1887

Abstract

HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. serovar Typhimurium ( Typhimurium) mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of in is governed by a single promoter that is dependent on (RpoE). Typhimurium also possesses a -dependent promoter; however, we found that the absence of had little effect on production of HtrA by Typhimurium. This suggests that additional promoters control expression of in Typhimurium. We identified three Typhimurium promoters. Only the most proximal promoter, , was dependent. The other promoters, and , are probably recognized by the principal sigma factor . These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of is different in Typhimurium and . The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to Typhimurium and . Point or deletion mutants of that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These variants were placed under the control of the Typhimurium promoters and expressed in a Typhimurium mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 °C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of HtrA to complement the growth defects of GVB1343 or .

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2009-03-01
2019-10-20
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References

  1. Baumler, A. J., Kusters, J. G., Stojiljkovic, I. & Heffron, F. ( 1994; ). Salmonella typhimurium loci involved in survival within macrophages. Infect Immun 62, 1623–1630.
    [Google Scholar]
  2. Bringer, M. A., Barnich, N., Glasser, A. L., Bardot, O. & Darfeuille-Michaud, A. ( 2005; ). HtrA stress protein is involved in intramacrophagic replication of adherent and invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease. Infect Immun 73, 712–721.[CrossRef]
    [Google Scholar]
  3. CastilloKeller, M & Misra, R. ( 2003; ). Protease-deficient DegP suppresses lethal effects of a mutant OmpC protein by its capture. J Bacteriol 185, 148–154.[CrossRef]
    [Google Scholar]
  4. Chatfield, S. N., Strahan, K., Pickard, D., Charles, I. G., Hormaeche, C. E. & Dougan, G. ( 1992; ). Evaluation of Salmonella typhimurium strains harbouring defined mutations in htrA and aroA in the murine salmonellosis model. Microb Pathog 12, 145–151.[CrossRef]
    [Google Scholar]
  5. Cortes, G., de Astorza, B., Benedi, V. J. & Alberti, S. ( 2002; ). Role of the htrA gene in Klebsiella pneumoniae virulence. Infect Immun 70, 4772–4776.[CrossRef]
    [Google Scholar]
  6. Danese, P. N., Snyder, W. B., Cosma, C. L., Davis, L. J. & Silhavy, T. J. ( 1995; ). The Cpx two-component signal transduction pathway of Escherichia coli regulates transcription of the gene specifying the stress-inducible periplasmic protease, DegP. Genes Dev 9, 387–398.[CrossRef]
    [Google Scholar]
  7. De Wulf, P., McGuire, A. M., Liu, X. & Lin, E. C. ( 2002; ). Genome-wide profiling of promoter recognition by the two-component response regulator CpxR-P in Escherichia coli. J Biol Chem 277, 26652–26661.[CrossRef]
    [Google Scholar]
  8. Eriksson, S., Lucchini, S., Thompson, A., Rhen, M. & Hinton, J. C. ( 2003; ). Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Mol Microbiol 47, 103–118.
    [Google Scholar]
  9. Everest, P., Frankel, G., Li, J., Lund, P., Chatfield, S. & Dougan, G. ( 1995; ). Expression of LacZ from the htrA, nirB and groE promoters in a Salmonella vaccine strain: influence of growth in mammalian cells. FEMS Microbiol Lett 126, 97–101.[CrossRef]
    [Google Scholar]
  10. Foster, J. W. & Spector, M. P. ( 1995; ). How Salmonella survive against the odds. Annu Rev Microbiol 49, 145–174.[CrossRef]
    [Google Scholar]
  11. Hoiseth, S. K. & Stocker, B. A. ( 1981; ). Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239.[CrossRef]
    [Google Scholar]
  12. Humphreys, S., Stevenson, A., Bacon, A., Weinhardt, A. B. & Roberts, M. ( 1999; ). The alternative sigma factor, σ E, is critically important for the virulence of Salmonella typhimurium. Infect Immun 67, 1560–1568.
