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T6SS contributes to gut microbiome invasion and killing of an herbivorous pest insect by plant-beneficial Pseudomonas protegens

The ISME Journal (2019) | Download Citation


Pseudomonas protegens are multi-talented plant-colonizing bacteria that suppress plant pathogens and stimulate plant defenses. In addition, they are capable of invading and killing agriculturally important plant pest insects that makes them promising candidates for biocontrol applications. Here we assessed the role of type VI secretion system (T6SS) components of type strain CHA0 during interaction with larvae of the cabbage pest Pieris brassicae. We show that the T6SS core apparatus and two VgrG modules, encompassing the respective T6SS spikes (VgrG1a and VgrG1b) and associated effectors (RhsA and Ghh1), contribute significantly to insect pathogenicity of P. protegens in oral infection assays but not when bacteria are injected directly into the hemolymph. Monitoring of the colonization levels of P. protegens in the gut, hemolymph, and excrements of the insect larvae revealed that the invader relies on T6SS and VgrG1a module function to promote hemocoel invasion. A 16S metagenomic analysis demonstrated that T6SS-supported invasion by P. protegens induces significant changes in the insect gut microbiome affecting notably Enterobacteriaceae, a dominant group of the commensal gut bacteria. Our study supports the concept that pathogens deploy T6SS-based strategies to disrupt the commensal microbiota in order to promote host colonization and pathogenesis.

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  1. 1.

    Garrido-Sanz D, Arrebola E, Martínez-Granero F, García-Méndez S, Muriel C, Blanco-Romero E, et al. Classification of isolates from the Pseudomonas fluorescens complex into phylogenomic groups based in group-specific markers. Front Microbiol. 2017;8:413.

  2. 2.

    Vacheron J, Desbrosses G, Bouffaud M-L, Touraine B, Moënne-Loccoz Y, Muller D, et al. Plant growth-promoting rhizobacteria and root system functioning. Front Plant Sci. 2013;4:356.

  3. 3.

    Mauchline TH, Malone JG. Life in earth - the root microbiome to the rescue? Curr Opin Microbiol. 2017;37:23–28.

  4. 4.

    Kupferschmied P, Maurhofer M, Keel C. Promise for plant pest control: root-associated pseudomonads with insecticidal activities. Front Plant Sci. 2013;4:287.

  5. 5.

    Flury P, Aellen N, Ruffner B, Péchy-Tarr M, Fataar S, Metla Z, et al. Insect pathogenicity in plant-beneficial pseudomonads: phylogenetic distribution and comparative genomics. ISME J. 2016;10:2527–42.

  6. 6.

    Haas D, Défago G. Biological control of soil-borne pathogens by fluorescent pseudomonads. Nat Rev Microbiol. 2005;3:307–19.

  7. 7.

    Haas D, Keel C. Regulation of antibiotic production in root-colonizing Pseudomonas spp. and relevance for biological control of plant disease. Annu Rev Phytopathol. 2003;41:117–53.

  8. 8.

    Péchy-Tarr M, Borel N, Kupferschmied P, Turner V, Binggeli O, Radovanovic D, et al. Control and host-dependent activation of insect toxin expression in a root-associated biocontrol pseudomonad. Environ Microbiol. 2013;15:736–50.

  9. 9.

    Ruffner B, Péchy-Tarr M, Ryffel F, Hoegger P, Obrist C, Rindlisbacher A, et al. Oral insecticidal activity of plant-associated pseudomonads. Environ Microbiol. 2013;15:751–63.

  10. 10.

    Keel C. A look into the toolbox of multi-talents: insect pathogenicity determinants of plant-beneficial pseudomonads. Environ Microbiol. 2016;18:3207–9.

  11. 11.

    Flury P, Vesga P, Péchy-Tarr M, Aellen N, Dennert F, Hofer N, et al. Antimicrobial and insecticidal: cyclic lipopeptides and hydrogen cyanide produced by plant-beneficial Pseudomonas strains CHA0, CMR12a, and PCL1391 contribute to insect killing. Front Microbiol. 2017;8:100.

  12. 12.

    Loper JE, Henkels MD, Rangel LI, Olcott MH, Walker FL, Bond KL, et al. Rhizoxin analogs, orfamide A and chitinase production contribute to the toxicity of Pseudomonas protegens strain Pf-5 to Drosophila melanogaster. Environ Microbiol. 2016;18:3509–21.

  13. 13.

    Péchy-Tarr M, Bruck DJ, Maurhofer M, Fischer E, Vogne C, Henkels MD, et al. Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens. Environ Microbiol. 2008;10:2368–86.

  14. 14.

