1887

Abstract

Bacterial pathogens use a range of protein secretion systems to colonize their host. One recent addition to this arsenal is the type VI secretion system (T6SS), which is found in many Gram-negative bacteria. The T6SS involves 12–15 components, including a ClpV-like AAA ATPase. Moreover, the VgrG and Hcp components have been proposed to form a puncturing device, based on structural similarity to the tail spike components gp5/gp27 and the tail tube component gp19 of the T4 bacteriophage, respectively. Another T6SS component shows similarity to a T4 phage protein, namely gp25. The gp25 protein has been proposed to have lysozyme activity. Other T6SS components do not exhibit obvious similarity to characterized T4 phage components. The genome of contains three T6SS gene clusters. In each cluster a gene encoding a putative member of the gp25-like protein family was identified, which we called HsiF. We confirmed this similarity by analysing the structure of the HsiF proteins using secondary and tertiary structure prediction tools. We demonstrated that HsiF1 is crucial for the T6SS-dependent secretion of Hcp and VgrG. Importantly, lysozyme activity of HsiF proteins was not detectable, and we related this observation to the demonstration that HsiF1 localizes to the cytoplasm of . Finally, our data showed that a conserved glutamate, predicted to be required for proper HsiF folding, is essential for its function. In conclusion, our data confirm the central role of HsiF in the T6SS mechanism, provide information on the predicted HsiF structure, and call for reconsideration of the function of gp25-like proteins.

Funding
This study was supported by the:
  • Wellcome Trust (Award WT091939)
  • Medical Research Council
  • MRC (Award G0800171/ID86344)
  • MRC (Award G0800171/ID86344)
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2011-12-01
2021-05-06
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References

  1. Aschtgen M. S., Bernard C. S., De Bentzmann S., Lloubès R., Cascales E. ( 2008). SciN is an outer membrane lipoprotein required for type VI secretion in enteroaggregative Escherichia coli . J Bacteriol 190:7523–7531 [CrossRef][PubMed]
    [Google Scholar]
  2. Aschtgen M. S., Gavioli M., Dessen A., Lloubès R., Cascales E. ( 2010a). The SciZ protein anchors the enteroaggregative Escherichia coli type VI secretion system to the cell wall. Mol Microbiol 75:886–899 [CrossRef][PubMed]
    [Google Scholar]
  3. Aschtgen M. S., Thomas M. S., Cascales E. ( 2010b). Anchoring the type VI secretion system to the peptidoglycan: TssL, TagL, TagP... what else?. Virulence 1:535–540 [CrossRef][PubMed]
    [Google Scholar]
  4. Ballister E. R., Lai A. H., Zuckermann R. N., Cheng Y., Mougous J. D. ( 2008). In vitro self-assembly of tailorable nanotubes from a simple protein building block. Proc Natl Acad Sci U S A 105:3733–3738 [CrossRef][PubMed]
    [Google Scholar]
  5. Bingle L. E., Bailey C. M., Pallen M. J. ( 2008). Type VI secretion: a beginner’s guide. Curr Opin Microbiol 11:3–8 [CrossRef][PubMed]
    [Google Scholar]
  6. Bladergroen M. R., Badelt K., Spaink H. P. ( 2003). Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion. Mol Plant Microbe Interact 16:53–64 [CrossRef][PubMed]
    [Google Scholar]
  7. Bleves S., Lazdunski A., Filloux A. ( 1996). Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa . J Bacteriol 178:4297–4300[PubMed]
    [Google Scholar]
