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

Amy Cotsonas King¹, Liping Wu¹

¹MaxThera, 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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Abstract

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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Introduction
Basic Protocol 1: The Macromolecular Synthesis (MMS) Assay in Cell Culture Tubes
Alternate Protocol: 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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Materials

Basic Protocol 1: The Macromolecular Synthesis (MMS) Assay in Cell Culture Tubes

Materials

Microorganism(s) to be used for the MMS assay (e.g., Staphylococcus aureus, ATCC #29213 or E. 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: Macromolecular Synthesis (MMS) Assay in 96-Well Microtiter Plates

Additional Materials (also see Basic Protocol 1)

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

Materials

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, 1999)
Appropriate agar medium, e.g., Trypticase soy agar (TSA) plates
Cation-adjusted Mueller-Hinton broth (CAMHB; see recipe)
3,3-dipropylthiadicarbocyanine iodide [DiSC₃(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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Figures

Figure 13A.7.1

Images of a glass microanalysis filter holder and two filtering flasks of different sizes (A) and a diagram showing the assembling of a 25-mm GF/C glass microfiber filter and the parts of the glass microanalysis filter holder (B). This figure is modified with permission from images and diagrams provided by Millipore Corporation (http://www.millipore.com/catalogue/module/C164).

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Figure 13A.7.2

An example plate layout for MMS assays in a 96-well format, showing the planned locations and the final treatment concentrations of compounds and control antibiotics in 2-fold serial dilutions. The plate is configured with three identical sets of assays, corresponding to three sets of samples to be taken consecutively at three different desired time points for precipitation of the radiolabeled macromolecules. For each set of assays, bacterial cells are treated with two test compounds and five control antibiotics, each at the final concentrations of 0, 0.5×, 1×, and 2× MIC. Each plate may be used for MMS labeling with one desired radiolabeled precursor (e.g., acetic acid, [1, 2-¹⁴C] for fatty acid). Some drug-free, cell-free blank controls (in Row H) that contain only medium and the relevant precursor are also included in each set of assays, which will be used for background correction of the scintillation counting.

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Figure 13A.7.3

Preparation of the master plate with 2-fold serially diluted compound working solutions.

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Figure 13A.7.4

An example dataset showing the effects of Triclosan on fatty acid synthesis in Staphylococcus aureus YCL316. Trisclosan specifically and effectively inhibited incorporation of [¹⁴C]-acetic acid into fatty acid (A and B), but had only a minor effect on incorporation of [¹⁴C]-N-acetyl-glucosamine into the cell wall (C and D) of the bacterium at 0.5× and 1× MIC. At 2× MIC, however, incorporation of [¹⁴C]-acetic acid and [¹⁴C]-N-acetyl-glucosamine were both inhibited due to cell death caused by overdosage of Triclosan. The data are expressed both as inhibition of the TCA-precipitable radioactivity in cpm per ml versus the treatment time (A and C) and as percentage inhibition of incorporation versus the treatment time (B and D), compared to the drug-free controls.

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Figure 13A.7.5

Relative fluorescence intensity of different concentrations of DiSC₃(5) in different bacterial cultures. Example standard curves of DiSC₃(5) fluorescence intensity as a function of concentration in CAMHB and two bacterial cell suspensions. The selected DiSC₃(5) concentrations are 10 to 12.5 nM for a S. aureus strain (ATCC29213) and 12.5 to 25 nM for an outer membrane-permeable E. coli mutant strain WO153 that contains a tolC deletion and an asmB1 mutation.

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Figure 13A.7.6

An example dataset from a membrane potential depolarization assay, showing the measured membrane potential expressed as both fluorescence intensity units as a function of the compound treatment time (A) and percent fluorescence reduction and cell viability as a function of the compound treatment time compared to pre-treatment control (B). The assay was conducted using an E. coli mutant strain that contains tolC and asmB1, enhancing outer membrane permeability. A test compound MT136 (at 4× MIC) did not affect membrane potential (panels A and B, diamonds) while killing the majority of E. coli cells (panel B, squares) during the 3-hr treatment time. In contrast, the membrane depolarizer Nisin effectively perturbed the membrane potential, causing rapid membrane depolarization within 5 to 15 min, as detected by the marked increase in fluorescence intensity (panel A, squares).

