1887

Abstract

Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggest that the flagellum-associated gene products have a differential effect on twitching motility, based on whether components are intra- or extracellular. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.

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2018-11-01
2019-10-18
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References

  1. Foundation CF Cystic fibrosis foundation - annual report. CDC 2012
    [Google Scholar]
  2. van Delden C, Iglewski BH. Cell-to-cell signaling and Pseudomonas aeruginosa infections. Emerg Infect Dis 1998;4:551–560 [CrossRef][PubMed]
    [Google Scholar]
  3. Hospenthal MK, Costa TRD, Waksman G. A comprehensive guide to pilus biogenesis in Gram-negative bacteria. Nat Rev Microbiol 2017;15:365–379 [CrossRef][PubMed]
    [Google Scholar]
  4. Whitchurch CB. Biogenesis and function of type IV pili in Pseudomonas species. In Pseudomonasvol. 4 Molecular Biology of Emerging Issues USA: Springer; 2006; pp.139–188
    [Google Scholar]
  5. Gloag ES, Turnbull L, Huang A, Vallotton P, Wang H et al. Self-organization of bacterial biofilms is facilitated by extracellular DNA. Proc Natl Acad Sci USA 2013;110:11541–11546 [CrossRef][PubMed]
    [Google Scholar]
  6. Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002;15:167–193 [CrossRef][PubMed]
    [Google Scholar]
  7. Sabbuba N, Hughes G, Stickler DJ. The migration of Proteus mirabilis and other urinary tract pathogens over Foley catheters. BJU Int 2002;89:55–60 [CrossRef][PubMed]
    [Google Scholar]
  8. Burrows LL. Pseudomonas aeruginosa twitching motility: type IV pili in action. Annu Rev Microbiol 2012;66:493–520 [CrossRef][PubMed]
    [Google Scholar]
  9. Chiang P, Habash M, Burrows LL. Disparate subcellular localization patterns of Pseudomonas aeruginosa Type IV pilus ATPases involved in twitching motility. J Bacteriol 2005;187:829–839 [CrossRef][PubMed]
    [Google Scholar]
  10. Takhar HK, Kemp K, Kim M, Howell PL, Burrows LL. The platform protein is essential for type IV pilus biogenesis. J Biol Chem 2013;288:9721–9728 [CrossRef][PubMed]
    [Google Scholar]
  11. Ayers M, Sampaleanu LM, Tammam S, Koo J, Harvey H et al. PilM/N/O/P proteins form an inner membrane complex that affects the stability of the Pseudomonas aeruginosa type IV pilus secretin. J Mol Biol 2009;394:128–142 [CrossRef][PubMed]
    [Google Scholar]
  12. Sampaleanu LM, Bonanno JB, Ayers M, Koo J, Tammam S et al. Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex. J Mol Biol 2009;394:143–159 [CrossRef][PubMed]
    [Google Scholar]
  13. Tammam S, Sampaleanu LM, Koo J, Manoharan K, Daubaras M et al. PilMNOPQ from the Pseudomonas aeruginosa type IV pilus system form a transenvelope protein interaction network that interacts with PilA. J Bacteriol 2013;195:2126–2135 [CrossRef][PubMed]
    [Google Scholar]
  14. Leighton TL, Dayalani N, Sampaleanu LM, Howell PL, Burrows LL. Novel role for PilNO in type IV pilus retraction revealed by alignment subcomplex mutations. J Bacteriol 2015;197:2229–2238 [CrossRef][PubMed]
    [Google Scholar]
  15. Nunn D, Bergman S, Lory S. Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili. J Bacteriol 1990;172:2911–2919 [CrossRef][PubMed]
    [Google Scholar]