  8. Bleves S., Viarre V., Salacha R., Michel G. P., Filloux A., Voulhoux R. ( 2010). Protein secretion systems in Pseudomonas aeruginosa: a wealth of pathogenic weapons. Int J Med Microbiol 300:534–543 [CrossRef][PubMed]
    [Google Scholar]
  9. Bönemann G., Pietrosiuk A., Diemand A., Zentgraf H., Mogk A. ( 2009). Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion. EMBO J 28:315–325 [CrossRef][PubMed]
    [Google Scholar]
  10. Bönemann G., Pietrosiuk A., Mogk A. ( 2010). Tubules and donuts: a type VI secretion story. Mol Microbiol 76:815–821 [CrossRef][PubMed]
    [Google Scholar]
  11. Cascales E. ( 2008). The type VI secretion toolkit. EMBO Rep 9:735–741 [CrossRef][PubMed]
    [Google Scholar]
  12. Das S., Chaudhuri K. ( 2003). Identification of a unique IAHP (IcmF associated homologous proteins) cluster in Vibrio cholerae and other proteobacteria through in silico analysis. In Silico Biol 3:287–300[PubMed]
    [Google Scholar]
  13. Das S., Chakrabortty A., Banerjee R., Roychoudhury S., Chaudhuri K. ( 2000). Comparison of global transcription responses allows identification of Vibrio cholerae genes differentially expressed following infection. FEMS Microbiol Lett 190:87–91 [CrossRef][PubMed]
    [Google Scholar]
  14. Filloux A. ( 2009). The type VI secretion system: a tubular story. EMBO J 28:309–310 [CrossRef][PubMed]
    [Google Scholar]
  15. Filloux A., Hachani A., Bleves S. ( 2008). The bacterial type VI secretion machine: yet another player for protein transport across membranes. Microbiology 154:1570–1583 [CrossRef][PubMed]
    [Google Scholar]
  16. Goodman A. L., Kulasekara B., Rietsch A., Boyd D., Smith R. S., Lory S. ( 2004). A signaling network reciprocally regulates genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa . Dev Cell 7:745–754 [CrossRef][PubMed]
    [Google Scholar]
  17. Gutierrez C., Devedjian J. C. ( 1989). A plasmid facilitating in vitro construction of phoA gene fusions in Escherichia coli . Nucleic Acids Res 17:3999 [CrossRef][PubMed]
    [Google Scholar]
  18. Hachani A., Lossi N. S., Hamilton A., Jones C., Bleves S., Albesa-Jové D., Filloux A. ( 2011). Type VI secretion system in Pseudomonas aeruginosa: secretion and multimerization of VgrG proteins. J Biol Chem 286:12317–12327 [CrossRef][PubMed]
    [Google Scholar]
  19. Hauser A. R. ( 2009). The type III secretion system of Pseudomonas aeruginosa: infection by injection. Nat Rev Microbiol 7:654–665 [CrossRef][PubMed]
    [Google Scholar]
  20. Hauser A. R., Cobb E., Bodi M., Mariscal D., Vallés J., Engel J. N., Rello J. ( 2002). Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa . Crit Care Med 30:521–528 [CrossRef][PubMed]
    [Google Scholar]
  21. Hood R. D., Singh P., Hsu F., Güvener T., Carl M. A., Trinidad R. R., Silverman J. M., Ohlson B. B., Hicks K. G. et al. & other authors ( 2010). A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe 7:25–37 [CrossRef][PubMed]
    [Google Scholar]
  22. Kaniga K., Delor I., Cornelis G. R. ( 1991). A wide-host-range suicide vector for improving reverse genetics in Gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica . Gene 109:137–141 [CrossRef][PubMed]
    [Google Scholar]
  23. Kelley L. A., Sternberg M. J. ( 2009). Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363–371 [CrossRef][PubMed]
    [Google Scholar]
  24. Kostyuchenko V. A., Leiman P. G., Chipman P. R., Kanamaru S., van Raaij M. J., Arisaka F., Mesyanzhinov V. V., Rossmann M. G. ( 2003). Three-dimensional structure of bacteriophage T4 baseplate. Nat Struct Biol 10:688–693 [CrossRef][PubMed]