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Literature Cited

Literature Cited
	Banerjee, A., Dubnau, E., Quemard, A., Balasubramanian, V., Um, K.S., Wilson, T., Collins, D., de Lisle, G., and Jacobs, W.R. Jr. 1994. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 263:227-230.
	Breeuwer, P. and Abee, T., 2004. "Assessment of the membrane potential, intracellular pH and respiration of bacteria employing fluorescence techniques". In Molecular Microbial Ecology Manual, 2nd ed. 8.01: pp. 1563-1580. Kluwer Academic Publishers, The Netherlands.
	Cho, H., Oh, Y., Park, S., and Lee, Y. 2001. Concentration of CCCP should be optimized to detect the efflux system in quinolone-susceptible Escherichia coli. J. Microbiol. 39:62-66.
	Clinical and Laboratory Standard Institute. 2006. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Seventh Edition. National Committee for Clinical and Laboratory Standards, Wayne, Penn.
	Friedrich, C.L., Moyles, D., Beveridge, T.J., and Hancock, R.E.W. 2000. Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria. Antimicrob. Agents Chemother. 44:2086-2092.
	Gao, F.H., Abee, T., and Konings, W.N. 1991. Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidase-containing proteoliposomes. Appl. Environ. Microb. 57:2164-2170.
	Ghoul, M., Pommepuy, M., Moillo-Batt, A., and Cormier, M. 1989. Effect of carbonyl cyanide m-chlorophenylhydrazone on Escherichia coli halotolerance. Appl. Environ. Microb. 55:1040-1043.
	Guan, L., Blumenthal, R.M., and Burnham, J.C. 1992. Analysis of macromolecular biosynthesis to define the quinolone-induced postantibiotic effect in Escherichia coli. Antimicrob. Agents Chemother. 36:2118-2124.
	Haenni, M. and Moreillon, P. 2006. Mutations in penicillin-binding protein (PBP) genes and in non-PBP genes during selection of penicillin-resistant Streptococcus gordonii. Antimicrob. Agents Chemother. 50:4053-4061.
	Herranz, C., Chen, Y., Chung, H.-J., Cintas, L.M., Hernández, P.E., Montville, T.J., and Chikindas, M.L. 2001. Enterocin P selectively dissipates the membrane potential of Enterococcus faecium T136. Appl. Environ. Microb. 67:1689-1692.
	Higgins, D.L., Chang, R., Debabov, D.V., Leung, J., Wu, T., Krause, K.M., Sandvik, E., Hubbard, J.M., Kaniga, K., Schmidt, D.E. Jr., Gao, Q., Cass, R.T., Karr, D.E., Benton, B.M., and Humphrey, P.P. 2005. Telavancin, a multifunctional lipoglycopeptide, disrupts both cell wall synthesis and cell membrane integrity in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 49:1127-1134.
	Hobbs, J.K., Miller K., O'Neill, A.J., and Chopra, I. 2008. Consequences of daptomycin-mediated membrane damage in Staphylococcus aureus. J. Antimicrob. Chemother. 62:1003-1008.
	Ince, D. and Hooper, D.C. 2003. Quinolone resistance due to reduced target enzyme expression. J. Bacteriol. 185:6883-6892.
	Jin, D.J. and Gross, C.A. 1989. Characterization of the pleiotropic phenotypes of rifampin-resistant rpoB mutants of Escherichia coli. J. Bacteriol. 171:5229-5231.
	Ling, L.L., Xian, J., Ali, S., Geng, B., Fan, J., Mills, D.M., Arvanites, A.C., Orgueira, H., Ashwell, M.A., Carmel, G., Xiang, Y., and Moir, D.T. 2004. Identification and characterization of inhibitors of bacterial enoyl-acyl carrier protein reductase. Antimicrob. Agents Chemother. 48:1541-1547.
	Marquardt, J.L., Siegele, D.A., Kolter, R., and Walsh, C.T. 1992. Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-N-acetylglucosamine enolpyruvyl transferase. J. Bacteriol. 174:5748-5752.
	McMurry, L.M., Oethinger, M., and Levy, S.B. 1998. Triclosan targets lipid synthesis. Nature 394:531-532.
	O'Neill, A.J. and Chopra, I. 2004. Preclinical evaluation of novel antibacterial agents by microbiological and molecular techniques. Expert Opin. Investig. Drugs 13:1045-1063.
	O'Neill, A.J., Miller, K., Oliva, B., and Chopra, I. 2004. Comparison of assays for detection of agents causing membrane damage in Staphylococcus aureus. J. Antimicrob. Chemother. 54:1127-1129.
	Rossman, T., Norris, C., and Troll, W. 1974. Inhibition of macromolecular synthesis in Escherichia coli by protease inhibitors. J. Biol. Chem. 249:3412-3417.
	Ruhr, E. and Sahl, H.G. 1985. Mode of action of the peptide antibiotic nisin and influence on the membrane potential of whole cells and on cytoplasmic and artificial membrane vesicles. Antimicrob. Agents Chemother. 27:841-845.
	Silverman, J.A., Perlmutter, N.G., and Shapiro, H.M. 2003. Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrob. Agents Chemother. 47:2538-2544.
	Singh, M.P., Petersen, P.J., Weiss, W.J., Janso, J.E., Luckman, S.W., Lenoy, E.B., Bradford, P.A., Testa, R.T., and Greenstein, M. 2003. Mannopeptimycins, new cyclic glycopeptide antibiotics produced by Streptomyces hygroscopicus LL-AC98: Antibacterial and mechanistic activities. Antimicrob. Agents Chemother. 47:62-69.
	Slater-Radosti, C., Van Aller, G., Greenwood, R., Nicholas, R., Keller, P.M., DeWolf, W.E. Jr., Fan, F., Payne, D.J., and Jaworski, D.D. 2001. Biochemical and genetic characterization of the action of triclosan on Staphylococcus aureus. J. Antimicrob. Chemother. 48:1-6.
	Takahata, S., Iida, M., Yoshida, T., Kumura, K., Kitagawa, H., and Hoshiko, S. 2007. Discovery of 4-pyridone derivatives as specific inhibitors of enoyl-acyl carrier protein reductase (FabI) with antibacterial activity against Staphylococcus aureus. J. Antibiot. 60:123-128.
	Wiedemann, I., Benz, R., and Sahl, H.-G. 2004. Lipid II-mediated pore formation by the peptide antibiotic nisin: A black lipid membrane study. J. Bacteriol. 186:3259-3261.
	Wu, M. and Hancock, R.E.W. 1999. Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane. J. Biol. Chem. 274:29-35.
	Xiong, Y., Bayer, A.S., and Yeaman, M.R. 2002. Inhibition of intracellular macromolecular synthesis in Staphylococcus aureus by thrombin-induced platelet microbicidal proteins. J. Infect. Dis. 185:348-356.
	Yenugu, S., Hamil, K.G., French, F.S., and Hall, S.H. 2004. Antimicrobial actions of the human epididymis 2 (HE2) protein isoforms, HE2alpha, HE2beta1 and HE2beta2. Reprod. Biol. Endocrin. 2:61.