The Type III Secretion Chaperone Slc1 Engages Multiple Early Effectors, Including TepP, a Tyrosine-phosphorylated Protein Required for the Recruitment of CrkI-II to Nascent Inclusions and Innate Immune Signaling

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Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. Like many T3S effectors, TARP requires a chaperone (Slc1) for efficient translocation into host cells. In this study, we defined proteins that associate with Slc1 in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry. We identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form large molecular weight complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to EBs at entry sites where it remained associated with nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia.

Vyšlo v časopise: The Type III Secretion Chaperone Slc1 Engages Multiple Early Effectors, Including TepP, a Tyrosine-phosphorylated Protein Required for the Recruitment of CrkI-II to Nascent Inclusions and Innate Immune Signaling. PLoS Pathog 10(2): e32767. doi:10.1371/journal.ppat.1003954
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1003954

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Zdroje

1. LowN, CassellJA, SpencerB, BenderN, HilberAM, et al. (2012) Chlamydia control activities in Europe: cross-sectional survey. Eur J Public Health 22 : 556–561.

2. PascoliniD, MariottiSP (2012) Global estimates of visual impairment: 2010. Br J Ophthalmol 96 : 614–618.

3. CDC (2011) CDC Grand Rounds: Chlamydia prevention: challenges and strategies for reducing disease burden and sequelae. MMWR Morb Mortal Wkly Rep 60 : 370–373.

4. MoulderJW (1991) Interaction of Chlamydiae and host cells in vitro. Microbiol Rev 55 : 143–190.

5. HeinzenRA, ScidmoreMA, RockeyDD, HackstadtT (1996) Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis. Infect Immun 64 : 796–809.

6. ClausenJD, ChristiansenG, HolstHU, BirkelundS (1997) Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection. Mol Microbiol 25 : 441–449.

7. GrieshaberSS, GrieshaberNA, HackstadtT (2003) Chlamydia trachomatis uses host cell dynein to traffic to the microtubule-organizing center in a p50 dynamitin-independent process. J Cell Sci 116 : 3793–3802.

8. AbdelrahmanYM, BellandRJ (2005) The chlamydial developmental cycle. FEMS Microbiol Rev 29 : 949–959.

9. HybiskeK, StephensRS (2007) Mechanisms of host cell exit by the intracellular bacterium Chlamydia. Proc Natl Acad Sci U S A 104 : 11430–11435.

10. PetersJ, WilsonDP, MyersG, TimmsP, BavoilPM (2007) Type III secretion a la Chlamydia. Trends Microbiol 15 : 241–251.

11. BettsHJ, WolfK, FieldsKA (2009) Effector protein modulation of host cells: examples in the Chlamydia spp. arsenal. Curr Opin Microbiol 12 : 81–87.

12. CliftonDR, FieldsKA, GrieshaberSS, DooleyCA, FischerER, et al. (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.

13. LaneBJ, MutchlerC, Al KhodorS, GrieshaberSS, CarabeoRA (2008) Chlamydial entry involves TARP binding of guanine nucleotide exchange factors. PLoS Pathog 4: e1000014.

14. JewettTJ, MillerNJ, DooleyCA, HackstadtT (2010) The conserved Tarp actin binding domain is important for chlamydial invasion. PLoS Pathog 6: e1000997.

15. HowerS, WolfK, FieldsKA (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.

16. RockeyDD, ScidmoreMA, BannantineJP, BrownWJ (2002) Proteins in the chlamydial inclusion membrane. Microbes Infect 4 : 333–340.

17. DelevoyeC, NilgesM, DehouxP, PaumetF, PerrinetS, et al. (2008) SNARE protein mimicry by an intracellular bacterium. PLoS Pathog 4: e1000022.

18. DerreI, SwissR, AgaisseH (2011) The lipid transfer protein CERT interacts with the Chlamydia inclusion protein IncD and participates to ER-Chlamydia inclusion membrane contact sites. PLoS Pathog 7: e1002092.

