A Substrate-Fusion Protein Is Trapped inside the Type III Secretion System Channel in


The Type III Secretion System (T3SS) is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion.


Vyšlo v časopise: A Substrate-Fusion Protein Is Trapped inside the Type III Secretion System Channel in. PLoS Pathog 10(1): e32767. doi:10.1371/journal.ppat.1003881
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1003881

Souhrn

The Type III Secretion System (T3SS) is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion.


Zdroje

1. HueckCJ (1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiology and molecular biology reviews 62: 379–433.

2. DeanP (2011) Functional domains and motifs of bacterial type III effector proteins and their roles in infection. FEMS microbiology reviews 35: 1100–25.

3. ButtnerD, HeSY (2009) Type III protein secretion in plant pathogenic bacteria. Plant physiology 150: 1656–64.

4. SotoMJ, SanjuanJ, OlivaresJ (2006) Rhizobia and plant-pathogenic bacteria: common infection weapons. Microbiology 152: 3167–74.

5. VenkatesanMM, GoldbergMB, RoseDJ, GrotbeckEJ, BurlandV, et al. (2001) Complete DNA sequence and analysis of the large virulence plasmid of Shigella exneri. Infection and immunity 69: 3271–85.

6. MaurelliAT, BaudryB, d'HautevilleH, HaleTL, SansonettiPJ (1985) Cloning of plasmid DNA sequences involved in invasion of HeLa cells by Shigella flexneri. Infection and immunity 49: 164–71.

7. MenardR, SansonettiPJ, ParsotC (1993) Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. Journal of bacteriology 175: 5899–906.

8. VeenendaalAK, HodgkinsonJL, SchwarzerL, StabatD, ZenkSF, et al. (2007) The type III secretion system needle tip complex mediates host cell sensing and translocon insertion. Molecular microbiology 63: 1719–30.

9. MenardR, SansonettiP, ParsotC (1994) The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD. The EMBO journal 13: 5293–302.

10. TroisfontainesP, CornelisGR (2005) Type III secretion: more systems than you think. Physiology 20: 326–39.

11. CornelisGR (2006) The type III secretion injectisome. Nature reviews Microbiology 4: 811–25.

12. BlockerA, JouihriN, LarquetE, GounonP, EbelF, et al. (2001) Structure and composition of the Shigella flexneri “needle complex”, a part of its type III secreton. Molecular microbiology 39: 652–63.

13. HodgkinsonJL, HorsleyA, StabatD, SimonM, JohnsonS, et al. (2009) Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout. Nature structural & molecular biology 16: 477–85.

14. LoquetA, SgourakisNG, GuptaR, GillerK, RiedelD, et al. (2012) Atomic model of the type III secretion system needle. Nature 486: 276–9.

15. DemersJP, SgourakisNG, GuptaR, LoquetA, GillerK, et al. (2013) The common structural architecture of Shigella flexneri and Salmonella typhimurium type three secretion needles. PLoS pathogens 9: e1003245.

16. MichielsT, CornelisGR (1991) Secretion of hybrid proteins by the Yersinia Yop export system. Journal of bacteriology 173: 1677–85.

17. FeldmanMF, MullerS, WuestE, CornelisGR (2002) SycE allows secretion of YopE-DHFR hybrids by the Yersinia enterocolitica type III Ysc system. Molecular microbiology 46: 1183–97.

18. LeeVT, SchneewindO (2002) Yop fusions to tightly folded protein domains and their effects on Yersinia enterocolitica type III secretion. Journal of bacteriology 184: 3740–5.

19. SorgJA, BlaylockB, SchneewindO (2006) Secretion signal recognition by YscN, the Yersinia type III secretion ATPase. Proceedings of the National Academy of Sciences of the United States of America 103: 16490–5.

20. RiordanKE, SorgJA, BerubeBJ, SchneewindO (2008) Impassable YscP substrates and their impact on the Yersinia enterocolitica type III secretion pathway. Journal of bacteriology 190: 6204–16.

21. GhoshP (2004) Process of protein transport by the type III secretion system. Microbiology and molecular biology reviews 68: 771–95.

22. EdgrenT, ForsbergA, RosqvistR, Wolf-WatzH (2012) Type III secretion in Yersinia: injectisome or not? PLoS pathogens 8: e1002669.

23. NurekiO, ShirouzuM, HashimotoK, IshitaniR, TeradaT, et al. (2002) An enzyme with a deep trefoil knot for the active-site architecture. Acta crystallographica Section D, Biological crystallography 58: 1129–37.

24. ZychlinskyA, KennyB, MenardR, PrevostMC, HollandIB, et al. (1994) IpaB mediates macrophage apoptosis induced by Shigella flexneri. Molecular microbiology 11: 619–27.

25. SenerovicL, TsunodaSP, GoosmannC, BrinkmannV, ZychlinskyA, et al. (2012) Spontaneous formation of IpaB ion channels in host cell membranes reveals how Shigella induces pyroptosis in macrophages. Cell death & disease 3: e384.

26. JungblutPR, SeifertR (1990) Analysis by high-resolution two-dimensional electrophoresis of differentiation-dependent alterations in cytosolic protein pattern of HL-60 leukemic cells. Journal of biochemical and biophysical methods 21: 47–58.

27. Zimny-ArndtU, SchmidM, AckermannR, JungblutPR (2009) Classical proteomics: two-dimensional electrophoresis/MALDI mass spectrometry. Methods in molecular biology 492: 65–91.

28. SansonettiPJ, KopeckoDJ, FormalSB (1982) Involvement of a plasmid in the invasive ability of Shigella flexneri. Infection and immunity 35: 852–60.

29. HendersonIR, Navarro-GarciaF, DesvauxM, FernandezRC, Ala'AldeenD (2004) Type V protein secretion pathway: the autotransporter story. Microbiology and molecular biology reviews : MMBR 68: 692–744.

30. SchroederGN, HilbiH (2008) Molecular pathogenesis of Shigella spp.: controlling host cell signaling, invasion, and death by type III secretion. Clinical microbiology reviews 21: 134–56.

31. BarzuS, NatoF, RouyreS, MazieJC, SansonettiP, et al. (1993) Characterization of B-cell epitopes on IpaB, an invasion-associated antigen of Shigella flexneri: identification of an immunodominant domain recognized during natural infection. Infection and immunity 61: 3825–31.

32. GalanJE, Wolf-WatzH (2006) Protein delivery into eukaryotic cells by type III secretion machines. Nature 444: 567–73.

33. DatsenkoKA, WannerBL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America 97: 6640–5.

34. SansonettiPJ, RyterA, ClercP, MaurelliAT, MounierJ (1986) Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infection and immunity 51: 461–9.

35. LunelliM, LokareddyRK, ZychlinskyA, KolbeM (2009) IpaB-IpgC interaction defines binding motif for type III secretion translocator. Proceedings of the National Academy of Sciences of the United States of America 106: 9661–6.

36. AbramoffMD, MagalhaesPJ, RamSJ (2004) Image Processing with ImageJ. Biophotonics International 11: 36–42.

37. Team RDC (2003) R: a language and environment for statistical computing.

38. GentlemanRC, CareyVJ, BatesDM, BolstadB, DettlingM, et al. (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome biology 5: R80.

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Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

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