1887

Microbiology Society logo

Volume 160, Issue 9

Characterization of a novel two-partner secretion system implicated in the virulence of Free

Abstract

is an opportunistic human pathogen implicated in nosocomial infection and infecting people with compromised immune systems such as cystic fibrosis patients. Although multiple genes involved in pathogenesis have been characterized, the overall mechanism of virulence is not fully understood. In this study, we identified a functional two-partner secretion (TPS) system, composed of the PdtA exoprotein and its cognate pore-forming β-barrel PdtB transporter, which is implicated in the virulence of . We found that the predicted PdtA exoprotein is related to the HMW-like adhesins subfamily TPS systems. We demonstrate here that limitation of inorganic phosphate (Pi) allows the production of PdtA protein. We show that PdtA is processed during its outer-membrane translocation, with an N-terminal domain released into the extracellular environment and a C-terminal domain associated with the outer membrane of the cell. We also obtained evidence that the transport of PdtA is strictly dependent on the production of PdtB, a result confirming that these proteins constitute a functional TPS system. Furthermore, using the model of infection, we show that a mutant is less virulent than the wild-type strain.

  • Received:
  • Accepted:
  • Published Online:
Funding
This study was supported by the:
  • French cystic fibrosis foundation (VLM) (Award FR20110600567/1/1/69)
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.079616-0
2014-09-01
2024-04-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/160/9/1940.html?itemId=/content/journal/micro/10.1099/mic.0.079616-0&mimeType=html&fmt=ahah

