Journal of Microbiology

, Volume 51, Issue 3, pp 367–372 | Cite as

Live and dead GFP-tagged bacteria showed indistinguishable fluorescence in Caenorhabditis elegans gut

  • Ju-Ya Hsiao
  • Chun-Yao Chen
  • Mei-Jun Yang
  • Han-Chen Ho
Microbial Pathogenesis and Host-Microbe Interaction

Abstract

Caenorhabditis elegans has been used for studying host-pathogen interactions since long, and many virulence genes of pathogens have been successfully identified. In several studies, fluorescent pathogens were fed to C. elegans and fluorescence observed in the gut was considered an indicator for bacterial colonization. However, the grinder in the pharynx of these nematodes supposedly crushes the bacterial cells, and the ground material is delivered to the intestine for nutrient absorption. Therefore, it remains unclear whether intact bacteria pass through the grinder and colonize in the intestine. Here we investigated whether the appearance of fluorescence is indicative of intact bacteria in the gut using both fluorescence microscopy and transmission electron microscopy. In wild-type N2 C. elegans, Escherichia coli DH5α, and Vibrio vulnificus 93U204, both of which express the green fluorescence protein, were found intact only proximal to the grinder, while crushed bacterial debris was found in the post-pharyngeal lumen. Nevertheless, the fluorescence was evident throughout the lumen of worm intestines irrespective of whether the bacteria were intact or not. We further investigated the interaction of the bacteria with C. elegans phm-2 mutant, which has a dysfunctional grinder. Both strains of bacteria were found to be intact and accumulated in the pharynx and intestine owing to the defective grinder. The fluorescence intensity of intact bacteria in phm-2 worms was indistinguishable from that of crushed bacterial debris in N2 worms. Therefore, appearance of fluorescence in the C. elegans intestine should not be directly interpreted as successful bacterial colonization in the intestine.