  16. Whitchurch CB, Hobbs M, Livingston SP, Krishnapillai V, Mattick JS. Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. Gene 1991;101:33–44 [CrossRef][PubMed]
    [Google Scholar]
  17. Whitchurch CB, Mattick JS. Characterization of a gene, pilU, required for twitching motility but not phage sensitivity in Pseudomonas aeruginosa. Mol Microbiol 1994;13:1079–1091 [CrossRef][PubMed]
    [Google Scholar]
  18. Alm RA, Bodero AJ, Free PD, Mattick JS. Identification of a novel gene, pilZ, essential for type 4 fimbrial biogenesis in Pseudomonas aeruginosa. J Bacteriol 1996;178:46–53 [CrossRef][PubMed]
    [Google Scholar]
  19. Kaiser D. Bacterial motility: how do pili pull?. Curr Biol 2000;10:R777–R780 [CrossRef][PubMed]
    [Google Scholar]
  20. Merz AJ, So M, Sheetz MP. Pilus retraction powers bacterial twitching motility. Nature 2000;407:98–102 [CrossRef][PubMed]
    [Google Scholar]
  21. Huang B, Whitchurch CB, Mattick JS. FimX, a multidomain protein connecting environmental signals to twitching motility in Pseudomonas aeruginosa. J Bacteriol 2003;185:7068–7076 [CrossRef][PubMed]
    [Google Scholar]
  22. Ishimoto KS, Lory S. Formation of pilin in Pseudomonas aeruginosa requires the alternative sigma factor (RpoN) of RNA polymerase. Proc Natl Acad Sci USA 1989;86:1954–1957 [CrossRef][PubMed]
    [Google Scholar]
  23. Hobbs M, Collie ES, Free PD, Livingston SP, Mattick JS. PilS and PilR, a two-component transcriptional regulatory system controlling expression of type 4 fimbriae in Pseudomonas aeruginosa. Mol Microbiol 1993;7:669–682 [CrossRef][PubMed]
    [Google Scholar]
  24. Whitchurch CB, Alm RA, Mattick JS. The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 1996;93:9839–9843 [CrossRef][PubMed]
    [Google Scholar]
  25. Darzins A. The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY. J Bacteriol 1993;175:5934–5944 [CrossRef][PubMed]
    [Google Scholar]
  26. Darzins A. Characterization of a Pseudomonas aeruginosa gene cluster involved in pilus biosynthesis and twitching motility: sequence similarity to the chemotaxis proteins of enterics and the gliding bacterium Myxococcus xanthus. Mol Microbiol 1994;11:137–153 [CrossRef][PubMed]
    [Google Scholar]
  27. Darzins A. The Pseudomonas aeruginosa pilK gene encodes a chemotactic methyltransferase (CheR) homologue that is translationally regulated. Mol Microbiol 1995;15:703–717 [CrossRef][PubMed]
    [Google Scholar]
  28. Whitchurch CB, Leech AJ, Young MD, Kennedy D, Sargent JL et al. Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa. Mol Microbiol 2004;52:873–893 [CrossRef][PubMed]
    [Google Scholar]
  29. Winther-Larsen HC, Koomey M. Transcriptional, chemosensory and cell-contact-dependent regulation of type IV pilus expression. Curr Opin Microbiol 2002;5:173–178 [CrossRef][PubMed]
    [Google Scholar]
  30. Wadhams GH, Armitage JP. Making sense of it all: bacterial chemotaxis. Nat Rev Mol Cell Biol 2004;5:1024–1037 [CrossRef][PubMed]
    [Google Scholar]
  31. Beatson SA, Whitchurch CB, Sargent JL, Levesque RC, Mattick JS. Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa. J Bacteriol 2002;184:3605–3613 [CrossRef][PubMed]
    [Google Scholar]
  32. Wolfgang MC, Lee VT, Gilmore ME, Lory S. Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent signaling pathway. Dev Cell 2003;4:253–263 [CrossRef][PubMed]
    [Google Scholar]