References

  1. Aoki S. K., Pamma R., Hernday A. D., Bickham J. E., Braaten B. A., Low D. A. ( 2005). Contact-dependent inhibition of growth in Escherichia coli . Science 309:1245–1248 [View Article][PubMed]
     [Google Scholar]
  2. 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 [View Article][PubMed]
     [Google Scholar]
  3. Borlee B. R., Goldman A. D., Murakami K., Samudrala R., Wozniak D. J., Parsek M. R. ( 2010). Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix. Mol Microbiol 75:827–842 [View Article][PubMed]
     [Google Scholar]
  4. Braun V., Hobbie S., Ondraczek R. ( 1992). Serratia marcescens forms a new type of cytolysin. FEMS Microbiol Lett 100:299–305 [View Article][PubMed]
     [Google Scholar]
  5. Brenner S. ( 1974). The genetics of Caenorhabditis elegans . Genetics 77:71–94
     [Google Scholar]
  6. Buscher A. Z., Grass S., Heuser J., Roth R., St Geme J. W. III ( 2006). Surface anchoring of a bacterial adhesin secreted by the two-partner secretion pathway. Mol Microbiol 61:470–483 [View Article][PubMed]
     [Google Scholar]
  7. Choi K. H., Schweizer H. P. ( 2006). mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa . Nat Protoc 1:153–161 [View Article][PubMed]
     [Google Scholar]
  8. Clantin B., Delattre A. S., Rucktooa P., Saint N., Méli A. C., Locht C., Jacob-Dubuisson F., Villeret V. ( 2007). Structure of the membrane protein FhaC: a member of the Omp85-TpsB transporter superfamily. Science 317:957–961 [View Article][PubMed]
     [Google Scholar]
  9. Coutte L., Antoine R., Drobecq H., Locht C., Jacob-Dubuisson F. ( 2001). Subtilisin-like autotransporter serves as maturation protease in a bacterial secretion pathway. EMBO J 20:5040–5048 [View Article][PubMed]
     [Google Scholar]
  10. Delattre A. S., Saint N., Clantin B., Willery E., Lippens G., Locht C., Villeret V., Jacob-Dubuisson F. ( 2011). Substrate recognition by the POTRA domains of TpsB transporter FhaC. Mol Microbiol 81:99–112 [View Article][PubMed]
     [Google Scholar]
  11. Faure L. M., Llamas M. A., Bastiaansen K. C., de Bentzmann S., Bigot S. ( 2013). Phosphate starvation relayed by PhoB activates the expression of the Pseudomonas aeruginosa σvreI ECF factor and its target genes. Microbiology 159:1315–1327 [View Article][PubMed]
     [Google Scholar]
  12. Figurski D. H., Helinski D. R. ( 1979). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. . Proc Natl Acad Sci U S A 76:1648–1652 [View Article][PubMed]
     [Google Scholar]
  13. Flemming H. C., Wingender J. ( 2010). The biofilm matrix. Nat Rev Microbiol 8:623–633[PubMed]
     [Google Scholar]
  14. Garvis S., Munder A., Ball G., de Bentzmann S., Wiehlmann L., Ewbank J. J., Tümmler B., Filloux A. ( 2009). Caenorhabditis elegans semi-automated liquid screen reveals a specialized role for the chemotaxis gene cheB2 in Pseudomonas aeruginosa virulence. Plos Pathog 5: [CrossRef]
     [Google Scholar]
  15. Grass S., St Geme J. W. III ( 2000). Maturation and secretion of the non-typable Haemophilus influenzae HMW1 adhesin: roles of the N-terminal and C-terminal domains. Mol Microbiol 36:55–67 [View Article][PubMed]
     [Google Scholar]
  16. Guédin S., Willery E., Locht C., Jacob-Dubuisson F. ( 1998). Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin. Mol Microbiol 29:763–774 [View Article][PubMed]
     [Google Scholar]
  17. Herrero M., de Lorenzo V., Timmis K. N. ( 1990). Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. J Bacteriol 172:6557–6567[PubMed]
     [Google Scholar]
  18. Hertle R. ( 2005). The family of Serratia type pore forming toxins. Curr Protein Pept Sci 6:313–325 [View Article][PubMed]
     [Google Scholar]
  19. Hoang T. T., Karkhoff-Schweizer R. R., Kutchma A. J., Schweizer H. P. ( 1998). A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86 [View Article][PubMed]
     [Google Scholar]
  20. Hoang T. T., Kutchma A. J., Becher A., Schweizer H. P. ( 2000). Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains. Plasmid 43:59–72 [View Article][PubMed]
     [Google Scholar]
  21. Ize B., Viarre V., Voulhoux R. ( 2014). Cell fractionation. Methods Mol Biol 1149:185–191 [View Article][PubMed]
