1887

Abstract

Using antibodies raised with fusion proteins, we localized a hypothetical protein encoded by the ORF ct622 in the cytoplasm of -infected mammalian cells. The detection was specific since the antibody labelling of CT622 protein was removed by preabsorption with CT622 but not other fusion proteins. We similarly confirmed that CT621, a known secretion protein encoded by a hypothetical ORF downstream of ct622, was secreted into host cell cytosol. Proteins CT622 and CT621 displayed a similar secretion pattern, with both intra-inclusion and host cell cytosol localization, that was distinct from that of CPAF (chlamydial protease/proteasome-like activity factor). However, the expression and secretion kinetics differed significantly between CT622 and CT621: CT622 mRNA was detected at 2 h, protein at 6 h and secretion of protein into host cell cytoplasm at 36 h post-infection, while CT621 mRNA was detected at 8 h, protein at 16 h and secretion at 24 h. The secretion of both CT622 and CT621 was blocked by ′-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide (compound 1), an inhibitor known to target the type III secretion system of bacteria. These results suggest that CT621 and CT622 may fulfil different functions during chlamydial intracellular growth. Further characterization of these proteins may generate important information for understanding chlamydial pathogenesis.

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2011-04-01
2024-12-13
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References

  1. Aiello D., Williams J. D., Majgier-Baranowska H., Patel I., Peet N. P., Huang J., Lory S., Bowlin T. L., Moir D. T. 2010; Discovery and characterization of inhibitors of Pseudomonas aeruginosa type III secretion. Antimicrob Agents Chemother 54:1988–1999
    [Google Scholar]
  2. Andersen J. N., Sathyanarayanan S., Di Bacco A., Chi A., Zhang T., Chen A. H., Dolinski B., Kraus M., Roberts B. other authors 2010; Pathway-based identification of biomarkers for targeted therapeutics: personalized oncology with PI3K pathway inhibitors. Sci Transl Med 2:ra55
    [Google Scholar]
  3. Arnold R., Brandmaier S., Kleine F., Tischler P., Heinz E., Behrens S., Niinikoski A., Mewes H. W., Horn M., Rattei T. 2009; Sequence-based prediction of type III secreted proteins. PLoS Pathog 5:e1000376
    [Google Scholar]
  4. Balsalobre C., Silván J. M., Berglund S., Mizunoe Y., Uhlin B. E., Wai S. N. 2006; Release of the type I secreted alpha-haemolysin via outer membrane vesicles from Escherichia coli . Mol Microbiol 59:99–112
    [Google Scholar]
  5. Belland R. J., Zhong G., Crane D. D., Hogan D., Sturdevant D., Sharma J., Beatty W. L., Caldwell H. D. 2003; Genomic transcriptional profiling of the developmental cycle of Chlamydia trachomatis . Proc Natl Acad Sci U S A 100:8478–8483
    [Google Scholar]
  6. Betts-Hampikian H., Fields K. 2010; The chlamydial type III secretion mechanism: revealing cracks in a tough nut. Front Cell Infect Microbiol 1:
    [Google Scholar]
  7. Chellas-Géry B., Linton C. N., Fields K. A. 2007; Human GCIP interacts with CT847, a novel Chlamydia trachomatis type III secretion substrate, and is degraded in a tissue-culture infection model. Cell Microbiol 9:2417–2430
    [Google Scholar]
  8. Chen C., Chen D., Sharma J., Cheng W., Zhong Y., Liu K., Jensen J., Shain R., Arulanandam B., Zhong G. 2006; The hypothetical protein CT813 is localized in the Chlamydia trachomatis inclusion membrane and is immunogenic in women urogenitally infected with C. trachomatis . Infect Immun 74:4826–4840
    [Google Scholar]
  9. Chen D., Chai J., Hart P. J., Zhong G. 2009; Identifying catalytic residues in CPAF, a Chlamydia-secreted protease. Arch Biochem Biophys 485:16–23
    [Google Scholar]
  10. Chen D., Lei L., Lu C., Flores R., DeLisa M. P., Roberts T. C., Romesberg F. E., Zhong G. 2010; Secretion of the chlamydial virulence factor CPAF requires the Sec-dependent pathway. Microbiology 156:3031–3040
    [Google Scholar]