19. RzompKA, MoorheadAR, ScidmoreMA (2006) The GTPase Rab4 interacts with Chlamydia trachomatis inclusion membrane protein CT229. Infect Immun 74 : 5362–5373.

20. ScidmoreMA, HackstadtT (2001) Mammalian 14-3-3beta associates with the Chlamydia trachomatis inclusion membrane via its interaction with IncG. Mol Microbiol 39 : 1638–1650.

21. SamudralaR, HeffronF, McDermottJE (2009) Accurate prediction of secreted substrates and identification of a conserved putative secretion signal for type III secretion systems. PLoS Pathog 5: e1000375.

22. CornelisGR (2006) The type III secretion injectisome. Nat Rev Microbiol 4 : 811–825.

23. ThomasNA, MaI, PrasadME, RafuseC (2012) Expanded roles for multicargo and class 1B effector chaperones in type III secretion. J Bacteriol 194 : 3767–3773.

24. IzoreT, JobV, DessenA (2011) Biogenesis, regulation, and targeting of the type III secretion system. Structure 19 : 603–612.

25. StephensRS, KalmanS, LammelC, FanJ, MaratheR, et al. (1998) Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282 : 754–759.

26. Fields KA HT (2006) The Chlamydia Type III Secretion System: Structure and Implications for Pathogenesis. In: Patrik M. Bavoil PBW, editor. Chlamydia: Genomics And Pathogenesis. Norfolk, UK: Horizon Bioscience. pp. 222–223.

27. BrinkworthAJ, MalcolmDS, PedrosaAT, RoguskaK, ShahbazianS, et al. (2011) Chlamydia trachomatis Slc1 is a type III secretion chaperone that enhances the translocation of its invasion effector substrate TARP. Mol Microbiol 82 : 131–144.

28. PaisSV, MilhoC, AlmeidaF, MotaLJ (2013) Identification of novel type III secretion chaperone-substrate complexes of Chlamydia trachomatis. PLoS One 8: e56292.

29. Silva-HerzogE, JosephSS, AveryAK, CobaJA, WolfK, et al. (2011) Scc1 (CP0432) and Scc4 (CP0033) function as a type III secretion chaperone for CopN of Chlamydia pneumoniae. J Bacteriol 193 : 3490–3496.

30. SpaethKE, ChenYS, ValdiviaRH (2009) The Chlamydia type III secretion system C-ring engages a chaperone-effector protein complex. PLoS Pathog 5: e1000579.

31. SakaHA, ThompsonJW, ChenYS, KumarY, DuboisLG, et al. (2011) Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms. Mol Microbiol 82 : 1185–1203.

32. TohH, MiuraK, ShiraiM, HattoriM (2003) In silico inference of inclusion membrane protein family in obligate intracellular parasites Chlamydiae. DNA Res 10 : 9–17.

33. LiZ, ChenC, ChenD, WuY, ZhongY, et al. (2008) Characterization of fifty putative inclusion membrane proteins encoded in the Chlamydia trachomatis genome. Infect Immun 76 : 2746–2757.

34. FeldmanMF, CornelisGR (2003) The multitalented type III chaperones: all you can do with 15 kDa. FEMS Microbiol Lett 219 : 151–158.

35. EricksonHP (2009) Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. Biol Proced Online 11 : 32–51.

36. ArnoldR, BrandmaierS, KleineF, TischlerP, HeinzE, et al. (2009) Sequence-based prediction of type III secreted proteins. PLoS Pathog 5: e1000376.

37. BirkelundS, JohnsenH, ChristiansenG (1994) Chlamydia trachomatis serovar L2 induces protein tyrosine phosphorylation during uptake by HeLa cells. Infect Immun 62 : 4900–4908.

38. FawazFS, van OoijC, HomolaE, MutkaSC, EngelJN (1997) Infection with Chlamydia trachomatis alters the tyrosine phosphorylation and/or localization of several host cell proteins including cortactin. Infect Immun 65 : 5301–5308.

