High‐Throughput Screening for Novel Anti‐Infectives Using a C. elegans Pathogenesis Model

Annie L. Conery1, Jonah Larkins‐Ford2, Frederick M. Ausubel1, Natalia V. Kirienko1

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts, 2 Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130160
Online Posting Date:  March, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


In recent history, the nematode Caenorhabditis elegans has provided a compelling platform for the discovery of novel antimicrobial drugs. In this protocol, we present an automated, high‐throughput C. elegans pathogenesis assay, which can be used to screen for anti‐infective compounds that prevent nematodes from dying due to Pseudomonas aeruginosa. New antibiotics identified from such screens would be promising candidates for treatment of human infections, and also can be used as probe compounds to identify novel targets in microbial pathogenesis or host immunity. Curr. Protoc. Chem. Biol. 6:25‐37. © 2014 by John Wiley & Sons, Inc.

Keywords: Caenorhabditis elegans; Pseudomonas aeruginosa; high‐throughput screening; pathogenesis model; antibiotic; antimicrobial; drug discovery; liquid killing

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: P. aeruginosa Liquid Killing Assay in C. elegans
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: P. aeruginosa Liquid Killing Assay in C. elegans

  • P. aeruginosa strain PA14 (Rahme et al., )
  • 10‐cm LB agar plates (see recipe)
  • LB broth (see recipe)
  • 10‐cm SK agar plates (see recipe)
  • Gravid C. elegans strain SS104 glp‐4(bn2ts) (Beanan and Strome, ); can be ordered from the Caenorhabditis Genetics Center (http://www.cbs.umn.edu/research/resources/cgc)
  • M9 buffer (see recipe)
  • 10‐cm OP50 NGM agar plates (see recipe)
  • Worm bleaching solution (see recipe)
  • S Basal+ buffer (see recipe)
  • SK medium
  • S Basal buffer (see recipe)
  • Sytox Orange solution (see recipe)
  • 37°C incubator
  • Sterile bacteria spreader
  • 15‐ml conical tubes
  • Clinical centrifuge (e.g., Thermo Scientific CL2 centrifuge)
  • Dissecting microscope (e.g., Nikon SMZ660)
  • Rotator (e.g., Thermo Scientific Labquake tube rotator)
  • 15°, 20°, and 25°C incubators with humidifying capability (e.g., Thermo Forma, model no. 3920)
  • Sterile cell scraper (e.g., Small Cell Scraper, Corning, cat. no. 3010)
  • Spectrophotometer (e.g., Beckman Coulter DU730 UV/Vis spectrophotometer)
  • Multichannel pipet or reagent dispenser (e.g., Thermo Scientific Multidrop Combi)
  • 384‐well microplate (e.g., Corning, cat. no. 3712)
  • Union Biometrica COPAS BioSort large particle sorter
  • Gas‐permeable membrane (e.g., Breathe‐Easy, Diversified Biotech)
  • Microplate washer (e.g., BioTek ELx405)
  • Microplate mixer (e.g., Eppendorf MixMate)
  • Automated microscope (e.g., Molecular Devices ImageXpress Micro)
  • Image analysis software (e.g., CellProfiler, http://www.cellprofiler.org)
PDF or HTML at Wiley Online Library



