Macromolecular Synthesis and Membrane Perturbation Assays for Mechanisms of Action Studies of Antimicrobial Agents

Amy Cotsonas King1, Liping Wu1

1 MaxThera, Beverly, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13A.7
DOI:  10.1002/0471141755.ph13a07s47
Online Posting Date:  December, 2009
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The definition and confirmation of the mechanism of action of an NCE is central to antimicrobial drug discovery. Most antibiotics currently in clinical use selectively target and block one or more bacterial macromolecular synthesis processes, e.g., DNA replication, RNA synthesis (transcription), protein synthesis (translation), cell wall (peptidoglycan) synthesis, and fatty acid (lipid) biosynthesis. This unit includes two protocols for determining the effect of test compounds on macromolecular synthesis, one in test tube format and the other in 96‐well plate format. A membrane potential depolarization protocol is also provided. Disruption of cell membrane integrity may be a legitimate mechanism of action for antibacterials, but it also may be the result of nonspecific cell membrane activity, an effect that must be ruled out for mammalian cells. These assays provide useful means for verifying inhibition of an intended target pathway with investigational antimicrobial compounds. They can also be used as valuable secondary assays for lead optimization to eliminate inhibitors that display nonselective toxicity. Curr. Protoc. Pharmacol. 47:13A.7.1‐13A.7.23. © 2009 by John Wiley & Sons, Inc.

Keywords: macromolecular synthesis; membrane potential depolarization; mechanism of action; antimicrobial

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Table of Contents

  • Introduction
  • Basic Protocol 1: The Macromolecular Synthesis (MMS) Assay in Cell Culture Tubes
  • Alternate Protocol 1: Macromolecular Synthesis (MMS) Assay in 96‐Well Microtiter Plates
  • Basic Protocol 2: Membrane Potential Depolarization Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: The Macromolecular Synthesis (MMS) Assay in Cell Culture Tubes

  • Microorganism(s) to be used for the MMS assay (e.g., Staphylococcus aureus, ATCC #29213 orE. coli, ATCC #25922)
  • Appropriate agar medium [e.g., Trypticase soy agar (TSA) plates (VWR‐BD)]
  • Appropriate liquid media, e.g.:
    • Trypticase soy broth (TSB; VWR‐BD) for the fatty acid, DNA, RNA, and cell wall biosynthesis assays
    • M5T (M9 containing 5% TSB; see recipe) for the protein biosynthesis assay
  • 5% and 10% ice‐cold trichloroacetic acid (TCA)
  • Control antibiotics (Sigma‐Aldrich) for relevant MMS pathways (see recipe), e.g.,
    • Triclosan for fatty acid biosynthesis
    • Novobiocin for DNA biosynthsis
    • Rifampin for RNA biosynthesis
    • Vancomycin for cell wall biosynthesis (for Gram‐positive bacteria)
    • Chloramphenicol for protein biosynthesis
  • Test compound(s), prepared in appropriate solvent (e.g., DMSO; see recipe) as 5 mg/ml stock(s)
  • Radiolabeled precursors for relevant MMS assays, e.g.:
    • Acetic acid, [1, 2‐14C], sodium salt for labeling fatty acid biosynthesis (MP Biomedicals, cat. no. 12019)
    • Thymidine, [2‐14C] for labeling DNA biosynthesis (MP Biomedicals, cat. no. 14036)
    • Uridine, [2‐14C] for labeling RNA biosynthesis (MP Biomedicals, cat. no. 14040)
    • N‐Acetyl glucosamine, [Glucosamine‐1‐14C] for labeling cell wall biosynthesis (MP Biomedicals, cat. no. 11145)
    • L‐Amino acid mixture, [14C] for labeling protein biosynthesis (MP Biomedicals, cat. no. 10147)
  • 75% (v/v) ethanol
  • ScintiSafe Plus 50% Cocktail (Fisher Scientific, cat. no. SX25‐5)
  • Inoculating loops, sterile
  • Incubator set at 37°C with 85% relative humidity
  • Spectrometer for culture turbidity measurement (600 nm)
  • 100‐ml flasks, sterile
  • Incubating shaker set at 37°C with shaking at 225 rpm
  • 5‐ and 14‐ml plastic culture tubes with snap caps, sterile (VWR)
  • 10‐ and 25‐ml disposable serological pipets, sterile
  • Centrifuge
  • 10‐µl, 200‐µl, and 1‐ml pipets and sterile pipet tips (Rainin or VWR)
  • Glass Microanalysis Vacuum Filter Holder for 25‐mm GF/C with Frit Support (Millipore, cat. no. XX1002500)
  • Filtering flask
  • Vacuum pump
  • 25‐mm GF/C Glass Microfiber Filters (Whatman, cat. no. 1822‐025)
  • Forceps
  • Paper towels
  • 7‐ml Scintillation vials with screwed caps (VWR, cat. no. 66022‐387)
  • Wallac Liquid Scintillation Counter (PerkinElmer)
CAUTION: Use TCA with extreme caution. It is fatal if inhaled and causes severe respiratory tract, eye, and skin burns. Since it is hygroscopic and readily soluble in cold water, containers for TCA should be kept tightly closed.

Alternate Protocol 1: Macromolecular Synthesis (MMS) Assay in 96‐Well Microtiter Plates

  • MicroScint 20 scintillation fluid (PerkinElmer‐Packard)
  • Water bath shaker set at 37°C
  • 96‐well microtiter plates
  • Glass tray
  • 10‐ and 200‐µl multichannel pipettors and sterile pipet tips (Rainin or VWR)
  • Vortex
  • MultiScreen Vacuum Manifold (e.g., Millipore, cat. no. MAEVM0960R)
  • Vacuum pump
  • MultiScreen GF/C filter plates (e.g., Millipore, cat. no. MAFCN0B50)
  • Wallac MicroBeta Liquid Scintillation and Luminescence Counter (PerkinElmer, cat. no. 6013621)
  • MicroBeta cassette for Millipore MultiScreen Plates (PerkinElmer, cat. no. 1450‐106)

Basic Protocol 2: Membrane Potential Depolarization Assay

  • Test microorganism(s): e.g., a Staphylococcus aureus (ATCC #29213) or the E. coli DC2 outer membrane barrier‐defective mutant strain with increased outer membrane permeability (Wu and Hancock, )
  • Appropriate agar medium, e.g., Trypticase soy agar (TSA) plates
  • Cation‐adjusted Mueller‐Hinton broth (CAMHB; see recipe)
  • 3,3‐dipropylthiadicarbocyanine iodide [DiSC 3(5),] stock solution (see recipe)
  • Test compound(s) prepared in DMSO (see recipe) as 5 mg/ml stock(s)
  • Membrane potential depolarizer(s) as positive control reagent(s): Nisin or CCCP (both are available from Sigma‐Aldrich)
  • Non‐membrane potential depolarizing antibiotic(s) as negative control(s), e.g.:
    • Ampicillin, chloramphenicol, ciprofloxacin, and vancomycin (all are available from Sigma‐Aldrich)
  • Sterile inoculating loops
  • Incubator, set at 37°C with 85% relative humidity
  • 150‐ and 200‐ml flasks
  • Incubating shaker set at 37°C with shaking at 225 rpm
  • Spectrometer for culture turbidity measurement (600 nm)
  • 14‐ml round‐bottom tubes
  • Fluorescence spectrophotometer
  • 50‐ml tubes
NOTE: Daptomycin is a membrane disruptor; therefore, it should not be used as a negative control.
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Literature Cited

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