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Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive Dyes.

Te Winkel JD, Gray DA, Seistrup KH, Hamoen LW, Strahl H - Front Cell Dev Biol (2016)

Bottom Line: The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes.Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential.At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Newcastle upon Tyne, UK.

ABSTRACT
The bacterial cytoplasmic membrane is a major inhibitory target for antimicrobial compounds. Commonly, although not exclusively, these compounds unfold their antimicrobial activity by disrupting the essential barrier function of the cell membrane. As a consequence, membrane permeability assays are central for mode of action studies analysing membrane-targeting antimicrobial compounds. The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes. Unfortunately, these assays are not sensitive to changes in membrane ion permeability which are sufficient to inhibit and kill bacteria by membrane depolarization. In this manuscript, we provide experimental advice how membrane potential, and its changes triggered by membrane-targeting antimicrobials can be accurately assessed in vivo. Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential. At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.

No MeSH data available.


Related in: MedlinePlus

Calibration of DiSC3(5) assay. (A) Fluorescence intensity of DiSC3(5) in B. subtilis cell suspensions in media with varying K+ concentrations. Upon addition of K+ carrier valinomycin (4 μM), membrane potential reaches stable levels pre-determined by the K+ gradient across the membrane (see Table 2). The time point of valinomycin addition is highlighted with an arrow. (B) The fluorescence levels obtained from the experiment shown in panel (A) can be used to calibrate the arbitrary DiSC3(5) fluorescence values. The estimated membrane potential for untreated B. subtilis cells (−110 mV) is indicated with a dashed line. Strain used: B. subtilis 168 (wild type).
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Figure 4: Calibration of DiSC3(5) assay. (A) Fluorescence intensity of DiSC3(5) in B. subtilis cell suspensions in media with varying K+ concentrations. Upon addition of K+ carrier valinomycin (4 μM), membrane potential reaches stable levels pre-determined by the K+ gradient across the membrane (see Table 2). The time point of valinomycin addition is highlighted with an arrow. (B) The fluorescence levels obtained from the experiment shown in panel (A) can be used to calibrate the arbitrary DiSC3(5) fluorescence values. The estimated membrane potential for untreated B. subtilis cells (−110 mV) is indicated with a dashed line. Strain used: B. subtilis 168 (wild type).

Mentions: Since DiSC3(5) follows a Nernstian distribution across the membrane, its fluorescence can also be used for a quantitative measurement of the membrane potential. For this aim, partial depolarization triggered by the K+ carrier valinomycin serves as a convenient method to generate a calibration curve. Following the Goldman-Hodgkin-Katz voltage equation, membrane potential is determined by the relative contribution of membrane gradients for each ion. The extent, to which an ion gradient contributes to the overall membrane potential, depends on the permeability (both passive and active) of the membrane to the individual ion species. Under normal conditions, the transport of H+ across the membrane greatly surpasses the contribution of other ions. This is mainly due to the high H+ transport activity of the respiratory chain. As a consequence, the membrane potential is normally dominated by the H+ gradient (Mitchell, 1961; Saraste, 1999). This changes upon addition of the K+-specific carrier valinomycin (Shapiro, 1994). When supplied at sufficient concentrations, the relative permeability of the cell membrane for K+ is increased to an extent in which the K+ gradient across the membrane becomes the predominant factor. Upon addition of valinomycin, the membrane potential now equilibrates with the K+ gradient resulting in a level which can be calculated using the Nernst-equation (see Table 2). K+ gradient as such can easily be modified by altering the medium K+/Na+-ratio since cells strive to maintain a stable cytoplasmic K+ concentration in media with comparable osmolality (Whatmore et al., 1990). This method provides the means to measure DiSC3(5) fluorescence at different membrane potential levels (Figure 4A), and thus to calibrate the DiSC3(5) assay (Figure 4B; Singh and Nicholls, 1985; Vecer et al., 1997; Breeuwer and Abee, 2004). Using this approach, we estimated the membrane potential of B. subtilis to reach ~ −110 mV under the growth conditions used in our experiments. This value is in good agreement with previously published estimates (Hosoi et al., 1980; Zaritsky et al., 1981).


Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive Dyes.

Te Winkel JD, Gray DA, Seistrup KH, Hamoen LW, Strahl H - Front Cell Dev Biol (2016)

Calibration of DiSC3(5) assay. (A) Fluorescence intensity of DiSC3(5) in B. subtilis cell suspensions in media with varying K+ concentrations. Upon addition of K+ carrier valinomycin (4 μM), membrane potential reaches stable levels pre-determined by the K+ gradient across the membrane (see Table 2). The time point of valinomycin addition is highlighted with an arrow. (B) The fluorescence levels obtained from the experiment shown in panel (A) can be used to calibrate the arbitrary DiSC3(5) fluorescence values. The estimated membrane potential for untreated B. subtilis cells (−110 mV) is indicated with a dashed line. Strain used: B. subtilis 168 (wild type).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4829611&req=5

Figure 4: Calibration of DiSC3(5) assay. (A) Fluorescence intensity of DiSC3(5) in B. subtilis cell suspensions in media with varying K+ concentrations. Upon addition of K+ carrier valinomycin (4 μM), membrane potential reaches stable levels pre-determined by the K+ gradient across the membrane (see Table 2). The time point of valinomycin addition is highlighted with an arrow. (B) The fluorescence levels obtained from the experiment shown in panel (A) can be used to calibrate the arbitrary DiSC3(5) fluorescence values. The estimated membrane potential for untreated B. subtilis cells (−110 mV) is indicated with a dashed line. Strain used: B. subtilis 168 (wild type).
Mentions: Since DiSC3(5) follows a Nernstian distribution across the membrane, its fluorescence can also be used for a quantitative measurement of the membrane potential. For this aim, partial depolarization triggered by the K+ carrier valinomycin serves as a convenient method to generate a calibration curve. Following the Goldman-Hodgkin-Katz voltage equation, membrane potential is determined by the relative contribution of membrane gradients for each ion. The extent, to which an ion gradient contributes to the overall membrane potential, depends on the permeability (both passive and active) of the membrane to the individual ion species. Under normal conditions, the transport of H+ across the membrane greatly surpasses the contribution of other ions. This is mainly due to the high H+ transport activity of the respiratory chain. As a consequence, the membrane potential is normally dominated by the H+ gradient (Mitchell, 1961; Saraste, 1999). This changes upon addition of the K+-specific carrier valinomycin (Shapiro, 1994). When supplied at sufficient concentrations, the relative permeability of the cell membrane for K+ is increased to an extent in which the K+ gradient across the membrane becomes the predominant factor. Upon addition of valinomycin, the membrane potential now equilibrates with the K+ gradient resulting in a level which can be calculated using the Nernst-equation (see Table 2). K+ gradient as such can easily be modified by altering the medium K+/Na+-ratio since cells strive to maintain a stable cytoplasmic K+ concentration in media with comparable osmolality (Whatmore et al., 1990). This method provides the means to measure DiSC3(5) fluorescence at different membrane potential levels (Figure 4A), and thus to calibrate the DiSC3(5) assay (Figure 4B; Singh and Nicholls, 1985; Vecer et al., 1997; Breeuwer and Abee, 2004). Using this approach, we estimated the membrane potential of B. subtilis to reach ~ −110 mV under the growth conditions used in our experiments. This value is in good agreement with previously published estimates (Hosoi et al., 1980; Zaritsky et al., 1981).

Bottom Line: The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes.Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential.At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Newcastle upon Tyne, UK.

ABSTRACT
The bacterial cytoplasmic membrane is a major inhibitory target for antimicrobial compounds. Commonly, although not exclusively, these compounds unfold their antimicrobial activity by disrupting the essential barrier function of the cell membrane. As a consequence, membrane permeability assays are central for mode of action studies analysing membrane-targeting antimicrobial compounds. The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes. Unfortunately, these assays are not sensitive to changes in membrane ion permeability which are sufficient to inhibit and kill bacteria by membrane depolarization. In this manuscript, we provide experimental advice how membrane potential, and its changes triggered by membrane-targeting antimicrobials can be accurately assessed in vivo. Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential. At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.

No MeSH data available.


Related in: MedlinePlus