Keywords

Vibri vulnificus} phm-2 electron microscopy 

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References

  1. Aballay, A., Yorgey, P., and Ausubel, F.M. 2000. Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans. Curr. Biol.10, 1539–1542.PubMedCrossRefGoogle Scholar
  2. Avery, L. 1993. The genetics of feeding in Caenorhabditis elegans. Genetics133, 897–917.PubMedGoogle Scholar
  3. Bolm, M., Jansen, W.T., Schnabel, R., and Chhatwal, G.S. 2004. Hydrogen peroxide-mediated killing of Caenorhabditis elegans: a common feature of different streptococcal species. Infect. Immun.72, 1192–1194.PubMedCrossRefGoogle Scholar
  4. Coenye, T., Vandamme, P., Govan, J.R., and LiPuma, J.J. 2001. Taxonomy and identification of the Burkholderia cepacia complex. J. Clin. Microbiol.39, 3427–3436.PubMedCrossRefGoogle Scholar
  5. Darby, C., Cosma, C.L., Thomas, J.H., and Manoil, C. 1999. Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA96, 15202–15207.PubMedCrossRefGoogle Scholar
  6. Dhakal, B.K., Lee, W., Kim, Y.R., Choy, H.E., Ahnn, J., and Rhee, J.H. 2006. Caenorhabditis elegans as a simple model host for Vibrio vulnificus infection. Biochem. Biophys. Res. Commun.346, 751–757.PubMedCrossRefGoogle Scholar
  7. Gan, Y.H., Chua, K.L., Chua, H.H., Liu, B., Hii, C.S., Chong, H.L., and Tan, P. 2002. Characterization of Burkholderia pseudomallei infection and identification of novel virulence factors using a Caenorhabditis elegans host system. Mol. Microbiol.44, 1185–1197.PubMedCrossRefGoogle Scholar
  8. Garigan, D., Hsu, A.L., Fraser, A.G., Kamath, R.S., Ahringer, J., and Kenyon, C. 2002. Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation. Genetics161, 1101–1112.PubMedGoogle Scholar
  9. Garsin, D.A., Sifri, C.D., Mylonakis, E., Qin, X., Singh, K.V., Murray, B.E., Calderwood, S.B., and Ausubel, F.M. 2001. A simple model host for identifying Gram-positive virulence factors. Proc. Natl. Acad. Sci. USA98, 10892–10897.PubMedCrossRefGoogle Scholar
  10. Hodgkin, J., Kuwabara, P.E., and Corneliussen, B. 2000. A novel bacterial pathogen, Microbacterium nematophilum, induces morphological change in the nematode C. elegans. Curr. Biol.10, 1615–1618.PubMedCrossRefGoogle Scholar
  11. Hor, L.I., Chang, Y.K., Chang, C.C., Lei, H.Y., and Ou, J.T. 2000. Mechanism of high susceptibility of iron-overloaded mouse to Vibrio vulnificus infection. Microbiol. Immunol.44, 871–878.PubMedGoogle Scholar
  12. Hsueh, P.R., Lin, C.Y., Tang, H.J., Lee, H.C., Liu, J.W., Liu, Y.C., and Chuang, Y.C. 2004. Vibrio vulnificus in Taiwan. Emerg. Infect. Dis.10, 1363–1368.PubMedCrossRefGoogle Scholar
  13. Jansen, W.T., Bolm, M., Balling, R., Chhatwal, G.S., and Schnabel, R. 2002. Hydrogen peroxide-mediated killing of Caenorhabditis elegans by Streptococcus pyogenes. Infect. Immun.70, 5202–5207.PubMedCrossRefGoogle Scholar
  14. Jones, M.K. and Oliver, J.D. 2009. Vibrio vulnificus: Disease and pathogenesis. Infect. Immun.77, 1723–1733.PubMedCrossRefGoogle Scholar
  15. Köthe, M., Antl, M., Huber, B., Stoecker, K., Ebrecht, D., Steinmetz, I., and Eberl, L. 2003. Killing of Caenorhabditis elegans by Burkholderia cepacia is controlled by the cep quorum-sensing system. Cell. Microbiol.5, 343–351.PubMedCrossRefGoogle Scholar
  16. Kurz, C.L., Chauvet, S., Andrès, E., Aurouze, M., Vallet, I., Michel, G.P., Uh, M., Celli, J., Filloux, A., De Bentzmann, andet al. 2003. Virulence factors of the human opportunistic pathogen Serratia marcescens identified by in vivo screening. EMBO J.22, 1451–1460.PubMedCrossRefGoogle Scholar
  17. Kurz, C.L. and Ewbank, J.J. 2000. Caenorhabditis elegans for the study of host-pathogen interactions. Trends Microbiol.8, 142–144.PubMedCrossRefGoogle Scholar
  18. Kurz, C.L. and Ewbank, J.J. 2007. Infection in a dish: high-throughput analyses of bacterial pathogenesis. Curr. Opin. Microbiol.10, 10–16.PubMedCrossRefGoogle Scholar
  19. Labrousse, A., Chauvet, S., Couillault, C., Kurz, C.L., and Ewbank, J.J. 2000. Caenorhabditis elegans is a model host for Salmonella typhimurium. Curr. Biol.10, 1543–1545.PubMedCrossRefGoogle Scholar
