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Role of Two Sets of RND-Type Multidrug Efflux Pump Transporter Genes, mexAB-oprM and mexEF-oprN, in Virulence of Pseudomonas syringae pv. tabaci 6605

  • Ichinose, Yuki (Graduate School of Environmental and Life Science, Okayama University) ;
  • Nishimura, Takafumi (Graduate School of Environmental and Life Science, Okayama University) ;
  • Harada, Minori (Faculty of Agriculture, Okayama University) ;
  • Kashiwagi, Ryota (Graduate School of Environmental and Life Science, Okayama University) ;
  • Yamamoto, Mikihiro (Graduate School of Environmental and Life Science, Okayama University) ;
  • Noutoshi, Yoshiteru (Graduate School of Environmental and Life Science, Okayama University) ;
  • Toyoda, Kazuhiro (Graduate School of Environmental and Life Science, Okayama University) ;
  • Taguchi, Fumiko (Graduate School of Environmental and Life Science, Okayama University) ;
  • Takemoto, Daigo (Graduate School of Bioagricultural Sciences, Nagoya University) ;
  • Matsui, Hidenori (Graduate School of Environmental and Life Science, Okayama University)
  • Received : 2019.11.14
  • Accepted : 2020.03.06
  • Published : 2020.04.01

Abstract

Pseudomonas syringae pv. tabaci 6605 has two multidrug resistance (MDR) efflux pump transporters, MexAB-OprM and MexEF-OprN. To understand the role of these MDR efflux pumps in virulence, we generated deletion mutants, ΔmexB, ΔmexF, and ΔmexBΔmexF, and investigated their sensitivity to plant-derived antimicrobial compounds, antibiotics, and virulence. Growth inhibition assays with KB soft agar plate showed that growth of the wild-type (WT) was inhibited by 5 μl of 1 M catechol and 1 M coumarin but not by other plant-derived potential antimicrobial compounds tested including phytoalexins. The sensitivity to these compounds tended to increase in ΔmexB and ΔmexBΔmexF mutants. The ΔmexBΔmexF mutant was also sensitive to 2 M acetovanillone. The mexAB-oprM was constitutively expressed, and activated in the ΔmexF and ΔmexBΔmexF mutant strains. The swarming and swimming motilities were impaired in ΔmexF and ΔmexBΔmexF mutants. The flood inoculation test indicated that bacterial populations in all mutant strains were significantly lower than that of WT, although all mutants and WT caused similar disease symptoms. These results indicate that MexAB-OprM extrudes plant-derived catechol, acetovanillone, or coumarin, and contributes to bacterial virulence. Furthermore, MexAB-OprM and MexEF-OprN complemented each other's functions to some extent.