    Rangel LI, Henkels MD, Shaffer BT, Walker FL, Davis EW, Stockwell VO, et al. Characterization of toxin complex gene clusters and insect toxicity of bacteria representing four subgroups of Pseudomonas fluorescens. PLoS ONE. 2016;11:e0161120.

  15. 15.

    Kupferschmied P, Chai T, Flury P, Blom J, Smits THM, Maurhofer M, et al. Specific surface glycan decorations enable antimicrobial peptide resistance in plant-beneficial pseudomonads with insect-pathogenic properties. Environ Microbiol. 2016;18:4265–81.

  16. 16.

    Kupferschmied P, Péchy-Tarr M, Imperiali N, Maurhofer M, Keel C. Domain shuffling in a sensor protein contributed to the evolution of insect pathogenicity in plant-beneficial Pseudomonas protegens. PLoS Pathog. 2014;10:e1003964.

  17. 17.

    Basler M. Type VI secretion system: secretion by a contractile nanomachine. Philos Trans R Soc Lond B Biol Sci. 2015;370:pii20150021.

  18. 18.

    Cianfanelli FR, Monlezun L, Coulthurst SJ. Aim, load, fire: The type VI secretion system, a bacterial nanoweapon. Trends Microbiol. 2016;24:51–62.

  19. 19.

    Hachani A, Wood TE, Filloux A. Type VI secretion and anti-host effectors. Curr Opin Microbiol. 2016;29:81–93.

  20. 20.

    Ho BT, Dong TG, Mekalanos JJ. A view to a kill: the bacterial type VI secretion system. Cell Host Microbe. 2014;15:9–21.

  21. 21.

    Nguyen VS, Douzi B, Durand E, Roussel A, Cascales E, Cambillau C. Towards a complete structural deciphering of type VI secretion system. Curr Opin Struct Biol. 2018;49:77–84.

  22. 22.

    Fu Y, Waldor MK, Mekalanos JJ. Tn-Seq analysis of Vibrio cholerae intestinal colonization reveals a role for T6SS-mediated antibacterial activity in the host. Cell Host Microbe. 2013;14:652–63.

  23. 23.

    Sana TG, Flaugnatti N, Lugo KA, Lam LH, Jacobson A, Baylot V, et al. Salmonella typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut. Proc Natl Acad Sci USA. 2016;113:E5044–51.

  24. 24.

    Bernal P, Llamas MA, Filloux A. Type VI secretion systems in plant-associated bacteria. Environ Microbiol. 2018;20:1–15.

  25. 25.

    Ma L-S, Hachani A, Lin J-S, Filloux A, Lai E-M. Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta. Cell Host Microbe. 2014;16:94–104.

  26. 26.

    Filloux A, Freemont P. Structural biology: baseplates in contractile machines. Nat Microbiol. 2016;1:16104.

  27. 27.

    Silverman JM, Brunet YR, Cascales E, Mougous JD. Structure and regulation of the type VI secretion system. Annu Rev Microbiol. 2012;66:453–72.

  28. 28.

    Alcoforado Diniz J, Liu Y-C, Coulthurst SJ. Molecular weaponry: diverse effectors delivered by the type VI secretion system. Cell Microbiol. 2015;17:1742–51.

  29. 29.

    Cianfanelli FR, Alcoforado Diniz J, Guo M, De Cesare V, Trost M, Coulthurst SJ. VgrG and PAAR proteins define distinct versions of a functional type VI secretion system. PLoS Pathog. 2016;12:e1005735.

  30. 30.

    Shneider MM, Buth SA, Ho BT, Basler M, Mekalanos JJ, Leiman PG. PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature. 2013;500:350–53.

  31. 31.

    Durand E, Cambillau C, Cascales E, Journet L. VgrG, Tae, Tle, and beyond: the versatile arsenal of type VI secretion effectors. Trends Microbiol. 2014;22:498–507.

  32. 32.

    Russell AB, Peterson SB, Mougous JD. Type VI secretion system effectors: poisons with a purpose. Nat Rev Microbiol. 2014;12:137–48.

  33. 33.

    Whitney JC, Beck CM, Goo YA, Russell AB, Harding B, De Leon JA, et al. Genetically distinct pathways guide effector export through the type VI secretion system. Mol Microbiol. 2014;92:529–42.

  34. 34.

    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;289:17872–84.

  35. 35.

    Winsor GL, Griffiths EJ, Lo R, Dhillon BK, Shay JA, Brinkman FSL. Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Nucleic Acids Res. 2016;44:D646–53.

  36. 36.

    Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, et al. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 2017;45:D200–D203.

  37. 37.