  11. Clifton D. R., Fields K. A., Grieshaber S. S., Dooley C. A., Fischer E. R., Mead D. J., Carabeo R. A., Hackstadt T. 2004; A chlamydial type III translocated protein is tyrosine-phosphorylated at the site of entry and associated with recruitment of actin. Proc Natl Acad Sci U S A 101:10166–10171
    [Google Scholar]
  12. Cocchiaro J. L., Kumar Y., Fischer E. R., Hackstadt T., Valdivia R. H. 2008; Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole. Proc Natl Acad Sci U S A 105:9379–9384
    [Google Scholar]
  13. den Hartog J. E., Morré S. A., Land J. A. 2006; Chlamydia trachomatis -associated tubal factor subfertility: immunogenetic aspects and serological screening. Hum Reprod Update 12:719–730
    [Google Scholar]
  14. Dong F., Su H., Huang Y., Zhong Y., Zhong G. 2004; Cleavage of host keratin 8 by a Chlamydia-secreted protease. Infect Immun 72:3863–3868
    [Google Scholar]
  15. Dong F., Flores R., Chen D., Luo J., Zhong Y., Wu Z., Zhong G. 2006; Localization of the hypothetical protein Cpn0797 in the cytoplasm of Chlamydia pneumoniae -infected host cells. Infect Immun 74:6479–6486
    [Google Scholar]
  16. Ellis T. N., Kuehn M. J. 2010; Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol Mol Biol Rev 74:81–94
    [Google Scholar]
  17. Fan T., Lu H., Hu H., Shi L., McClarty G. A., Nance D. M., Greenberg A. H., Zhong G. 1998; Inhibition of apoptosis in chlamydia-infected cells: blockade of mitochondrial cytochrome c release and caspase activation. J Exp Med 187:487–496
    [Google Scholar]
  18. Fan P., Dong F., Huang Y., Zhong G. 2002; Chlamydia pneumoniae secretion of a protease-like activity factor for degrading host cell transcription factors is required for major histocompatibility complex antigen expression. Infect Immun 70:345–349
    [Google Scholar]
  19. Fields K. A., Mead D. J., Dooley C. A., Hackstadt T. 2003; Chlamydia trachomatis type III secretion: evidence for a functional apparatus during early-cycle development. Mol Microbiol 48:671–683
    [Google Scholar]
  20. Galka F., Wai S. N., Kusch H., Engelmann S., Hecker M., Schmeck B., Hippenstiel S., Uhlin B. E., Steinert M. 2008; Proteomic characterization of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles. Infect Immun 76:1825–1836
    [Google Scholar]
  21. Giles D. K., Whittimore J. D., LaRue R. W., Raulston J. E., Wyrick P. B. 2006; Ultrastructural analysis of chlamydial antigen-containing vesicles everting from the Chlamydia trachomatis inclusion. Microbes Infect 8:1579–1591
    [Google Scholar]
  22. Hackstadt T., Scidmore M. A., Rockey D. D. 1995; Lipid metabolism in Chlamydia trachomatis -infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion. Proc Natl Acad Sci U S A 92:4877–4881
    [Google Scholar]
  23. Hackstadt T., Fischer E. R., Scidmore M. A., Rockey D. D., Heinzen R. A. 1997; Origins and functions of the chlamydial inclusion. Trends Microbiol 5:288–293
    [Google Scholar]
  24. Hackstadt T., Scidmore-Carlson M. A., Shaw E. I., Fischer E. R. 1999; The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion. Cell Microbiol 1:119–130
    [Google Scholar]
  25. Harmon D. E., Davis A. J., Castillo C., Mecsas J. 2010; Identification and characterization of small-molecule inhibitors of Yop translocation in Yersinia pseudotuberculosis . Antimicrob Agents Chemother 54:3241–3254
    [Google Scholar]
  26. Hobolt-Pedersen A. S., Christiansen G., Timmerman E., Gevaert K., Birkelund S. 2009; Identification of Chlamydia trachomatis CT621, a protein delivered through the type III secretion system to the host cell cytoplasm and nucleus. FEMS Immunol Med Microbiol 57:46–58
    [Google Scholar]
  27. Hower S., Wolf K., Fields K. A. 2009; Evidence that CT694 is a novel Chlamydia trachomatis T3S substrate capable of functioning during invasion or early cycle development. Mol Microbiol 72:1423–1437
    [Google Scholar]
  28. Huang Z., Feng Y., Chen D., Wu X., Huang S., Wang X., Xiao X., Li W., Huang N., Gu L. 2008; Structural basis for activation and inhibition of the secreted chlamydia protease CPAF. Cell Host Microbe 4:529–542
    [Google Scholar]