    [Google Scholar]
  25. Laemmli U. K. ( 1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef][PubMed]
    [Google Scholar]
  26. Leiman P. G., Basler M., Ramagopal U. A., Bonanno J. B., Sauder J. M., Pukatzki S., Burley S. K., Almo S. C., Mekalanos J. J. ( 2009). Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin. Proc Natl Acad Sci U S A 106:4154–4159 [CrossRef][PubMed]
    [Google Scholar]
  27. Leiman P. G., Arisaka F., van Raaij M. J., Kostyuchenko V. A., Aksyuk A. A., Kanamaru S., Rossmann M. G. ( 2010). Morphogenesis of the T4 tail and tail fibers. Virol J 7:355 [CrossRef][PubMed]
    [Google Scholar]
  28. Lesic B., Starkey M., He J., Hazan R., Rahme L. G. ( 2009). Quorum sensing differentially regulates Pseudomonas aeruginosa type VI secretion locus I and homologous loci II and III, which are required for pathogenesis. Microbiology 155:2845–2855 [CrossRef][PubMed]
    [Google Scholar]
  29. Lory S., Jin S., Boyd J. M., Rakeman J. L., Bergman P. ( 1996). Differential gene expression by Pseudomonas aeruginosa during interaction with respiratory mucus. Am J Respir Crit Care Med 154:S183–S186[PubMed] [CrossRef]
    [Google Scholar]
  30. Ma A. T., McAuley S., Pukatzki S., Mekalanos J. J. ( 2009). Translocation of a Vibrio cholerae type VI secretion effector requires bacterial endocytosis by host cells. Cell Host Microbe 5:234–243 [CrossRef][PubMed]
    [Google Scholar]
  31. Matthews B. W., Remington S. J. ( 1974). The three dimensional structure of the lysozyme from bacteriophage T4. Proc Natl Acad Sci U S A 71:4178–4182 [CrossRef][PubMed]
    [Google Scholar]
  32. Mattinen L., Nissinen R., Riipi T., Kalkkinen N., Pirhonen M. ( 2007). Host-extract induced changes in the secretome of the plant pathogenic bacterium Pectobacterium atrosepticum . Proteomics 7:3527–3537 [CrossRef][PubMed]
    [Google Scholar]
  33. Merrell D. S., Falkow S. ( 2004). Frontal and stealth attack strategies in microbial pathogenesis. Nature 430:250–256 [CrossRef][PubMed]
    [Google Scholar]
  34. Michel G., Bleves S., Ball G., Lazdunski A., Filloux A. ( 1998). Mutual stabilization of the XcpZ and XcpY components of the secretory apparatus in Pseudomonas aeruginosa . Microbiology 144:3379–3386 [CrossRef][PubMed]
    [Google Scholar]
  35. Mougous J. D., Cuff M. E., Raunser S., Shen A., Zhou M., Gifford C. A., Goodman A. L., Joachimiak G., Ordoñez C. L. et al. & other authors ( 2006). A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312:1526–1530 [CrossRef][PubMed]
    [Google Scholar]
  36. Mougous J. D., Gifford C. A., Ramsdell T. L., Mekalanos J. J. ( 2007). Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa . Nat Cell Biol 9:797–803 [CrossRef][PubMed]
    [Google Scholar]
  37. Nakagawa H., Arisaka F., Ishii S. ( 1985). Isolation and characterization of the bacteriophage T4 tail-associated lysozyme. J Virol 54:460–466[PubMed]
    [Google Scholar]
  38. Nakimbugwe D., Masschalck B., Deckers D., Callewaert L., Aertsen A., Michiels C. W. ( 2006). Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts. FEMS Microbiol Lett 259:41–46 [CrossRef][PubMed]
    [Google Scholar]
  39. Parsons D. A., Heffron F. ( 2005). sciS, an icmF homolog in Salmonella enterica serovar Typhimurium, limits intracellular replication and decreases virulence. Infect Immun 73:4338–4345 [CrossRef][PubMed]
    [Google Scholar]
  40. Potvin E., Lehoux D. E., Kukavica-Ibrulj I., Richard K. L., Sanschagrin F., Lau G. W., Levesque R. C. ( 2003). In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets. Environ Microbiol 5:1294–1308 [CrossRef][PubMed]
