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
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Abstract

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

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: P. aeruginosa Liquid Killing Assay in C. elegans
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

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

  Materials
  • 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)
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Figures

Videos

Literature Cited

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  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.
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  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.
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  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.
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  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.
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  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.
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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
  http://www.wormbase.org/
  WormBase is a compendium of useful information about the genome and biology of C. elegans.
  http://www.wormbook.org/
  WormBook is a comprehensive review of C. elegans biology with a section devoted to methods used in nematode experimentation.
  http://www.cellprofiler.org/
  CellProfiler is a free, open‐source image analysis software. The software and example pipelines can be downloaded from the CellProfiler Web site.
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