    [Google Scholar]
  13. Humphreys, S., Rowley, G., Stevenson, A., Kenyon, W. J., Spector, M. P. & Roberts, M. ( 2003; ). Role of periplasmic peptidylprolyl isomerases in Salmonella enterica serovar Typhimurium virulence. Infect Immun 71, 5386–5388.[CrossRef]
    [Google Scholar]
  14. Ibrahim, Y. M., Kerr, A. R., McCluskey, J. & Mitchell, T. J. ( 2004; ). Role of HtrA in the virulence and competence of Streptococcus pneumoniae. Infect Immun 72, 3584–3591.[CrossRef]
    [Google Scholar]
  15. Jiang, J., Zhang, X., Chen, Y., Wu, Y., Zhou, Z. H., Chang, Z. & Sui, S. F. ( 2008; ). Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins. Proc Natl Acad Sci U S A 105, 11939–11944.[CrossRef]
    [Google Scholar]
  16. Johnson, K., Charles, I., Dougan, G., Pickard, D., O'Gaora, P., Costa, G., Ali, T., Miller, I. & Hormaeche, C. ( 1991; ). The role of a stress-response protein in Salmonella typhimurium virulence. Mol Microbiol 5, 401–407.[CrossRef]
    [Google Scholar]
  17. Jones, C. H., Bolken, T. C., Jones, K. F., Zeller, G. O. & Hruby, D. E. ( 2001; ). Conserved DegP protease in gram-positive bacteria is essential for thermal and oxidative tolerance and full virulence in Streptococcus pyogenes. Infect Immun 69, 5538–5545.[CrossRef]
    [Google Scholar]
  18. Kenyon, W. J., Sayers, D. G., Humphreys, S., Roberts, M. & Spector, M. P. ( 2002; ). The starvation-stress response of Salmonella enterica serovar Typhimurium requires σ E-, but not CpxR-regulated extracytoplasmic functions. Microbiology 148, 113–122.
    [Google Scholar]
  19. Kormanec, J. ( 2001; ). Analyzing the developmental expression of sigma factors with S1-nuclease mapping. In Nuclease Methods and Protocols, pp. 481–494. Edited by C. H. Chein: Humana Press.
  20. Krojer, T., Sawa, J., Schäfer, E., Saibil, H. R., Ehrmann, M. & Clausen, T. ( 2008; ). Structural basis for the regulated protease and chaperone function of DegP. Nature 453, 885–890.[CrossRef]
    [Google Scholar]
  21. Lewis, C., Skovierova, H., Rowley, G., Rezuchova, B., Homerova, D., Stevenson, A., Sherry, A., Kormanec, J. & Roberts, M. ( 2008; ). Small outer-membrane lipoprotein, SmpA, is regulated by σ E and has a role in cell envelope integrity and virulence of Salmonella enterica serovar Typhimurium. Microbiology 154, 979–988.[CrossRef]
    [Google Scholar]
  22. Lipinska, B., Sharma, S. & Georgopoulos, C. ( 1988; ). Sequence analysis and regulation of the htrA gene of Escherichia coli: a σ 32-independent mechanism of heat-inducible transcription. Nucleic Acids Res 16, 10053–10067.[CrossRef]
    [Google Scholar]
  23. Lipinska, B., Fayet, O., Baird, L. & Georgopoulos, C. ( 1989; ). Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures. J Bacteriol 171, 1574–1584.
    [Google Scholar]