    [Google Scholar]
  41. Pukatzki S., Ma A. T., Revel A. T., Sturtevant D., Mekalanos J. J. ( 2007). Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. Proc Natl Acad Sci U S A 104:15508–15513 [CrossRef][PubMed]
    [Google Scholar]
  42. Pukatzki S., McAuley S. B., Miyata S. T. ( 2009). The type VI secretion system: translocation of effectors and effector-domains. Curr Opin Microbiol 12:11–17 [CrossRef][PubMed]
    [Google Scholar]
  43. Records A. R., Gross D. C. ( 2010). Sensor kinases RetS and LadS regulate Pseudomonas syringae type VI secretion and virulence factors. J Bacteriol 192:3584–3596 [CrossRef][PubMed]
    [Google Scholar]
  44. Rossmann M. G., Mesyanzhinov V. V., Arisaka F., Leiman P. G. ( 2004). The bacteriophage T4 DNA injection machine. Curr Opin Struct Biol 14:171–180 [CrossRef][PubMed]
    [Google Scholar]
  45. Roy-Burman A., Savel R. H., Racine S., Swanson B. L., Revadigar N. S., Fujimoto J., Sawa T., Frank D. W., Wiener-Kronish J. P. ( 2001). Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J Infect Dis 183:1767–1774 [CrossRef][PubMed]
    [Google Scholar]
  46. Russell A. B., Hood R. D., Bui N. K., LeRoux M., Vollmer W., Mougous J. D. ( 2011). Type VI secretion delivers bacteriolytic effectors to target cells. Nature 475:343–347 [CrossRef][PubMed]
    [Google Scholar]
  47. Schwarz S., Hood R. D., Mougous J. D. ( 2010). What is type VI secretion doing in all those bugs?. Trends Microbiol 18:531–537 [CrossRef][PubMed]
    [Google Scholar]
  48. Suarez G., Sierra J. C., Erova T. E., Sha J., Horneman A. J., Chopra A. K. ( 2010). A type VI secretion system effector protein, VgrG1, from Aeromonas hydrophila that induces host cell toxicity by ADP ribosylation of actin. J Bacteriol 192:155–168 [CrossRef][PubMed]
    [Google Scholar]
  49. Szewczyk B., Bienkowska-Szewczyk K., Kozloff L. M. ( 1986). Identification of T4 gene 25 product, a component of the tail baseplate, as a 15K lysozyme. Mol Gen Genet 202:363–367 [CrossRef][PubMed]
    [Google Scholar]
  50. Takeda S., Hoshida K., Arisaka F. ( 1998). Mapping of functional sites on the primary structure of the tail lysozyme of bacteriophage T4 by mutational analysis. Biochim Biophys Acta 1384:243–252 [CrossRef][PubMed]
    [Google Scholar]
  51. Urban A., Leipelt M., Eggert T., Jaeger K. E. ( 2001). DsbA and DsbC affect extracellular enzyme formation in Pseudomonas aeruginosa . J Bacteriol 183:587–596 [CrossRef][PubMed]
    [Google Scholar]
  52. Vasseur P., Vallet-Gely I., Soscia C., Genin S., Filloux A. ( 2005). The pel genes of the Pseudomonas aeruginosa PAK strain are involved at early and late stages of biofilm formation. Microbiology 151:985–997 [CrossRef][PubMed]
    [Google Scholar]
  53. Ventre I., Goodman A. L., Vallet-Gely I., Vasseur P., Soscia C., Molin S., Bleves S., Lazdunski A., Lory S., Filloux A. ( 2006). Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes. Proc Natl Acad Sci U S A 103:171–176 [CrossRef][PubMed]
    [Google Scholar]
  54. Yap M. L., Mio K., Leiman P. G., Kanamaru S., Arisaka F. ( 2010). The baseplate wedges of bacteriophage T4 spontaneously assemble into hubless baseplate-like structure in vitro. J Mol Biol 395:349–360 [CrossRef][PubMed]
    [Google Scholar]
  55. Zheng J., Leung K. Y. ( 2007). Dissection of a type VI secretion system in Edwardsiella tarda . Mol Microbiol 66:1192–1206 [CrossRef][PubMed]
    [Google Scholar]
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