Novel Approaches to Bacterial Infection Therapy by Interfering with Cell‐to‐Cell Signaling

David A. Rasko1, Vanessa Sperandio1

1 University of Texas Southwestern Medical Center at Dallas, Dallas, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 17.3
DOI:  10.1002/9780471729259.mc1703s12
Online Posting Date:  February, 2009
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The identification of cell‐to‐cell signaling inhibitors among bacteria is a novel mechanism used to control the virulence of pathogens. Signaling inhibitors have a significant potential to be used as therapeutics in that they will exert less biological pressure to develop resistance than classic antibiotics, which attempt to eradicate or significantly inhibit bacterial growth. Decreasing the virulence of pathogens by inhibiting signaling provides a mechanism by which the bacteria will be “misled” into not activating virulence factors. If virulence factors are not activated, these pathogens will either pass through the host without incidence or be controlled by the host's immune system. In this unit, we discuss the general principles for the design and implementation of a high‐throughput screen to identify inhibitors of bacterial cell‐to‐cell communication. We also provide a detailed protocol using a specific example of signaling inhibitor identification using enterohemorrhagic Escherichia coli as a model system. Curr. Protoc. Microbiol. 12:17.3.1‐17.3.11. © 2009 by John Wiley & Sons, Inc.

Keywords: cell‐to‐cell signaling; autoinducer; virulence; inhibitor; Escherichia coli

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Identification of E. coli TEVS232 Inhibitory Compounds via a High‐Throughput Screening
  • Support Protocol 1: Preparing Preconditioned DMEM
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Identification of E. coli TEVS232 Inhibitory Compounds via a High‐Throughput Screening

  • E. coli TEVS232 (contains LEE1::lacZ chromosomal fusion)
  • Luria broth (Miller base; Invitrogen)
  • Dulbecco's modified Eagle medium (DMEM; Invitrogen)
  • EHEC‐preconditioned medium (see Support Protocol)
  • Dimethyl sulfoxide (DMSO)
  • Small molecule library (this study utilized the UT Southwestern Medical Center, Department of Biochemistry)
  • Lysozyme
  • Beta‐Glo reagent (Promega)
  • 37°C, 5% CO 2 incubator
  • 37°C shaking incubator
  • 384‐well plates
  • Biomek FX liquid handler
  • Additional reagents for growing E. coli in liquid medium (Elbing and Brent, )

Support Protocol 1: Preparing Preconditioned DMEM

  • E. coli 86‐24 (other E. coli strains could be used if they have been shown to produce AI‐3; Walters et al., )
  • Dulbecco's modified Eagle medium (DMEM; Invitrogen)
  • 37°C shaking incubator
  • Centrifuge
  • 0.22‐µm filter
  • Additional reagents and equipment for growing E. coli in a liquid medium (Elbing and Brent, )
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Literature Cited

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