Keywords

References

  1. Alcalde-Rico, M., Hernando-Amado, S., Blanco, P. and Martinez, J. L. 2016. Multidrug efflux pumps at the crossroad between antibiotic resistance and bacterial virulence. Front. Microbiol. 7:1483.
  2. Alvarez-Ortega, C., Olivares, J. and Martinez, J. L. 2013. RND multidrug efflux pumps: what are they good for? Front. Microbiol. 4:7. https://doi.org/10.3389/fmicb.2013.00007
  3. Baker, C. J., Mock, N. M., Smith, J. M. and Aver'yanov, A. A. 2015. The dynamics of apoplast phenolics in tobacco leaves following inoculation with bacteria. Front. Plant Sci. 6:649. https://doi.org/10.3389/fpls.2015.00649
  4. Egea, C., Alcazar, M. D. and Candela, M. E. 1996. Capsidiol: its role in the resistance of Capsicum annuum to Phytophthora capsici. Physiol. Plant. 98:737-742. https://doi.org/10.1111/j.1399-3054.1996.tb06679.x
  5. Fan, J., Crooks, C., Creissen, G., Hill, L., Fairhurst, S., Doerner, P. and Lamb, C. 2011. Pseudomonas sax genes overcome aliphatic isothiocyanate-mediated non-host resistance in Arabidopsis. Science 331:1185-1188. https://doi.org/10.1126/science.1199707
  6. Fernando, D. M. and Kumar, A. 2013. Resistance-nodulationdivision multidrug efflux pumps in Gram-negative bacteria:role in virulence. Antibiotics 2:163-181. https://doi.org/10.3390/antibiotics2010163
  7. Glazebrook, J. and Ausubel, F. M. 1994. Isolation of phytoalexindeficient mutants of Arabidopsis thaliana and characterization of their interactions with bacterial pathogens. Proc. Natl. Acad. Sci. U. S. A. 91:8955-8959. https://doi.org/10.1073/pnas.91.19.8955
  8. Gnanamanickam, S. S. and Mansfield, J. W. 1981. Selective toxicity of wyerone and other phytoalexins to Gram-positive bacteria. Phytochemistry 20:997-1000. https://doi.org/10.1016/0031-9422(81)83016-6
  9. Hernando-Amado, S., Blanco, P., Alcalde-Rico, M., Corona, F., Reales-Calderon, J. A., Sanchez, M. B. and Martinez, J. L. 2016. Multidrug efflux pumps as main players in intrinsic and acquired resistance to antimicrobials. Drug Resist. Updat. 28:13-27. https://doi.org/10.1016/j.drup.2016.06.007
  10. Ichinose, Y., Tasaka, Y., Yamamoto, S., Inoue, Y., Takata, M., Nakatsu, Y., Taguchi, F., Yamamoto, M., Toyoda, K., Noutoshi, Y. and Matsui, H. 2020. PsyR, a transcriptional regulator in quorum sensing system, binds lux box-like sequence in psyI promoter without AHL quorum sensing molecule and activates psyI transcription with AHL in Pseudomonas syringae pv. tabaci 6605. J. Gen. Plant Pathol. 86:124-133. https://doi.org/10.1007/s10327-019-00893-3
  11. Ishiga, Y., Ishiga, T., Uppalapati, S. R. and Mysore, K. S. 2011. Arabidopsis seedling flood-inoculation technique: a rapid and reliable assay for studying plant-bacterial interactions. Plant Methods 7:32. https://doi.org/10.1186/1746-4811-7-32
  12. Kang, H. and Gross, D. C. 2005. Characterization of a resistancenodulation-cell division transporter system associated with the syr-syp genomic island of Pseudomonas syringae pv. syringae. Appl. Environ. Microbiol. 71:5056-5065. https://doi.org/10.1128/AEM.71.9.5056-5065.2005
  13. Li, X.-Z., Plesiat, P. and Nikaido, H. 2015. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin. Microbiol. Rev. 28:337-418. https://doi.org/10.1128/CMR.00117-14
  14. Marutani, M., Taguchi, F., Shimizu, R., Inagaki, Y., Toyoda, K., Shiraishi, T. and Ichinose, Y. 2005. Flagellin from Pseudomonas syringae pv. tabaci induced hrp-independent HR in tomato. J. Gen. Plant Pathol. 71:289-295. https://doi.org/10.1007/s10327-005-0200-9
  15. McClean, K. H., Winson, M. K., Fish, L., Taylor, A., Chhabra, S. R., Camara, M., Daykin, M., Lamb, J. H., Swift, S., Bycroft, B. W., Stewart, G. S. A. B. and Williams, P. 1997. Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of Nacylhomoserine lactones. Microbiology 143:3703-3711. https://doi.org/10.1099/00221287-143-12-3703