  24. Maxam, A. M. & Gilbert, W. ( 1980; ). Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65, 499–560.
    [Google Scholar]
  25. Miller, J. H. ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
  26. Misra, R., CastilloKeller, M. & Deng, M. ( 2000; ). Overexpression of protease-deficient DegP(S210A) rescues the lethal phenotype of Escherichia coli OmpF assembly mutants in a degP background. J Bacteriol 182, 4882–4888.[CrossRef]
    [Google Scholar]
  27. Miticka, H., Rowley, G., Rezuchova, B., Homerova, D., Humphreys, S., Farn, J., Roberts, M. & Kormanec, J. ( 2003; ). Transcriptional analysis of the rpoE gene encoding extracytoplasmic stress response sigma factor σ E in Salmonella enterica serovar Typhimurium. FEMS Microbiol Lett 226, 307–314.[CrossRef]
    [Google Scholar]
  28. Pallen, M. J. & Wren, B. W. ( 1997; ). The HtrA family of serine proteases. Mol Microbiol 26, 209–221.[CrossRef]
    [Google Scholar]
  29. Pedersen, L. L., Radulic, M., Doric, M. & Abu, K. Y. ( 2001; ). HtrA homologue of Legionella pneumophila: an indispensable element for intracellular infection of mammalian but not protozoan cells. Infect Immun 69, 2569–2579.[CrossRef]
    [Google Scholar]
  30. Pogliano, J., Lynch, A. S., Belin, D., Lin, E. C. & Beckwith, J. ( 1997; ). Regulation of Escherichia coli cell envelope proteins involved in protein folding and degradation by the Cpx two-component system. Genes Dev 11, 1169–1182.[CrossRef]
    [Google Scholar]
  31. Pribnow, D. ( 1975; ). Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter. Proc Natl Acad Sci U S A 72, 784–788.[CrossRef]
    [Google Scholar]
  32. Resto-Ruiz, S. I., Sweger, D., Widen, R. H., Valkov, N. & Anderson, B. E. ( 2000; ). Transcriptional activation of the htrA (high-temperature requirement A) gene from Bartonella henselae. Infect Immun 68, 5970–5978.[CrossRef]
    [Google Scholar]
  33. Rezuchova, B. & Kormanec, J. ( 2001; ). A two-plasmid system for identification of promoters recognized by RNA polymerase containing extracytoplasmic stress response σ E in Escherichia coli. J Microbiol Methods 45, 103–111.[CrossRef]
    [Google Scholar]
  34. Rhodius, V. A., Suh, W. C., Nonaka, G., West, J. & Gross, C. A. ( 2006; ). Conserved and variable functions of the σ E stress response in related genomes. PLoS Biol 4, e2 [CrossRef]
    [Google Scholar]
  35. Rigoulay, C., Entenza, J. M., Halpern, D., Widmer, E., Moreillon, P., Poquet, I. & Gruss, A. ( 2005; ). Comparative analysis of the roles of HtrA-like surface proteases in two virulent Staphylococcus aureus strains. Infect Immun 73, 563–572.[CrossRef]
    [Google Scholar]
  36. Rowley, G. ( 2005; ). Characterisation of the S. Typhimurium σE regulon. PhD thesis, University of Glasgow.
  37. Rowley, G., Stevenson, A., Kormanec, J. & Roberts, M. ( 2005; ). Effect of inactivation of degS on Salmonella enterica serovar Typhimurium in vitro and in vivo. Infect Immun 73, 459–463.[CrossRef]
    [Google Scholar]
  38. Rowley, G., Spector, M., Kormanec, J. & Roberts, M. ( 2006; ). Pushing the envelope: extracytoplasmic stress responses in bacterial pathogens. Nat Rev Microbiol 4, 383–394.[CrossRef]
    [Google Scholar]
  39. Skorko-Glonek, J., Laskowska, E., Sobiecka-Szkatula, A. & Lipinska, B. ( 2007; ). Characterization of the chaperone-like activity of HtrA (DegP) protein from Escherichia coli under the conditions of heat shock. Arch Biochem Biophys 464, 80–89.[CrossRef]
    [Google Scholar]
  40. Skovierova, H., Rowley, G., Rezuchova, B., Homerova, D., Lewis, C., Roberts, M. & Kormanec, J. ( 2006; ). Identification of the σ E regulon of Salmonella enterica serovar Typhimurium. Microbiology 152, 1347–1359.[CrossRef]
    [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. Testerman, T. L., Vazquez-Torres, A., Xu, Y., Jones-Carson, J., Libby, S. J. & Fang, F. C. ( 2002; ). The alternative sigma factor σ E controls antioxidant defences required for Salmonella virulence and stationary-phase survival. Mol Microbiol 43, 771–782.[CrossRef]
    [Google Scholar]
  43. Wang, R. F. & Kushner, S. R. ( 1991; ). Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene 100, 195–199.[CrossRef]
    [Google Scholar]
  44. Williams, K., Oyston, P. C., Dorrell, N., Li, S., Titball, R. W. & Wren, B. W. ( 2000; ). Investigation into the role of the serine protease HtrA in Yersinia pestis pathogenesis. FEMS Microbiol Lett 186, 281–286.[CrossRef]
    [Google Scholar]
  45. Yamamoto, T., Hanawa, T., Ogata, S. & Kamiya, S. ( 1997; ). The Yersinia enterocolitica GsrA stress protein, involved in intracellular survival, is induced by macrophage phagocytosis. Infect Immun 65, 2190–2196.
    [Google Scholar]
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