    Finn RD, Attwood TK, Babbitt PC, Bateman A, Bork P, Bridge AJ, et al. InterPro in 2017—beyond protein family and domain annotations. Nucleic Acids Res. 2017;45:D190–D199.

  38. 38.

    Hachani A, Lossi NS, Hamilton A, Jones C, Bleves S, Albesa-Jové D, et al. Type VI secretion system in Pseudomonas aeruginosa secretion and multimerization of VgrG proteins. J Biol Chem. 2011;286:12317–27.

  39. 39.

    Hood RD, Singh P, Hsu F, Güvener T, Carl MA, Trinidad RRS, et al. A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe. 2010;7:25–37.

  40. 40.

    Pissaridou P, Allsopp LP, Wettstadt S, Howard SA, Mavridou DAI, Filloux A. The Pseudomonas aeruginosa T6SS-VgrG1b spike is topped by a PAAR protein eliciting DNA damage to bacterial competitors. PNAS. 2018;115:12519–12524.

  41. 41.

    Martínez-García E, de Lorenzo V. Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ Microbiol. 2011;13:2702–16.

  42. 42.

    David WAL, Gardiner BOC. Rearing Pieris brassicae L. larvae on a semi-synthetic diet. Nature. 1965;207:882–883.

  43. 43.

    Zakrzewski M, Proietti C, Ellis JJ, Hasan S, Brion M-J, Berger B, et al. Calypso: a user-friendly web-server for mining and visualizing microbiome–environment interactions. Bioinformatics. 2017;33:782–3.

  44. 44.

    Paulson JN, Stine OC, Bravo HC, Pop M. Robust methods for differential abundance analysis in marker gene surveys. Nat Methods. 2013;10:1200–2.

  45. 45.

    Filloux A, Hachani A, Bleves S. The bacterial type VI secretion machine: yet another player for protein transport across membranes. Microbiology. 2008;154:1570–83.

  46. 46.

    Mougous JD, Cuff ME, Raunser S, Shen A, Zhou M, Gifford CA, et al. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science. 2006;312:1526–30.

  47. 47.

    Hassan KA, Johnson A, Shaffer BT, Ren Q, Kidarsa TA, Elbourne LDH, et al. Inactivation of the GacA response regulator in Pseudomonas fluorescens Pf-5 has far-reaching transcriptomic consequences. Environ Microbiol. 2010;12:899–915.

  48. 48.

    Loper JE, Hassan KA, Mavrodi DV, Davis EW, Lim CK, Shaffer BT, et al. Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions. PLoS Genet. 2012;8:e1002784.

  49. 49.

    Basler M, Ho BT, Mekalanos JJ. Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions. Cell. 2013;152:884–94.

  50. 50.

    Cascales E, Cambillau C. Structural biology of type VI secretion systems. Philos Trans R Soc Lond B Biol Sci. 2012;367:1102–11.

  51. 51.

    Zoued A, Brunet YR, Durand E, Aschtgen M-S, Logger L, Douzi B, et al. Architecture and assembly of the type VI secretion system. Biochim Biophys Acta. 2014;1843:1664–73.

  52. 52.

    Ruffner B, Péchy-Tarr M, Höfte M, Bloemberg G, Grunder J, Keel C, et al. Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus. BMC Genomics. 2015;16:609.

  53. 53.

    Ma J, Pan Z, Huang J, Sun M, Lu C, Yao H. The Hcp proteins fused with diverse extended-toxin domains represent a novel pattern of antibacterial effectors in type VI secretion systems. Virulence. 2017;8:1189–1202.

  54. 54.

    Tang JY, Bullen NP, Ahmad S, Whitney JC. Diverse NADase effector families mediate interbacterial antagonism via the type VI secretion system. J Biol Chem. 2018;293:1504–14.

  55. 55.

    Unterweger D, Kostiuk B, Pukatzki S. Adaptor proteins of type VI secretion system effectors. Trends Microbiol. 2017;25:8–10.

  56. 56.

    Bondage DD, Lin J-S, Ma L-S, Kuo C-H, Lai E-M. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex. Proc Natl Acad Sci USA. 2016;113:E3931–40.

  57. 57.

    Kapitein N, Mogk A. Deadly syringes: type VI secretion system activities in pathogenicity and interbacterial competition. Curr Opin Microbiol. 2013;16:52–8.

  58. 58.

    Anderson MC, Vonaesch P, Saffarian A, Marteyn BS, Sansonetti PJ. Shigella sonnei encodes a functional T6SS used for interbacterial competition and niche occupancy. Cell Host Microbe. 2017;21:769–776.e3.

  59. 59.