     [Google Scholar]
  22. Jacob-Dubuisson F., Buisine C., Willery E., Renauld-Mongénie G., Locht C. ( 1997). Lack of functional complementation between Bordetella pertussis filamentous hemagglutinin and Proteus mirabilis HpmA hemolysin secretion machineries. J Bacteriol 179:775–783[PubMed]
     [Google Scholar]
  23. Jacob-Dubuisson F., El-Hamel C., Saint N., Guédin S., Willery E., Molle G., Locht C. ( 1999). Channel formation by FhaC, the outer membrane protein involved in the secretion of the Bordetella pertussis filamentous hemagglutinin. J Biol Chem 274:37731–37735 [View Article][PubMed]
     [Google Scholar]
  24. Jacob-Dubuisson F., Locht C., Antoine R. ( 2001). Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins. Mol Microbiol 40:306–313 [View Article][PubMed]
     [Google Scholar]
  25. Kajava A. V., Cheng N., Cleaver R., Kessel M., Simon M. N., Willery E., Jacob-Dubuisson F., Locht C., Steven A. C. ( 2001). Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins. Mol Microbiol 42:279–292 [View Article][PubMed]
     [Google Scholar]
  26. 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 [View Article][PubMed]
     [Google Scholar]
  27. Kida Y., Higashimoto Y., Inoue H., Shimizu T., Kuwano K. ( 2008). A novel secreted protease from Pseudomonas aeruginosa activates NF-κB through protease-activated receptors. Cell Microbiol 10:1491–1504 [View Article][PubMed]
     [Google Scholar]
  28. Kida Y., Shimizu T., Kuwano K. ( 2011). Cooperation between LepA and PlcH contributes to the in vivo virulence and growth of Pseudomonas aeruginosa in mice. Infect Immun 79:211–219 [View Article][PubMed]
     [Google Scholar]
  29. Llamas M. A., van der Sar A., Chu B. C., Sparrius M., Vogel H. J., Bitter W. ( 2009). A novel extracytoplasmic function (ECF) sigma factor regulates virulence in Pseudomonas aeruginosa . PLoS Pathog 5:e1000572 [View Article][PubMed]
     [Google Scholar]
  30. Makhov A. M., Hannah J. H., Brennan M. J., Trus B. L., Kocsis E., Conway J. F., Wingfield P. T., Simon M. N., Steven A. C. ( 1994). Filamentous hemagglutinin of Bordetella pertussis. A bacterial adhesin formed as a 50-nm monomeric rigid rod based on a 19-residue repeat motif rich in beta strands and turns. J Mol Biol 241:110–124 [View Article][PubMed]
     [Google Scholar]
  31. Mazar J., Cotter P. A. ( 2006). Topology and maturation of filamentous haemagglutinin suggest a new model for two-partner secretion. Mol Microbiol 62:641–654 [View Article][PubMed]
     [Google Scholar]
  32. Mazar J., Cotter P. A. ( 2007). New insight into the molecular mechanisms of two-partner secretion. Trends Microbiol 15:508–515 [View Article][PubMed]
     [Google Scholar]
  33. Miller J. H. ( 1972). Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  34. Newman J. R., Fuqua C. ( 1999). Broad-host-range expression vectors that carry the l-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene 227:197–203 [View Article][PubMed]
     [Google Scholar]
  35. Noël C. R., Mazar J., Melvin J. A., Sexton J. A., Cotter P. A. ( 2012). The prodomain of the Bordetella two-partner secretion pathway protein FhaB remains intracellular yet affects the conformation of the mature C-terminal domain. Mol Microbiol 86:988–1006 [View Article][PubMed]
     [Google Scholar]
  36. Ostroff R. M., Wretlind B., Vasil M. L. ( 1989). Mutations in the hemolytic-phospholipase C operon result in decreased virulence of Pseudomonas aeruginosa PAO1 grown under phosphate-limiting conditions. Infect Immun 57:1369–1373[PubMed]
     [Google Scholar]
  37. Petrova O. E., Schurr J. R., Schurr M. J., Sauer K. ( 2011). The novel Pseudomonas aeruginosa two-component regulator BfmR controls bacteriophage-mediated lysis and DNA release during biofilm development through PhdA. Mol Microbiol 81:767–783 [View Article][PubMed]
     [Google Scholar]
  38. Relman D. A., Domenighini M., Tuomanen E., Rappuoli R., Falkow S. ( 1989). Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc Natl Acad Sci U S A 86:2637–2641 [View Article][PubMed]
     [Google Scholar]
