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Volume 158, Issue 3

YgfZ contributes to secretion of cytotoxic necrotizing factor 1 into outer-membrane vesicles in Free

Abstract

Cytotoxic necrotizing factor 1 (CNF1), a Rho GTPase-activating bacterial toxin, has been shown to contribute to invasion by meningitis-causing K1 of human brain microvascular endothelial cells (HBMEC), which constitute the blood–brain barrier. However, CNF1 is a cytosolic protein and it remains unclear how its secretion occurs, contributing to invasion of HBMEC. To investigate the genetic requirement for CNF1 secretion in K1 strain RS218, we performed mini-Tn mutagenesis and constructed a transposon mutant library of strain NBC, in which β-lactamase was fused to the C-terminus of CNF1 in the chromosome of strain RS218. We identified a transposon mutant (NBC-1E6) that exhibited reduced β-lactamase activity in its culture supernatant and had the transposon inserted into the gene. When was deleted from the genome of strain RS218 (Δ), the translocation of CNF1 into HBMEC was impaired. Subcellular localization analysis of CNF1 demonstrated that YgfZ, a periplasmic protein, contributes to secretion of CNF1 into outer-membrane vesicles (OMVs). The Δ mutant was significantly defective in invasion of HBMEC compared to the parent K1 strain. The defects of the Δ mutant in CNF1 secretion into OMVs and translocation into HBMEC as well as invasion of HBMEC were abrogated by complementation with . Taken together, our findings demonstrate that YgfZ contributes to CNF1 secretion into OMVs in meningitis-causing K1.

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© 2012 SGM
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2012-03-01
2024-04-18
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References