    Joshi A, Kostiuk B, Rogers A, Teschler J, Pukatzki S, Yildiz FH. Rules of engagement: the type VI secretion system in Vibrio cholerae. Trends Microbiol. 2017;25:267–79.

  60. 60.

    Sana TG, Berni B, Bleves S. The T6SSs of Pseudomonas aeruginosa strain PAO1 and their effectors: beyond bacterial-cell targeting. Front Cell Infect Microbiol. 2016;6:61.

  61. 61.

    Ma J, Sun M, Pan Z, Lu C, Yao H. Diverse toxic effectors are harbored by vgrG islands for interbacterial antagonism in type VI secretion system. Biochim Biophys Acta. 2018;1862:1635–43.

  62. 62.

    Broderick NA, Robinson CJ, McMahon MD, Holt J, Handelsman J, Raffa KF. Contributions of gut bacteria to Bacillus thuringiensis-induced mortality vary across a range of Lepidoptera. BMC Biol. 2009;7:11.

  63. 63.

    Robinson CJ, Schloss P, Ramos Y, Raffa K, Handelsman J. Robustness of the bacterial community in the cabbage white butterfly larval midgut. Microb Ecol. 2010;59:199–211.

  64. 64.

    Tang X, Freitak D, Vogel H, Ping L, Shao Y, Cordero EA, et al. Complexity and variability of gut commensal microbiota in polyphagous lepidopteran larvae. PLoS ONE. 2012;7:e36978.

  65. 65.

    Paniagua Voirol LR, Frago E, Kaltenpoth M, Hilker M, Fatouros NE. Bacterial symbionts in Lepidoptera: their diversity, transmission, and impact on the host. Front Microbiol. 2018;9:556.

  66. 66.

    Xia X, Gurr GM, Vasseur L, Zheng D, Zhong H, Qin B, et al. Metagenomic sequencing of diamondback moth gut microbiome unveils key holobiont adaptations for herbivory. Front Microbiol. 2017;8:663.

  67. 67.

    Shao Y, Chen B, Sun C, Ishida K, Hertweck C, Boland W. Symbiont-derived antimicrobials contribute to the control of the Lepidopteran gut microbiota. Cell Chem Biol. 2017;24:66–75.

  68. 68.

    Engel P, Moran NA. The gut microbiota of insects – diversity in structure and function. FEMS Microbiol Rev. 2013;37:699–735.

  69. 69.

    Chou S, Bui NK, Russell AB, Lexa KW, Gardiner TE, LeRoux M, et al. Structure of a peptidoglycan amidase effector targeted to Gram-negative bacteria by the type VI secretion system. Cell Rep. 2012;1:656–64.

  70. 70.

    MacIntyre DL, Miyata ST, Kitaoka M, Pukatzki S. The Vibrio cholerae type VI secretion system displays antimicrobial properties. Proc Natl Acad Sci USA. 2010;107:19520–4.

  71. 71.

    Schwarz S, West TE, Boyer F, Chiang W-C, Carl MA, Hood RD, et al. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions. PLoS Pathog. 2010;6:e1001068.

  72. 72.

    Fast D, Kostiuk B, Foley E, Pukatzki S. Commensal pathogen competition impacts host viability. Proc Natl Acad Sci USA. 2018;115:7099–104.

  73. 73.

    Logan SL, Thomas J, Yan J, Baker RP, Shields DS, Xavier JB, et al. The Vibrio cholerae type VI secretion system can modulate host intestinal mechanics to displace gut bacterial symbionts. Proc Natl Acad Sci USA. 2018;115:3779–87.

  74. 74.

    Zhao W, Caro F, Robins W, Mekalanos JJ. Antagonism toward the intestinal microbiota and its effect on Vibrio cholerae virulence. Science. 2018;359:210–3.

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We gratefully acknowledge the group of Philippe Reymond at the Department of Plant Molecular Biology of the University of Lausanne for help with rearing Pieris brassicae and with the development of the artificial diet-based feeding assay. We thank the Biocommunications group (Consuelo De Moraes), Department of Environmental System Science, ETH Zurich for providing eggs of Pieris brassicae.

Sources of support (grants/equipment)

This study was supported by grant 31003A-159520 from the Swiss National Foundation for Scientific Research.

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  1. Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland

    • Jordan Vacheron
    • , Maria Péchy-Tarr
    • , Silvia Brochet
    • , Clara Margot Heiman
    • , Marina Stojiljkovic
    •  & Christoph Keel
  2. Plant Pathology, Institute of Integrative Biology, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland

    • Monika Maurhofer


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Correspondence to Monika Maurhofer or Christoph Keel.

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