  39. Renauld-Mongénie G., Cornette J., Mielcarek N., Menozzi F. D., Locht C. ( 1996). Distinct roles of the N-terminal and C-terminal precursor domains in the biogenesis of the Bordetella pertussis filamentous hemagglutinin. J Bacteriol 178:1053–1060[PubMed]
     [Google Scholar]
  40. Sana T. G., Hachani A., Bucior I., Soscia C., Garvis S., Termine E., Engel J., Filloux A., Bleves S. ( 2012). The second type VI secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum sensing and Fur and modulates internalization in epithelial cells. J Biol Chem 287:27095–27105 [View Article][PubMed]
     [Google Scholar]
  41. Schleiff E., Soll J. ( 2005). Membrane protein insertion: mixing eukaryotic and prokaryotic concepts. EMBO Rep 6:1023–1027 [View Article][PubMed]
     [Google Scholar]
  42. Spagnolo J., Bigot S., Denis Y., Bordi C., de Bentzmann S. ( 2012). Development of a genetic tool for activating chromosomal expression of cryptic or tightly regulated loci in Pseudomonas aeruginosa. Plasmid . 67:245–251[PubMed]
     [Google Scholar]
  43. St Geme J. W. III, Yeo H. J. ( 2009). A prototype two-partner secretion pathway: the Haemophilus influenzae HMW1 and HMW2 adhesin systems. Trends Microbiol 17:355–360 [View Article][PubMed]
     [Google Scholar]
  44. Tamura K., Stecher G., Peterson D., Filipski A., Kumar S. ( 2013). mega6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729 [View Article][PubMed]
     [Google Scholar]
  45. Tan M. W., Rahme L. G., Sternberg J. A., Tompkins R. G., Ausubel F. M. ( 1999). Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. Proc Natl Acad Sci U S A 96:2408–2413 [View Article][PubMed]
     [Google Scholar]
  46. Thanassi D. G., Stathopoulos C., Karkal A., Li H. ( 2005). Protein secretion in the absence of ATP: the autotransporter, two-partner secretion and chaperone/usher pathways of Gram-negative bacteria (review). Mol Membr Biol 22:63–72 [View Article][PubMed]
     [Google Scholar]
  47. Voulhoux R., Bos M. P., Geurtsen J., Mols M., Tommassen J. ( 2003). Role of a highly conserved bacterial protein in outer membrane protein assembly. Science 299:262–265 [View Article][PubMed]
     [Google Scholar]
  48. Webb J. S., Nikolakakis K. C., Willett J. L., Aoki S. K., Hayes C. S., Low D. A. ( 2013). Delivery of CdiA nuclease toxins into target cells during contact-dependent growth inhibition. PLoS ONE 8:e57609 [View Article][PubMed]
     [Google Scholar]
  49. Willems R. J., van der Heide H. G., Mooi F. R. ( 1992). Characterization of a Bordetella pertussis fimbrial gene cluster which is located directly downstream of the filamentous haemagglutinin gene. Mol Microbiol 6:2661–2671 [View Article][PubMed]
     [Google Scholar]
  50. Willems R. J., Geuijen C., van der Heide H. G., Renauld G., Bertin P., van den Akker W. M., Locht C., Mooi F. R. ( 1994). Mutational analysis of the Bordetella pertussis fim/fha gene cluster: identification of a gene with sequence similarities to haemolysin accessory genes involved in export of FHA. Mol Microbiol 11:337–347 [View Article][PubMed]
     [Google Scholar]
  51. Winsor G. L., Lam D. K., Fleming L., Lo R., Whiteside M. D., Yu N. Y., Hancock R. E., Brinkman F. S. ( 2011). Pseudomonas Genome Database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Res 39:Database issueD596–D600 [View Article][PubMed]
     [Google Scholar]
  52. Zaborin A., Romanowski K., Gerdes S., Holbrook C., Lepine F., Long J., Poroyko V., Diggle S. P., Wilke A. & other authors ( 2009). Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1. Proc Natl Acad Sci U S A 106:6327–6332 [View Article][PubMed]
     [Google Scholar]
  53. Zaborina O., Holbrook C., Chen Y., Long J., Zaborin A., Morozova I., Fernandez H., Wang Y., Turner J. R., Alverdy J. C. ( 2008). Structure–function aspects of PstS in multi-drug-resistant Pseudomonas aeruginosa . PLoS Pathog 4:e43 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.079616-0
Loading
Characterization of a novel two-partner secretion system implicated in the virulence of Pseudomonas aeruginosa
Microbiology 160, 1940 (2014); https://doi.org/10.1099/mic.0.079616-0
/content/journal/micro/10.1099/mic.0.079616-0
/content/journal/micro/10.1099/mic.0.079616-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error