  1. Aldick T., Bielaszewska M., Uhlin B. E., Humpf H. U., Wai S. N., Karch H. ( 2009). Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin. Mol Microbiol 71:1496–1508 [View Article][PubMed]
     [Google Scholar]
  2. Boquet P. ( 2001). The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli . Toxicon 39:1673–1680 [View Article][PubMed]
     [Google Scholar]
  3. Caprioli A., Falbo V., Roda L. G., Ruggeri F. M., Zona C. ( 1983). Partial purification and characterization of an Escherichia coli toxic factor that induces morphological cell alterations. Infect Immun 39:1300–1306[PubMed]
     [Google Scholar]
  4. Castro-Guerrero N., Sinha P. K., Torres-Bacete J., Matsuno-Yagi A., Yagi T. ( 2010). Pivotal roles of three conserved carboxyl residues of the NuoC (30k) segment in the structural integrity of proton-translocating NADH-quinone oxidoreductase from Escherichia coli . Biochemistry 49:10072–10080 [View Article][PubMed]
     [Google Scholar]
  5. Charpentier X., Oswald E. ( 2004). Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 β-lactamase as a new fluorescence-based reporter. J Bacteriol 186:5486–5495 [View Article][PubMed]
     [Google Scholar]
  6. Chen J. W., Sun C. M., Sheng W. L., Wang Y. C., Syu W. J. ( 2006). Expression analysis of up-regulated genes responding to plumbagin in Escherichia coli . J Bacteriol 188:456–463 [View Article][PubMed]
     [Google Scholar]
  7. Chung J. W., Hong S. J., Kim K. J., Goti D., Stins M. F., Shin S., Dawson V. L., Dawson T. M., Kim K. S. ( 2003). 37-kDa laminin receptor precursor modulates cytotoxic necrotizing factor 1-mediated RhoA activation and bacterial uptake. J Biol Chem 278:16857–16862 [View Article][PubMed]
     [Google Scholar]
  8. Datsenko K. A., Wanner B. L. ( 2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645 [View Article][PubMed]
     [Google Scholar]
  9. Davis J. M., Carvalho H. M., Rasmussen S. B., O’Brien A. D. ( 2006). Cytotoxic necrotizing factor type 1 delivered by outer membrane vesicles of uropathogenic Escherichia coli attenuates polymorphonuclear leukocyte antimicrobial activity and chemotaxis. Infect Immun 74:4401–4408 [View Article][PubMed]
     [Google Scholar]
  10. Economou A., Christie P. J., Fernandez R. C., Palmer T., Plano G. V., Pugsley A. P. ( 2006). Secretion by numbers: protein traffic in prokaryotes. Mol Microbiol 62:308–319 [View Article][PubMed]
     [Google Scholar]
  11. Falzano L., Fiorentini C., Donelli G., Michel E., Kocks C., Cossart P., Cabanié L., Oswald E., Boquet P. ( 1993). Induction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor type 1. Mol Microbiol 9:1247–1254 [View Article][PubMed]
     [Google Scholar]
  12. Flatau G., Lemichez E., Gauthier M., Chardin P., Paris S., Fiorentini C., Boquet P. ( 1997). Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine. Nature 387:729–733 [View Article][PubMed]
     [Google Scholar]
  13. Geddes K., Worley M., Niemann G., Heffron F. ( 2005). Identification of new secreted effectors in Salmonella enterica serovar Typhimurium. Infect Immun 73:6260–6271 [View Article][PubMed]
     [Google Scholar]
  14. Haurat M. F., Aduse-Opoku J., Rangarajan M., Dorobantu L., Gray M. R., Curtis M. A., Feldman M. F. ( 2011). Selective sorting of cargo proteins into bacterial membrane vesicles. J Biol Chem 286:1269–1276 [View Article][PubMed]
     [Google Scholar]
  15. Housden N. G., Wojdyla J. A., Korczynska J., Grishkovskaya I., Kirkpatrick N., Brzozowski A. M., Kleanthous C. ( 2010). Directed epitope delivery across the Escherichia coli outer membrane through the porin OmpF. Proc Natl Acad Sci U S A 107:21412–21417 [View Article][PubMed]
     [Google Scholar]
  16. Hu P., Janga S. C., Babu M., Díaz-Mejía J. J., Butland G., Yang W., Pogoutse O., Guo X., Phanse S. & other authors ( 2009). Global functional atlas of Escherichia coli encompassing previously uncharacterized proteins. PLoS Biol 7:e96 [View Article][PubMed]
     [Google Scholar]
  17. James R., Kleanthous C., Moore G. R. ( 1996). The biology of E colicins: paradigms and paradoxes. Microbiology 142:1569–1580 [View Article][PubMed]
     [Google Scholar]
  18. Kesty N. C., Mason K. M., Reedy M., Miller S. E., Kuehn M. J. ( 2004). Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells. EMBO J 23:4538–4549 [View Article][PubMed]
     [Google Scholar]
  19. Khan N. A., Wang Y., Kim K. J., Chung J. W., Wass C. A., Kim K. S. ( 2002). Cytotoxic necrotizing factor-1 contributes to Escherichia coli K1 invasion of the central nervous system. J Biol Chem 277:15607–15612 [View Article][PubMed]
     [Google Scholar]
  20. Kim K. S. ( 2003). Pathogenesis of bacterial meningitis: from bacteraemia to neuronal injury. Nat Rev Neurosci 4:376–385 [View Article][PubMed]
     [Google Scholar]
  21. Kim K. S. ( 2008). Mechanisms of microbial traversal of the blood-brain barrier. Nat Rev Microbiol 6:625–634 [View Article][PubMed]
     [Google Scholar]
  22. Kim K. J., Chung J. W., Kim K. S. ( 2005). 67-kDa laminin receptor promotes internalization of cytotoxic necrotizing factor 1-expressing Escherichia coli K1 into human brain microvascular endothelial cells. J Biol Chem 280:1360–1368 [View Article][PubMed]