39. BackertS, SelbachM (2005) Tyrosine-phosphorylated bacterial effector proteins: the enemies within. Trends Microbiol 13 : 476–484.

40. MillerML, JensenLJ, DiellaF, JorgensenC, TintiM, et al. (2008) Linear motif atlas for phosphorylation-dependent signaling. Sci Signal 1: ra2.

41. SongyangZ, ShoelsonSE, ChaudhuriM, GishG, PawsonT, et al. (1993) SH2 domains recognize specific phosphopeptide sequences. Cell 72 : 767–778.

42. BirgeRB, KalodimosC, InagakiF, TanakaS (2009) Crk and CrkL adaptor proteins: networks for physiological and pathological signaling. Cell Commun Signal 7 : 13.

43. NguyenBD, ValdiviaRH (2012) Virulence determinants in the obligate intracellular pathogen Chlamydia trachomatis revealed by forward genetic approaches. Proc Natl Acad Sci U S A 109 : 1263–1268.

44. WangY, KahaneS, CutcliffeLT, SkiltonRJ, LambdenPR, et al. (2011) Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathog 7: e1002258.

45. BucknerLR, SchustDJ, DingJ, NagamatsuT, BeattyW, et al. (2011) Innate immune mediator profiles and their regulation in a novel polarized immortalized epithelial cell model derived from human endocervix. J Reprod Immunol 92 : 8–20.

46. ValdiviaRH (2008) Chlamydia effector proteins and new insights into chlamydial cellular microbiology. Curr Opin Microbiol 11 : 53–59.

47. DarwinKH, MillerVL (2001) Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium. EMBO J 20 : 1850–1862.

48. FrancisMS, LloydSA, Wolf-WatzH (2001) The type III secretion chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis. Mol Microbiol 42 : 1075–1093.

49. RaoX, DeighanP, HuaZ, HuX, WangJ, et al. (2009) A regulator from Chlamydia trachomatis modulates the activity of RNA polymerase through direct interaction with the beta subunit and the primary sigma subunit. Genes Dev 23 : 1818–1829.

50. FahertyCS, MaurelliAT (2009) Spa15 of Shigella flexneri is secreted through the type III secretion system and prevents staurosporine-induced apoptosis. Infect Immun 77 : 5281–5290.

51. ParsotC, HamiauxC, PageAL (2003) The various and varying roles of specific chaperones in type III secretion systems. Curr Opin Microbiol 6 : 7–14.

52. BoydAP, LambermontI, CornelisGR (2000) Competition between the Yops of Yersinia enterocolitica for delivery into eukaryotic cells: role of the SycE chaperone binding domain of YopE. J Bacteriol 182 : 4811–4821.

53. ThomasNA, DengW, BakerN, PuenteJ, FinlayBB (2007) Hierarchical delivery of an essential host colonization factor in enteropathogenic Escherichia coli. J Biol Chem 282 : 29634–29645.

54. MillsE, BaruchK, CharpentierX, KobiS, RosenshineI (2008) Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli. Cell Host Microbe 3 : 104–113.

55. SuzukiM, MimuroH, SuzukiT, ParkM, YamamotoT, et al. (2005) Interaction of CagA with Crk plays an important role in Helicobacter pylori-induced loss of gastric epithelial cell adhesion. J Exp Med 202 : 1235–1247.

56. DengQ, SunJ, BarbieriJT (2005) Uncoupling Crk signal transduction by Pseudomonas exoenzyme T. J Biol Chem 280 : 35953–35960.

57. KiyokawaE, HashimotoY, KobayashiS, SugimuraH, KurataT, et al. (1998) Activation of Rac1 by a Crk SH3-binding protein, DOCK180. Genes Dev 12 : 3331–3336.

58. NimnualAS, YatsulaBA, Bar-SagiD (1998) Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos. Science 279 : 560–563.