Literature Cited

  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.
  Beanan, M.J. and Strome, S. 1992. Characterization of a germ‐line proliferation mutation in C. elegans. Development 116:755‐766.
  Breger, J., Fuchs, B.B., Aperis, G., Moy, T.I., Ausubel, F.M., and Mylonakis, E. 2007. Antifungal chemical compounds identified using a C. elegans pathogenicity assay. PLoS Pathogens 3:e18.
  Burns, A.R., Kwok, T.C., Howard, A., Houston, E., Johanson, K., Chan, A., Cutler, S.R., McCourt, P., and Roy, P.J. 2006. High‐throughput screening of small molecules for bioactivity and target identification in Caenorhabditis elegans. Nat. Protoc. 1:1906‐1914.
  Carpenter, A.E., Jones, T.R., Lamprecht, M.R., Clarke, C., Kang, I.H., Friman, O., Guertin, D.A., Chang, J.H., Lindquist, R.A., Moffat, J., Golland, P., and Sabatini, D.M. 2006. CellProfiler: Image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7:R100.
  Couillault, C., Pujol, N., Reboul, J., Sabatier, L., Guichou, J.F., Kohara, Y., and Ewbank, J.J. 2004. TLR‐independent control of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR‐1, an ortholog of human SARM. Nat. Immunol. 5:488‐494.
  Darby, C., Hsu, J.W., Ghori, N., and Falkow, S. 2002. Caenorhabditis elegans: Plague bacteria biofilm blocks food intake. Nature 417:243‐244.
  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. U.S.A. 98:10892‐10897.
  Girard, L.R., Fiedler, T.J., Harris, T.W., Carvalho, F., Antoshechkin, I., Han, M., Sternberg, P.W., Stein, L.D., and Chalfie, M. 2007. WormBook: The online review of Caenorhabditis elegans biology. Nucleic Acids Res. 35:472‐475.
  Kim, D.H., Feinbaum, R., Alloing, G., Emerson, F.E., Garsin, D.A., Inoue, H., Tanaka‐Hino, M., Hisamoto, N., Matsumoto, K., Tan, M.W., and Ausubel, F.M. 2002. A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity. Science 297:623‐626.
  Kirienko, N.V., Kirienko, D.R., Larkins‐Ford, J., Wahlby, C., Ruvkun, G., and Ausubel, F.M. 2013. Pseudomonas aeruginosa disrupts Caenorhabditis elegans iron homeostasis, causing a hypoxic response and death. Cell Host Microbe 13:406‐416.
  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.
  Lewis, K. 2013. Platforms for antibiotic discovery. Nat. Rev. Drug Discov. 12:371‐387.
  Liberati, N.T., Fitzgerald, K.A., Kim, D.H., Feinbaum, R., Golenbock, D.T., and Ausubel, F.M. 2004. Requirement for a conserved Toll/interleukin‐1 resistance domain protein in the Caenorhabditis elegans immune response. Proc. Natl. Acad. Sci. U.S.A. 101:6593‐6598.
  Mahajan‐Miklos, S., Tan, M.W., Rahme, L.G., and Ausubel, F.M. 1999. Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa‐Caenorhabditis elegans pathogenesis model. Cell 96:47‐56.
  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.
  Moy, T.I., Conery, A.L., Larkins‐Ford, J., Wu, G., Mazitschek, R., Casadei, G., Lewis, K., Carpenter, A.E., and Ausubel, F.M. 2009. High‐throughput screen for novel antimicrobials using a whole animal infection model. ACS Chem. Biol. 4:527‐533.
  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. U.S.A. 99:15675‐15680.
  Powell, J.R. and Ausubel, F.M. 2008. Models of Caenorhabditis elegans infection by bacterial and fungal pathogens. Methods Mol. Biol. 415:403‐427.
  Pukkila‐Worley, R. and Ausubel, F.M. 2012. Immune defense mechanisms in the Caenorhabditis elegans intestinal epithelium. Curr. Opin. Immunol. 24:3‐9.
  Rahme, L.G., Stevens, E.J., Wolfort, S.F., Shao, J., Tompkins, R.G., and Ausubel, F.M. 1995. Common virulence factors for bacterial pathogenicity in plants and animals. Science 268:1899‐1902.
  Riddle, D.L., Blumenthal, T., Meyer, B.J., and Priess, J.R. 1997. Introduction to C. elegans. In C. elegans II, 2nd ed. (D.L. Riddle, T. Blumenthal, B.J. Meyer, and J.R. Priess, eds.). Cold Spring Harbor Press, New York.
  Sifri, C.D., Begun, J., and Ausubel, F.M. 2005. The worm has turned–microbial virulence modeled in Caenorhabditis elegans. Trends Microbiol. 13:119‐127.
  Simonsen, K.T., Nielsen, G., Bjerrum, J.V., Kruse, T., Kallipolitis, B.H., and Moller‐Jensen, J. 2011. A role for the RNA chaperone Hfq in controlling adherent‐invasive Escherichia coli colonization and virulence. PloS One 6:e16387.
  Stein, L., Sternberg, P., Durbin, R., Thierry‐Mieg, J., and Spieth, J. 2001. WormBase: Network access to the genome and biology of Caenorhabditis elegans. Nucleic Acids Res. 29:82‐86.
  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. U.S.A. 96:715‐720.
  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. U.S.A. 96:2408‐2413.
  Vokes, M.S. and Carpenter, A.E. 2008. Using CellProfiler for automatic identification and measurement of biological objects in images. Curr. Protoc. Mol. Biol. 82:14.17.1‐14.17.12.
  Wahlby, C., Kamentsky, L., Liu, Z.H., Riklin‐Raviv, T., Conery, A.L., O'Rourke, E.J., Sokolnicki, K.L., Visvikis, O., Ljosa, V., Irazoqui, J.E., Golland, P., Ruvkun, G., Ausubel, F.M., and Carpenter, A.E. 2012. An image analysis toolbox for high‐throughput C. elegans assays. Nat. Methods 9:714‐716.
  Wood, W.B. 1988. The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  Zhang, J.H., Chung, T.D., and Oldenburg, K.R. 1999. A simple statistical parameter for use in evaluation and validation of high‐throughput screening assays. J. Biomol. Screen. 4:67‐73.
Key Reference
  Kirienko et al., 2013. See above.
  This manuscript describes the main component of the mechanism by which P. aeruginosa kills C. elegans in the liquid killing assay, and also shows the application of the liquid killing assay in a small molecule screen.
Internet Resources
  WormBase is a compendium of useful information about the genome and biology of C. elegans.
  WormBook is a comprehensive review of C. elegans biology with a section devoted to methods used in nematode experimentation.
  CellProfiler is a free, open‐source image analysis software. The software and example pipelines can be downloaded from the CellProfiler Web site.
PDF or HTML at Wiley Online Library