  29. Kauppi A. M., Nordfelth R., Uvell H., Wolf-Watz H., Elofsson M. 2003; Targeting bacterial virulence: inhibitors of type III secretion in Yersinia. Chem Biol 10:241–249
    [Google Scholar]
  30. Kim J. Y., Doody A. M., Chen D. J., Cremona G. H., Shuler M. L., Putnam D., DeLisa M. P. 2008; Engineered bacterial outer membrane vesicles with enhanced functionality. J Mol Biol 380:51–66
    [Google Scholar]
  31. Kouokam J. C., Wai S. N., Fällman M., Dobrindt U., Hacker J., Uhlin B. E. 2006; Active cytotoxic necrotizing factor 1 associated with outer membrane vesicles from uropathogenic Escherichia coli . Infect Immun 74:2022–2030
    [Google Scholar]
  32. Kuehn M. J., Kesty N. C. 2005; Bacterial outer membrane vesicles and the host–pathogen interaction. Genes Dev 19:2645–2655
    [Google Scholar]
  33. Kumar Y., Valdivia R. H. 2008; Actin and intermediate filaments stabilize the Chlamydia trachomatis vacuole by forming dynamic structural scaffolds. Cell Host Microbe 4:159–169
    [Google Scholar]
  34. Li Z., Chen C., Chen D., Wu Y., Zhong Y., Zhong G. 2008a; Characterization of fifty putative inclusion membrane proteins encoded in the Chlamydia trachomatis genome. Infect Immun 76:2746–2757
    [Google Scholar]
  35. Li Z., Chen D., Zhong Y., Wang S., Zhong G. 2008b; The chlamydial plasmid-encoded protein pgp3 is secreted into the cytosol of Chlamydia-infected cells. Infect Immun 76:3415–3428
    [Google Scholar]
  36. McClarty G. 1994; Chlamydiae and the biochemistry of intracellular parasitism. Trends Microbiol 2:157–164
    [Google Scholar]
  37. Mertz K. J., McQuillan G. M., Levine W. C., Candal D. H., Bullard J. C., Johnson R. E., St Louis M. E., Black C. M. 1998; A pilot study of the prevalence of chlamydial infection in a national household survey. Sex Transm Dis 25:225–228
    [Google Scholar]
  38. Misaghi S., Balsara Z. R., Catic A., Spooner E., Ploegh H. L., Starnbach M. N. 2006; Chlamydia trachomatis -derived deubiquitinating enzymes in mammalian cells during infection. Mol Microbiol 61:142–150
    [Google Scholar]
  39. Mullaney E., Brown P. A., Smith S. M., Botting C. H., Yamaoka Y. Y., Terres A. M., Kelleher D. P., Windle H. J. 2009; Proteomic and functional characterization of the outer membrane vesicles from the gastric pathogen Helicobacter pylori . Proteomics Clin Appl 3:785–796
    [Google Scholar]
  40. Nordfelth R., Kauppi A. M., Norberg H. A., Wolf-Watz H., Elofsson M. 2005; Small-molecule inhibitors specifically targeting type III secretion. Infect Immun 73:3104–3114
    [Google Scholar]
  41. Peterman T. A., Tian L. H., Metcalf C. A., Satterwhite C. L., Malotte C. K., DeAugustine N., Paul S. M., Cross H., Rietmeijer C. A. other authors 2006; High incidence of new sexually transmitted infections in the year following a sexually transmitted infection: a case for rescreening. Ann Intern Med 145:564–572
    [Google Scholar]
  42. Pirbhai M., Dong F., Zhong Y., Pan K. Z., Zhong G. 2006; The secreted protease factor CPAF is responsible for degrading pro-apoptotic BH3-only proteins in Chlamydia trachomatis -infected cells. J Biol Chem 281:31495–31501
    [Google Scholar]
  43. Rockey D. D., Scidmore M. A., Bannantine J. P., Brown W. J. 2002; Proteins in the chlamydial inclusion membrane. Microbes Infect 4:333–340
    [Google Scholar]
  44. Rockey D. D., Wang J., Lei L., Zhong G. 2009; Chlamydia vaccine candidates and tools for chlamydial antigen discovery. Expert Rev Vaccines 8:1365–1377
    [Google Scholar]
  45. Samudrala R., Heffron F., McDermott J. E. 2009; Accurate prediction of secreted substrates and identification of a conserved putative secretion signal for type III secretion systems. PLoS Pathog 5:e1000375
    [Google Scholar]
  46. Scidmore M. 2008; Chlamydia weave a protective cloak spun of actin and intermediate filaments. Cell Host Microbe 4:93–95
    [Google Scholar]
  47. Sharma J., Bosnic A. M., Piper J. M., Zhong G. 2004; Human antibody responses to a Chlamydia-secreted protease factor. Infect Immun 72:7164–7171
    [Google Scholar]
  48. Sharma J., Zhong Y., Dong F., Piper J. M., Wang G., Zhong G. 2006; Profiling of human antibody responses to Chlamydia trachomatis urogenital tract infection using microplates arrayed with 156 chlamydial fusion proteins. Infect Immun 74:1490–1499