59. CarabeoRA, GrieshaberSS, HasenkrugA, DooleyC, HackstadtT (2004) Requirement for the Rac GTPase in Chlamydia trachomatis invasion of non-phagocytic cells. Traffic 5 : 418–425.

60. ElwellCA, CeesayA, KimJH, KalmanD, EngelJN (2008) RNA interference screen identifies Abl kinase and PDGFR signaling in Chlamydia trachomatis entry. PLoS Pathog 4: e1000021.

61. HuiDJ, TerenziF, MerrickWC, SenGC (2005) Mouse p56 blocks a distinct function of eukaryotic initiation factor 3 in translation initiation. J Biol Chem 280 : 3433–3440.

62. TerenziF, HuiDJ, MerrickWC, SenGC (2006) Distinct induction patterns and functions of two closely related interferon-inducible human genes, ISG54 and ISG56. J Biol Chem 281 : 34064–34071.

63. PichlmairA, LassnigC, EberleCA, GornaMW, BaumannCL, et al. (2011) IFIT1 is an antiviral protein that recognizes 5′-triphosphate RNA. Nat Immunol 12 : 624–630.

64. StawowczykM, Van ScoyS, KumarKP, ReichNC (2011) The interferon stimulated gene 54 promotes apoptosis. J Biol Chem 286 : 7257–7266.

65. BerchtoldS, MannckeB, KlenkJ, GeiselJ, AutenriethIB, et al. (2008) Forced IFIT-2 expression represses LPS induced TNF-alpha expression at posttranscriptional levels. BMC Immunol 9 : 75.

66. SiegfriedA, BerchtoldS, MannckeB, DeuschleE, ReberJ, et al. (2013) IFIT2 is an effector protein of type I IFN-mediated amplification of lipopolysaccharide (LPS)-induced TNF-alpha secretion and LPS-induced endotoxin shock. J Immunol 191 : 3913–3921.

67. LadSP, FukudaEY, LiJ, de la MazaLM, LiE (2005) Up-regulation of the JAK/STAT1 signal pathway during Chlamydia trachomatis infection. J Immunol 174 : 7186–7193.

68. DerSD, ZhouA, WilliamsBR, SilvermanRH (1998) Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays. Proc Natl Acad Sci U S A 95 : 15623–15628.

69. ReichNC (2013) A death-promoting role for ISG54/IFIT2. J Interferon Cytokine Res 33 : 199–205.

70. BartraSS, StyerKL, O'BryantDM, NillesML, HinnebuschBJ, et al. (2008) Resistance of Yersinia pestis to complement-dependent killing is mediated by the Ail outer membrane protein. Infect Immun 76 : 612–622.

71. UneT, BrubakerRR (1984) In vivo comparison of avirulent Vwa -⁠ and Pgm -⁠ or Pstr phenotypes of Yersiniae. Infect Immun 43 : 895–900.

72. KellerA, NesvizhskiiAI, KolkerE, AebersoldR (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74 : 5383–5392.

73. NesvizhskiiAI, KellerA, KolkerE, AebersoldR (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75 : 4646–4658.

74. JorgensenI, BednarMM, AminV, DavisBK, TingJP, et al. (2011) The Chlamydia protease CPAF regulates host and bacterial proteins to maintain pathogen vacuole integrity and promote virulence. Cell Host Microbe 10 : 21–32.

75. TsaiA, CarstensRP (2006) An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach. Nat Protoc 1 : 2820–2827.

76. WilmM, ShevchenkoA, HouthaeveT, BreitS, SchweigererL, et al. (1996) Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379 : 466–469.

77. BeausoleilSA, VillenJ, GerberSA, RushJ, GygiSP (2006) A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol 24 : 1285–1292.

78. AgaisseH, DerreI (2013) A C. trachomatis cloning vector and the generation of C. trachomatis strains expressing fluorescent proteins under the control of a C. trachomatis promoter. PLoS One 8: e57090.