  20. Mallo, G.V., Kurz, C.L., Couillault, C., Pujol, N., Granjeaud, S., Kohara, Y., and Ewbank, J.J. 2002. Inducible antibacterial defense system in C. elegans. Curr. Biol.12, 1209–1214.PubMedCrossRefGoogle Scholar
  21. Mylonakis, E., Ausubel, F.M., Perfect, J.R., Heitman, J., and Calderwood, S.B. 2002. Killing of Caenorhabditis elegans by Cryptococcus neoformans as a model of yeast pathogenesis. Proc. Natl. Acad. Sci. USA99, 15675–15680.PubMedCrossRefGoogle Scholar
  22. O’Quinn, A.L., Wiegand, E.M., and Jeddeloh, J.A. 2001. Burkholderia pseudomallei kills the nematode Caenorhabditis elegans using an endotoxin-mediated paralysis. Cell. Microbiol.3, 381–393.PubMedCrossRefGoogle Scholar
  23. Portal-Celhay, C. and Blaser, M.J. 2012. Competition and resilience between founder and introduced bacteria in the Caenorhabditis elegans gut. Infect. Immun.80, 1288–1299.PubMedCrossRefGoogle Scholar
  24. Ruiz-Diez, B., Sanchez, P., Baquero, F., Martinez, J.L., and Navas, A. 2003. Differential interactions within the Caenorhabditis elegans-Pseudomonas aeruginosa pathogenesis model. J. Theor. Biol.225, 469–476.PubMedCrossRefGoogle Scholar
  25. Schulenburg, H. and Ewbank, J.J. 2004. Diversity and specificity in the interaction between Caenorhabditis elegans and the pathogen Serratia marcescens. BMC Evol. Biol.4, 49.PubMedCrossRefGoogle Scholar
  26. Spanier, B., Starke, M., Higel, F., Scherer, S., and Fuchs, T.M. 2010. Yersinia enterocolitica infection and tcaA-dependent killing of Caenorhabditis elegans. Appl. Environ. Microbiol.76, 6277–6285.PubMedCrossRefGoogle Scholar
  27. Strom, M.S. and Paranjpye, R.N. 2000. Epidemiology and pathogenesis of Vibrio vulnificus. Microbes Infect.2, 177–188.PubMedCrossRefGoogle Scholar
  28. Styer, K.L., Hopkins, G.W., Bartra, S.S., Plano, G.V., Frothingham, R., and Aballay, A. 2005. Yersinia pestis kills Caenorhabditis elegans by a biofilm-independent process that involves novel virulence factors. EMBO Rep.6, 992–997.PubMedCrossRefGoogle Scholar
  29. Tan, M.W. and Ausubel, F.M. 2000. Caenorhabditis elegans: A model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr. Opin. Microbiol.3, 29–34.PubMedCrossRefGoogle Scholar
  30. Tan, M.W., Mahajan-Miklos, S., and Ausubel, F.M. 1999a. Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proc. Natl. Acad. Sci. USA96, 715–720.PubMedCrossRefGoogle Scholar
  31. Tan, M.W., Rahme, L.G., Sternberg, J.A., Tompkins, R.G., and Ausubel, F.M. 1999b. Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. Proc. Natl. Acad. Sci. USA96, 2408–2413.PubMedCrossRefGoogle Scholar
  32. Tan, M.W. and Shapira, M. 2011. Genetic and molecular analysis of nematode-microbe interactions. Cell. Microbiol.13, 497–507.PubMedCrossRefGoogle Scholar
  33. Tang, R.J., Breger, J., Idnurm, A., Gerik, K.J., Lodge, J.K., Heitman, J., Calderwood, S.B., and Mylonakis, E. 2005. Cryptococcus neoformans gene involved in mammalian pathogenesis identified by a Caenorhabditis elegans progeny-based approach. Infect. Immun.73, 8219–8225.PubMedCrossRefGoogle Scholar
  34. Taylor, R.G., Walker, D.C., and McInnes, R.R. 1993. E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing. Nucleic Acids Res.21, 1677–1678.PubMedCrossRefGoogle Scholar
  35. Thomsen, L.E., Slutz, S.S., Tan, M.W., and Ingmer, H. 2006. Caenorhabditis elegans is a model host for Listeria monocytogenes. Appl. Environ. Microbiol.72, 1700–1701.PubMedCrossRefGoogle Scholar
  36. Vaitkevicius, K., Lindmark, B., Ou, G., Song, T., Toma, C., Iwanaga, M., Zhu, J., Andersson, A., Hammarström, M.L., Tuck, S., andet al. 2006. A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing. Proc. Natl. Acad. Sci. USA103, 9280–9285.PubMedCrossRefGoogle Scholar
  37. Zaborin, A., Romanowski, K., Gerdes, S., Holbrook, C., Lepine, F., Long, J., Poroyko, V., Diggle, S.P., Wilke, A., Righetti, K., andet al. 2009. Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1. Proc. Natl. Acad. Sci. USA106, 6327–6332.PubMedCrossRefGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Ju-Ya Hsiao
    • 1
  • Chun-Yao Chen
    • 2
  • Mei-Jun Yang
    • 1
  • Han-Chen Ho
    • 1
  1. 1.Department of AnatomyTzu Chi UniversityHualienTaiwan
  2. 2.Department of Life ScienceTzu Chi UniversityHualienTaiwan

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