dependent proteolysis down-regulates central metabolic pathways in glucose-starved Bacillus subtilis. J Bacteriol 190, 321–331.[CrossRef]
    [Google Scholar]
  20. Gottesman, S. ( 2003; ). Proteolysis in bacterial regulatory circuits. Annu Rev Cell Dev Biol 19, 565–587.[CrossRef]
    [Google Scholar]
  21. Gottesman, S., Wickner, S. & Maurizi, M. R. ( 1997; ). Protein quality control: triage by chaperones and proteases. Genes Dev 11, 815–823.[CrossRef]
    [Google Scholar]
  22. Ibrahim, Y. M., Kerr, A. R., Silva, N. A. & Mitchell, T. J. ( 2005; ). Contribution of the ATP-dependent protease ClpCP to the autolysis and virulence of Streptococcus pneumoniae. Infect Immun 73, 730–740.[CrossRef]
    [Google Scholar]
  23. Iwanczyk, J., Damjanovic, D., Kooistra, J., Leong, V., Jomaa, A., Ghirlando, R. & Ortega, J. ( 2007; ). Role of the PDZ domains in Escherichia coli DegP protein. J Bacteriol 189, 3176–3186.[CrossRef]
    [Google Scholar]
  24. Kajfasz, J. K., Martinez, A. R., Rivera-Ramos, I., Abranches, J., Koo, H., Quivey, R. G., Jr & Lemos, J. A. ( 2009; ). Role of Clp proteins in expression of virulence properties of Streptococcus mutans. J Bacteriol 191, 2060–2068.[CrossRef]
    [Google Scholar]
  25. Kock, H., Gerth, U. & Hecker, M. ( 2004; ). The ClpP peptidase is the major determinant of bulk protein turnover in Bacillus subtilis. J Bacteriol 186, 5856–5864.[CrossRef]
    [Google Scholar]
  26. Krüger, E., Zuhlke, D., Witt, E., Ludwig, H. & Hecker, M. ( 2001; ). Clp-mediated proteolysis in Gram-positive bacteria is autoregulated by the stability of a repressor. EMBO J 20, 852–863.[CrossRef]
    [Google Scholar]
  27. Manganelli, R., Voskuil, M. I., Schoolnik, G. K. & Smith, I. ( 2001; ). The Mycobacterium tuberculosis ECF sigma factor σ E: role in global gene expression and survival in macrophages. Mol Microbiol 41, 423–437.[CrossRef]
    [Google Scholar]
  28. Manganelli, R., Voskuil, M. I., Schoolnik, G. K., Dubnau, E., Gomez, M. & Smith, I. ( 2002; ). Role of the extracytoplasmic-function sigma factor σ H (H) in Mycobacterium tuberculosis global gene expression. Mol Microbiol 45, 365–374.[CrossRef]
    [Google Scholar]
  29. Mehra, S. & Kaushal, D. ( 2009; ). Functional genomics reveals extended roles of the Mycobacterium tuberculosis stress response factor σ H. J Bacteriol 191, 3965–3980.[CrossRef]
    [Google Scholar]
  30. Meltzer, M., Hasenbein, S., Hauske, P. & other authors ( 2008; ). Allosteric activation of HtrA protease DegP by stress signals during bacterial protein quality control. Angew Chem Int Ed Engl 47, 1332–1334.[CrossRef]
    [Google Scholar]
  31. O'Connor, S. P., Rumschlag, H. S. & Mayer, L. W. ( 1990; ). Nucleotide sequence of the gene encoding the 35-kDa protein of Mycobacterium tuberculosis. Res Microbiol 141, 407–423.[CrossRef]
    [Google Scholar]
  32. Pang, X. & Howard, S. T. ( 2007; ). Regulation of the alpha-crystallin gene acr2 by the MprAB two-component system of Mycobacterium tuberculosis. J Bacteriol 189, 6213–6221.[CrossRef]
    [Google Scholar]
  33. Ribeiro-Guimarães, M. L. & Pessolani, M. C. ( 2007; ). Comparative genomics of mycobacterial proteases. Microb Pathog 43, 173–178.[CrossRef]
    [Google Scholar]
  34. Roback, P., Beard, J., Baumann, D., Gille, C., Henry, K., Krohn, S., Wiste, H., Voskuil, M. I., Rainville, C. & Rutherford, R. ( 2007; ). A predicted operon map for Mycobacterium tuberculosis. Nucleic Acids Res 35, 5085–5095.[CrossRef]
    [Google Scholar]
  35. Russell, D. G. ( 2001; ). Mycobacterium tuberculosis: here today, and here tomorrow. Nat Rev Mol Cell Biol 2, 569–577.[CrossRef]
    [Google Scholar]