  33. Whitchurch CB, Beatson SA, Comolli JC, Jakobsen T, Sargent JL et al. Pseudomonas aeruginosa fimL regulates multiple virulence functions by intersecting with Vfr-modulated pathways. Mol Microbiol 2005;55:1357–1378 [CrossRef][PubMed]
    [Google Scholar]
  34. O'Toole GA, Gibbs KA, Hager PW, Phibbs PV, Kolter R. The global carbon metabolism regulator Crc is a component of a signal transduction pathway required for biofilm development by Pseudomonas aeruginosa. J Bacteriol 2000;182:425–431 [CrossRef][PubMed]
    [Google Scholar]
  35. Jain R, Sliusarenko O, Kazmierczak BI. Interaction of the cyclic-di-GMP binding protein FimX and the Type 4 pilus assembly ATPase promotes pilus assembly. PLoS Pathog 2017;13:e1006594 [CrossRef][PubMed]
    [Google Scholar]
  36. Buensuceso RNC, Daniel-Ivad M, Kilmury SLN, Leighton TL, Harvey H et al. Cyclic AMP-independent control of twitching motility in Pseudomonas aeruginosa. J Bacteriol 2017;199: [CrossRef][PubMed]
    [Google Scholar]
  37. Cowles KN, Moser TS, Siryaporn A, Nyakudarika N, Dixon W et al. The putative Poc complex controls two distinct Pseudomonas aeruginosa polar motility mechanisms. Mol Microbiol 2013;90:923–938 [CrossRef][PubMed]
    [Google Scholar]
  38. Huang B, Ru K, Yuan Z, Whitchurch CB, Mattick JS. tonB3 is required for normal twitching motility and extracellular assembly of type IV pili. J Bacteriol 2004;186:4387–4389 [CrossRef][PubMed]
    [Google Scholar]
  39. Inclan YF, Huseby MJ, Engel JN. FimL regulates cAMP synthesis in Pseudomonas aeruginosa. PLoS One 2011;6:e15867 [CrossRef][PubMed]
    [Google Scholar]
  40. Nolan LM, Beatson SA, Croft L, Jones PM, George AM et al. Extragenic suppressor mutations that restore twitching motility to fimL mutants of Pseudomonas aeruginosa are associated with elevated intracellular cyclic AMP levels. Microbiologyopen 2012;1:490–501 [CrossRef][PubMed]
    [Google Scholar]
  41. Langridge GC, Phan MD, Turner DJ, Perkins TT, Parts L et al. Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Res 2009;19:2308–2316 [CrossRef][PubMed]
    [Google Scholar]
  42. Barquist L, Mayho M, Cummins C, Cain AK, Boinett CJ et al. The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries. Bioinformatics 2016;32:1109–1111 [CrossRef][PubMed]
    [Google Scholar]
  43. Barquist L, Boinett CJ, Cain AK. Approaches to querying bacterial genomes with transposon-insertion sequencing. RNA Biol 2013;10:1161–1169 [CrossRef][PubMed]
    [Google Scholar]
  44. Gallagher LA, Shendure J, Manoil C. Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq. MBio 2011;2:e00315 [CrossRef][PubMed]
    [Google Scholar]
  45. Lee SA, Gallagher LA, Thongdee M, Staudinger BJ, Lippman S et al. General and condition-specific essential functions of Pseudomonas aeruginosa. Proc Natl Acad Sci USA 2015;112:5189–5194 [CrossRef][PubMed]
    [Google Scholar]
  46. Turner KH, Everett J, Trivedi U, Rumbaugh KP, Whiteley M. Requirements for Pseudomonas aeruginosa acute burn and chronic surgical wound infection. PLoS Genet 2014;10:e1004518 [CrossRef][PubMed]
    [Google Scholar]
  47. Liberati NT, Urbach JM, Miyata S, Lee DG, Drenkard E et al. An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc Natl Acad Sci USA 2006;103:2833–2838 [CrossRef][PubMed]
    [Google Scholar]