     [Google Scholar]
  23. Knust Z., Schmidt G. ( 2010). Cytotoxic necrotizing factors (CNFs) – a growing toxin family. Toxins (Basel) 2:116–127[PubMed] [CrossRef]
     [Google Scholar]
  24. 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 [View Article][PubMed]
     [Google Scholar]
  25. Kume K., Nakai T., Samejima Y., Sugimoto C. ( 1986). Properties of dermonecrotic toxin prepared from sonic extracts Bordetella bronchiseptica . Infect Immun 52:370–377[PubMed]
     [Google Scholar]
  26. Landraud L., Gauthier M., Fosse T., Boquet P. ( 2000). Frequency of Escherichia coli strains producing the cytotoxic necrotizing factor (CNF1) in nosocomial urinary tract infections. Lett Appl Microbiol 30:213–216 [View Article][PubMed]
     [Google Scholar]
  27. Lemonnier M., Landraud L., Lemichez E. ( 2007). Rho GTPase-activating bacterial toxins: from bacterial virulence regulation to eukaryotic cell biology. FEMS Microbiol Rev 31:515–534 [View Article][PubMed]
     [Google Scholar]
  28. Lin C. N., Syu W. J., Sun W. S., Chen J. W., Chen T. H., Don M. J., Wang S. H. ( 2010). A role of ygfZ in the Escherichia coli response to plumbagin challenge. J Biomed Sci 17:84 [View Article][PubMed]
     [Google Scholar]
  29. Lockman H. A., Gillespie R. A., Baker B. D., Shakhnovich E. ( 2002). Yersinia pseudotuberculosis produces a cytotoxic necrotizing factor. Infect Immun 70:2708–2714 [View Article][PubMed]
     [Google Scholar]
  30. Mashburn-Warren L. M., Whiteley M. ( 2006). Special delivery: vesicle trafficking in prokaryotes. Mol Microbiol 61:839–846 [View Article][PubMed]
     [Google Scholar]
  31. Mashburn-Warren L., Howe J., Garidel P., Richter W., Steiniger F., Roessle M., Brandenburg K., Whiteley M. ( 2008). Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation. Mol Microbiol 69:491–502 [View Article][PubMed]
     [Google Scholar]
  32. McKenzie G. J., Craig N. L. ( 2006). Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn7 without need for selection of the insertion event. BMC Microbiol 6:39 [View Article][PubMed]
     [Google Scholar]
  33. Meysick K. C., Mills M., O’Brien A. D. ( 2001). Epitope mapping of monoclonal antibodies capable of neutralizing cytotoxic necrotizing factor type 1 of uropathogenic Escherichia coli . Infect Immun 69:2066–2074 [View Article][PubMed]
     [Google Scholar]
  34. Mills E., Baruch K., Charpentier X., Kobi S., Rosenshine I. ( 2008). Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli . Cell Host Microbe 3:104–113 [View Article][PubMed]
     [Google Scholar]
  35. Ote T., Hashimoto M., Ikeuchi Y., Su’etsugu M., Suzuki T., Katayama T., Kato J. ( 2006). Involvement of the Escherichia coli folate-binding protein YgfZ in RNA modification and regulation of chromosomal replication initiation. Mol Microbiol 59:265–275 [View Article][PubMed]
     [Google Scholar]
  36. Schmidt G., Sehr P., Wilm M., Selzer J., Mann M., Aktories K. ( 1997). Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature 387:725–729 [View Article][PubMed]
     [Google Scholar]
  37. Teplyakov A., Obmolova G., Sarikaya E., Pullalarevu S., Krajewski W., Galkin A., Howard A. J., Herzberg O., Gilliland G. L. ( 2004). Crystal structure of the YgfZ protein from Escherichia coli suggests a folate-dependent regulatory role in one-carbon metabolism. J Bacteriol 186:7134–7140 [View Article][PubMed]
     [Google Scholar]
  38. Vouret-Craviari V., Grall D., Flatau G., Pouysségur J., Boquet P., Van Obberghen-Schilling E. ( 1999). Effects of cytotoxic necrotizing factor 1 and lethal toxin on actin cytoskeleton and VE-cadherin localization in human endothelial cell monolayers. Infect Immun 67:3002–3008[PubMed]
     [Google Scholar]
  39. Wai S. N., Lindmark B., Söderblom T., Takade A., Westermark M., Oscarsson J., Jass J., Richter-Dahlfors A., Mizunoe Y., Uhlin B. E. ( 2003). Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin. Cell 115:25–35 [View Article][PubMed]
     [Google Scholar]
  40. Waller J. C., Alvarez S., Naponelli V., Lara-Nuñez A., Blaby I. K., Da Silva V., Ziemak M. J., Vickers T. J., Beverley S. M. & other authors ( 2010). A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life. Proc Natl Acad Sci U S A 107:10412–10417 [View Article][PubMed]
     [Google Scholar]
  41. Yu H., Kim K. S. ( 2010). Ferredoxin is involved in secretion of cytotoxic necrotizing factor 1 across the cytoplasmic membrane in Escherichia coli K1. Infect Immun 78:838–844 [View Article][PubMed]
     [Google Scholar]
  42. Zlokarnik G., Negulescu P. A., Knapp T. E., Mere L., Burres N., Feng L., Whitney M., Roemer K., Tsien R. Y. ( 1998). Quantitation of transcription and clonal selection of single living cells with β-lactamase as reporter. Science 279:84–88 [View Article][PubMed]
     [Google Scholar]
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YgfZ contributes to secretion of cytotoxic necrotizing factor 1 into outer-membrane vesicles in Escherichia coli
Microbiology 158, 612 (2012); https://doi.org/10.1099/mic.0.054122-0
/content/journal/micro/10.1099/mic.0.054122-0
/content/journal/micro/10.1099/mic.0.054122-0
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