    [Google Scholar]
  49. Sherman K. J., Daling J. R., Stergachis A., Weiss N. S., Foy H. M., Wang S. P., Grayston J. T. 1990; Sexually transmitted diseases and tubal pregnancy. Sex Transm Dis 17:115–121
    [Google Scholar]
  50. Slepenkin A., de la Maza L. M., Peterson E. M. 2005; Interaction between components of the type III secretion system of Chlamydiaceae. J Bacteriol 187:473–479
    [Google Scholar]
  51. Slepenkin A., Enquist P. A., Hägglund U., de la Maza L. M., Elofsson M., Peterson E. M. 2007; Reversal of the antichlamydial activity of putative type III secretion inhibitors by iron. Infect Immun 75:3478–3489
    [Google Scholar]
  52. Spaeth K. E., Chen Y. S., Valdivia R. H. 2009; The Chlamydia type III secretion system C-ring engages a chaperone-effector protein complex. PLoS Pathog 5:e1000579
    [Google Scholar]
  53. Stephens R. S. 2003; The cellular paradigm of chlamydial pathogenesis. Trends Microbiol 11:44–51
    [Google Scholar]
  54. Su H., McClarty G., Dong F., Hatch G. M., Pan Z. K., Zhong G. 2004; Activation of Raf/MEK/ERK/cPLA2 signaling pathway is essential for chlamydial acquisition of host glycerophospholipids. J Biol Chem 279:9409–9416
    [Google Scholar]
  55. Subtil A., Delevoye C., Balañá M. E., Tastevin L., Perrinet S., Dautry-Varsat A. 2005; A directed screen for chlamydial proteins secreted by a type III mechanism identifies a translocated protein and numerous other new candidates. Mol Microbiol 56:1636–1647
    [Google Scholar]
  56. Toye B., Laferrière C., Claman P., Jessamine P., Peeling R. 1993; Association between antibody to the chlamydial heat-shock protein and tubal infertility. J Infect Dis 168:1236–1240
    [Google Scholar]
  57. Valdivia R. H. 2008; Chlamydia effector proteins and new insights into chlamydial cellular microbiology. Curr Opin Microbiol 11:53–59
    [Google Scholar]
  58. Vandahl B. B., Stensballe A., Roepstorff P., Christiansen G., Birkelund S. 2005; Secretion of Cpn0796 from Chlamydia pneumoniae into the host cell cytoplasm by an autotransporter mechanism. Cell Microbiol 7:825–836
    [Google Scholar]
  59. Wolf K., Betts H. J., Chellas-Géry B., Hower S., Linton C. N., Fields K. A. 2006; Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle. Mol Microbiol 61:1543–1555
    [Google Scholar]
  60. Wright H. R., Turner A., Taylor H. R. 2008; Trachoma. Lancet 371:1945–1954
    [Google Scholar]
  61. Xiao Y., Zhong Y., Su H., Zhou Z., Chiao P., Zhong G. 2005; NF-kappa B activation is not required for Chlamydia trachomatis inhibition of host epithelial cell apoptosis. J Immunol 174:1701–1708
    [Google Scholar]
  62. Zhong G. 2009; Killing me softly: chlamydial use of proteolysis for evading host defenses. Trends Microbiol 17:467–474
    [Google Scholar]
  63. Zhong G., Reis, Germain R. N., e Sousa C. 1997; Production, specificity, and functionality of monoclonal antibodies to specific peptide-major histocompatibility complex class II complexes formed by processing of exogenous protein. Proc Natl Acad Sci U S A 94:13856–13861
    [Google Scholar]
  64. Zhong G., Fan T., Liu L. 1999; Chlamydia inhibits interferon gamma-inducible major histocompatibility complex class II expression by degradation of upstream stimulatory factor 1. J Exp Med 189:1931–1938
    [Google Scholar]
  65. Zhong G., Liu L., Fan T., Fan P., Ji H. 2000; Degradation of transcription factor RFX5 during the inhibition of both constitutive and interferon gamma-inducible major histocompatibility complex class I expression in chlamydia-infected cells. J Exp Med 191:1525–1534
    [Google Scholar]
  66. Zhong G., Fan P., Ji H., Dong F., Huang Y. 2001; Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors. J Exp Med 193:935–942
    [Google Scholar]
  67. Zhong G., Lei L., Gong S., Lu C., Qi M., Chen D. 2011; Chlamydia-secreted proteins in chlamydial interactions with host cells. Current Chemical Biology 5:29–37
    [Google Scholar]
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