  16. Morrissey, J. P. and Osbourn, A. E. 1999. Fungal resistance to plant antibiotics as a mechanism of pathogenesis. Microbiol. Mol. Biol. Rev. 63:708-724. https://doi.org/10.1128/MMBR.63.3.708-724.1999
  17. Sawada, T., Eguchi, M., Asaki, S., Kashiwagi, R., Shimomura, K., Taguchi, F., Matsui, H., Yamamoto, M., Noutoshi, Y., Toyoda, K. and Ichinose, Y. 2018. MexEF-OprN multidrug efflux pump transporter negatively controls N-acyl-homoserine lactone accumulation in Pseudomonas syringae pv. tabaci 6605. Mol. Genet. Genomics 293:907-917. https://doi.org/10.1007/s00438-018-1430-9
  18. Schafer, A., Tauch, A., Jaeger, W., Kalinowski, J., Thierbach, G. and Puhler, A. 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69-73. https://doi.org/10.1016/0378-1119(94)90324-7
  19. Stoitsova, S. O., Braun, Y., Ullrich, M. S. and Weingart, H. 2008. Characterization of the RND-type multidrug efflux pump MexAB-OprM of the plant pathogen Pseudomonas syringae. Appl. Environ. Microbiol. 74:3387-3393. https://doi.org/10.1128/AEM.02866-07
  20. Taguchi, F. and Ichinose, Y. 2011. Role of type IV pili in virulence of Pseudomonas syringae pv. tabaci 6605: correlation of motility, multidrug resistance, and HR-inducing activity on a nonhost plant. Mol. Plant-Microbe Interact. 24:1001-1011. https://doi.org/10.1094/MPMI-02-11-0026
  21. Taguchi, F. and Ichinose, Y. 2013. Virulence factor regulator (Vfr) controls virulence-associated phenotypes in Pseudomonas syringae pv. tabaci 6605 by a quorum sensing-independent mechanism. Mol. Plant Pathol. 14:279-292. https://doi.org/10.1111/mpp.12003
  22. Taguchi, F., Inoue, Y., Suzuki, T., Inagaki, Y., Yamamoto, M., Toyoda, K., Noutoshi, Y., Shiraishi, T. and Ichinose, Y. 2015. Characterization of quorum sensing-controlled transcriptional regulator MarR and Rieske (2Fe-2S) cluster-containing protein (Orf5), which are involved in resistance to environmental stresses in Pseudomonas syringae pv. tabaci 6605. Mol. Plant Pathol. 16:376-387. https://doi.org/10.1111/mpp.12187
  23. Taguchi, F., Ogawa, Y., Takeuchi, K., Suzuki, T., Toyoda, K., Shiraishi, T. and Ichinose, Y. 2006. A homologue of the 3-oxoacyl-(acyl carrier protein) synthase III gene located in the glycosylation island of Pseudomonas syringae pv. tabaci regulates virulence factors via N-acyl homoserine lactone and fatty acid synthesis. J. Bacteriol. 188:8376-8384. https://doi.org/10.1128/JB.00763-06
  24. Tsuji, J., Jackson, E. P., Gage, D. A., Hammerschmidt, R. and Somerville, S. C. 1992. Phytoalexin accumulation in Arabidopsis thaliana during the hypersensitive reaction to Pseudomonas syringae pv syringae. Plant Physiol. 98:1304-1309. https://doi.org/10.1104/pp.98.4.1304
  25. VanEtten, H., Temporini, E. and Wasmann, C. 2001. Phytoalexin (and phytoanticipin) tolerance as a virulence trait: why is it not required by all pathogens? Physiol. Mol. Plant Pathol. 59:83-93. https://doi.org/10.1006/pmpp.2001.0350
  26. Vargas, P., Farias, G. A., Nogales, J., Prada, H., Carvajal, V., Baron, M., Rivilla, R., Martin, M., Olmedilla, A. and Gallegos, M.-T. 2013. Plant flavonoids target Pseudomonas syringae pv. tomato DC3000 flagella and type III secretion system. Environ. Microbiol. Rep. 5:841-850. https://doi.org/10.1111/1758-2229.12086
  27. Vargas, P., Felipe, A., Michan, C. and Gallegos, M.-T. 2011. Induction of Pseudomonas syringae pv. tomato DC3000 MexAB-OprM multidrug efflux pump by flavonoids is mediated by the repressor PmeR. Mol. Plant-Microbe Interact. 24:1207-1219. https://doi.org/10.1094/MPMI-03-11-0077