  48. Siehnel R, Traxler B, An DD, Parsek MR, Schaefer AL et al. A unique regulator controls the activation threshold of quorum-regulated genes in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 2010;107:7916–7921 [CrossRef][PubMed]
    [Google Scholar]
  49. Kaniga K, Delor I, Cornelis GR. A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene 1991;109:137–141 [CrossRef][PubMed]
    [Google Scholar]
  50. Hachani A, Allsopp LP, Oduko Y, Filloux A. The VgrG proteins are "à la carte" delivery systems for bacterial type VI effectors. J Biol Chem 2014;289:17872–17884 [CrossRef][PubMed]
    [Google Scholar]
  51. Watson AA, Mattick JS, Alm RA. Functional expression of heterologous type 4 fimbriae in Pseudomonas aeruginosa. Gene 1996;175:143–150 [CrossRef][PubMed]
    [Google Scholar]
  52. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989
    [Google Scholar]
  53. O'Toole GA. Microtiter dish biofilm formation assay. J Vis Exp 2011;47: [CrossRef][PubMed]
    [Google Scholar]
  54. Semmler AB, Whitchurch CB, Mattick JS. A re-examination of twitching motility in Pseudomonas aeruginosa. Microbiology 1999;145:2863–2873 [CrossRef][PubMed]
    [Google Scholar]
  55. Robinson MD, Mccarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010;26:139–140 [CrossRef][PubMed]
    [Google Scholar]
  56. Chand NS, Lee JS, Clatworthy AE, Golas AJ, Smith RS et al. The sensor kinase KinB regulates virulence in acute Pseudomonas aeruginosa infection. J Bacteriol 2011;193:2989–2999 [CrossRef][PubMed]
    [Google Scholar]
  57. Winsor GL, Griffiths EJ, Lo R, Dhillon BK, Shay JA et al. Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Nucleic Acids Res 2016;44:D646–D653 [CrossRef][PubMed]
    [Google Scholar]
  58. Li G, Miller A, Bull H, Howard SP. Assembly of the type II secretion system: identification of ExeA residues critical for peptidoglycan binding and secretin multimerization. J Bacteriol 2011;193:197–204 [CrossRef][PubMed]
    [Google Scholar]
  59. Kanehisa M, Furumichi M, Tanabe M, Sato Y, Morishima K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res 2017;45:D353–D361 [CrossRef][PubMed]
    [Google Scholar]
  60. Fernández L, Breidenstein EB, Song D, Hancock RE. Role of intracellular proteases in the antibiotic resistance, motility, and biofilm formation of Pseudomonas aeruginosa. Antimicrob Agents Chemother 2012;56:1128–1132 [CrossRef][PubMed]
    [Google Scholar]
  61. Jain R, Chan MK. Support for a potential role of E. coli oligopeptidase A in protein degradation. Biochem Biophys Res Commun 2007;359:486–490 [CrossRef][PubMed]
    [Google Scholar]
  62. du H, Pang M, Dong Y, Wu Y, Wang N et al. Identification and characterization of an Aeromonas hydrophila oligopeptidase gene pepF negatively related to biofilm formation. Front Microbiol 2016;7:1497 [CrossRef][PubMed]
    [Google Scholar]
  63. Doyle TB, Hawkins AC, McCarter LL. The complex flagellar torque generator of Pseudomonas aeruginosa. J Bacteriol 2004;186:6341–6350 [CrossRef][PubMed]
    [Google Scholar]
  64. Li N, Kojima S, Homma M. Sodium-driven motor of the polar flagellum in marine bacteria Vibrio. Genes Cells 2011;16:985–999 [CrossRef][PubMed]
    [Google Scholar]
  65. Lloyd SA, Tang H, Wang X, Billings S, Blair DF. Torque generation in the flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM or FliN. J Bacteriol 1996;178:223–231 [CrossRef][PubMed]
    [Google Scholar]
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