  36. Sassetti, C. M., Boyd, D. H. & Rubin, E. J. ( 2001; ). Comprehensive identification of conditionally essential genes in mycobacteria. Proc Natl Acad Sci U S A 98, 12712–12717.[CrossRef]
    [Google Scholar]
  37. 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]
  38. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. ( 1996; ). Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68, 850–858.[CrossRef]
    [Google Scholar]
  39. Smyth, G. K. ( 2005; ). Limma: linear models for microarray data. In Bioinformatics and Computational Biology Solutions using R and Bioconductor, pp. 397–420. Edited by R. Gentleman, V. Carey, W. Huber, R. Irizarry & S. Dudoit. New York. : Springer.
    [Google Scholar]
  40. Smyth, G. K., Yang, Y. H. & Speed, T. ( 2003; ). Statistical issues in cDNA microarray data analysis. Methods Mol Biol 224, 111–136.
    [Google Scholar]
  41. Spiess, C., Beil, A. & Ehrmann, M. ( 1999; ). A temperature-dependent switch from chaperone to protease in a widely conserved heat-shock protein. Cell 97, 339–347.[CrossRef]
    [Google Scholar]
  42. Stewart, G. R., Snewin, V. A., Walzl, G., Hussell, T., Tormay, P., O'Gaora, P., Goyal, M., Betts, J., Brown, I. N. & Young, D. B. ( 2001; ). Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection. Nat Med 7, 732–737.[CrossRef]
    [Google Scholar]
  43. 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]
  44. Stewart, G. R., Newton, S. M., Wilkinson, K. A., Humphreys, I. R., Murphy, H. N., Robertson, B. D., Wilkinson, R. J. & Young, D. B. ( 2005; ). The stress-responsive chaperone alpha-crystallin 2 is required for pathogenesis of Mycobacterium tuberculosis. Mol Microbiol 55, 1127–1137.
    [Google Scholar]
  45. Tailleux, L., Waddell, S. J., Pelizzola, M., Mortellaro, A., Withers, M., Tanne, A., Castagnoli, P. R., Gicquel, B., Stoker, N. G. & other authors ( 2008; ). Probing host pathogen cross-talk by transcriptional profiling of both Mycobacterium tuberculosis and infected human dendritic cells and macrophages. PLoS ONE 3, e1403.[CrossRef]
    [Google Scholar]
  46. Van Montfort, R., Slingsby, C. & Vierling, E. ( 2001; ). Structure and function of the small heat-shock protein/alpha-crystallin family of molecular chaperones. Adv Protein Chem 59, 105–156.
    [Google Scholar]
  47. Ventura, M., Zhang, Z., Cronin, M., Canchaya, C., Kenny, J. G., Fitzgerald, G. F. & van Sinderen, D. ( 2005; ). The ClgR protein regulates transcription of the clpP operon in Bifidobacterium breve UCC 2003. J Bacteriol 187, 8411–8426.[CrossRef]
    [Google Scholar]
  48. Wilkinson, K. A., Stewart, G. R., Newton, S. M., Vordermeier, H. M., Wain, J. R., Murphy, H. N., Horner, K., Young, D. B. & Wilkinson, R. J. ( 2005; ). Infection biology of a novel alpha-crystallin of Mycobacterium tuberculosis: Acr2. J Immunol 174, 4237–4243.[CrossRef]
    [Google Scholar]
  49. Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J. & Speed, T. P. ( 2002; ). Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30, e15.[CrossRef]
    [Google Scholar]
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Expression ratios of , and compared by microarray and qRT-PCR. Supplementary Figs [PDF]

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Primers and probes used for quantitative real-time PCR of gene targets. [PDF]

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Downregulated genes in the Δ mutant versus wild-type . [Excel file]

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Transcription of genes in complemented Δ mutant versus wild-type . [Excel file]

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LC-MS/MS proteomic data of Δ mutant versus wild-type . [Excel file]

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