Name: monitoring changes in membrane polarity, membrane integrity, and intracellular ion concentrations in streptococcus pneumoniae using fluorescent dyes
Uploaded: 2014-02-17T22:30:00
Description: Watch this Scientific Journal Video about Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes at JoVE.c

This work was supported by the Bill and Melinda Gates Foundation (Grant 53085), the JR Oishei Foundation, and The American Lung Association (Grant RG-123721-N) to APH, and NIH (NIDCD) fellowship F31DC011218 to EAC.

1. Preparing Bacterial&#160;Cultures
<ol>
	<li>Growing culture for use in experiments
	<ol>
		<li>In a 37 &#176;C heating block, thaw a frozen stock-vial of S. pneumoniae and add its content to 9 ml of fresh, prewarmed Todd-Hewitt broth with 0.5% yeast extract (THY) for a total volume of 10 ml in a glass culture tube.</li>
		<li>Incubate statically at 37 &#176;C until the culture reaches mid-log phase (Abs600nm &#8776;0.5-0.6).</li>
	</ol>
	</li>
</ol>
2. Detec...

<ol>
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Despite the limitation that size presents for using classic electrophysiology methods to detect changes in polarity and integrity of the membrane and changes in ion concentrations within bacteria, we have described a way to measure these events in S. pneumoniae using fluorescent dyes. Our protocols are the first of their kind described for the pneumococcus and one of the few described for bacterial species in general. By using a fluorescence detection plate reader, these events can be measured in small, 200 &#18...

Our lab has identified a protein-lipid complex from human milk named HAMLET (for Human Alpha-lactalbumin Made LEthal to Tumor cells) that induces apoptosis in tumor cells, but is also able to kill a variety of bacterial species1,2. The species that were found to be particularly sensitive were those that target the respiratory tract, with Streptococcus pneumoniae (the pneumococcus) displaying the greatest sensitivity and an apoptosis-like phenotype of death2,3. Membrane depolarization and specific ion transport events are well-described and crucial events during apoptosis in eukaryotic cells, particular in the mitochondria, where radioactive TPP+ ions and fluorescent dyes including JC-1 and TMRE have been used to demonstrate depolarization of the mitochondrial membrane3-5. Thus, we sought to learn more about the effect of HAMLET on these membrane-related features in the pneumococcus as we focused our efforts to develop a better understanding of the mechanistic components of the apoptosis-like phenotype in bacteria, with great potential for identifying novel antibacterial therapies or vaccine candidates in the process.
In seeking to establish protocols for our mechanistic studies, we discovered that in contrast to the well-described methodology in eukaryotic systems, there are very few published studies detailing electrophysiology and ion transport mechanisms of the bacterial membrane6,7. This is primarily attributed to the smaller size of microorganisms and their surface architecture, particularly the presence of cell wall, that restricts accessibility of the membrane for use of conventional eukaryotic methods like patch clamping, although some studies using giant protoplasts have been performed with mixed success8,9. As work with these giant protoplasts is not an ideal or even practical method for most bacterial species since it requires manipulated bacteria in an unnatural and abiotic state, the limited studies of bacterial membrane polarity that have been performed have primarily employed flow cytometry and the use of cyanine and oxonol fluorescent dyes10-16.
Instead of flow cytometry, which gathers individual fluorescence readings from one bacterium at a single time point, we chose to use a fluorescence detection plate reader to detect fluorescence intensity of bacterial suspensions in a 96-well plate format over time. This enabled us to treat a population of bacteria at various time points with much greater simplicity and ease, and continuously monitor the fluorescence kinetics of the entire population for extended periods of time, which is difficult to achieve using flow cytometry. After testing a wide variety of the potential-sensitive fluorescent dyes (including those mentioned above for use with the mitochondria), we achieved the best technical and practical success using the bis-oxonol dye called DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol) to monitor changes in polarity.
We also found it valuable to concurrently monitor disruptions in the integrity of the membrane using propidium iodide (PI). This dye fluoresces upon binding to nucleic acid, but is only able to do so when the integrity of the bacterial membrane is compromised, making it the popular component used to detect dead cells in live-dead staining assays. In addition to PI, SYTOX green and TO-PRO-1 are fluorescent dyes that are similar in action and have been previously used for bacteria in a few studies using flow cytometry detection methods17. We chose to use PI due to its excitation wavelength that allowed us to monitor its fluorescence concurrently with DiBAC in a given sample.
In our studies, we have observed that HAMLET, as well as another related protein-lipid complex with bactericidal activity known as ELOA, induced depolarization and rupture of the bacterial membrane as indicated by increases in the fluorescence of both dyes upon treatment of the pneumococci3,18,26. For both complexes, we observed that the fluorescence intensity of DiBAC4(3) increased prior to the increase in intensity of PI, indicating that depolarization occurred prior to rupture and is, therefore, a specific event induced by our bactericidal protein-lipid complexes of interest. This distinction is important to make, as rupture of the membrane can itself cause nonspecific depolarization. Measuring and analyzing both DiBAC4(3) and PI fluorescence kinetics concurrently allowed us to examine this relationship between the two membrane events, which is an additional advantage of using fluorometry instead of flow cytometry.
To monitor bacterial ion flux, there has been some previous success with using radioisotopes, including measuring uptake of 45Ca2+ in the pneumococcus19,20, which we have also used in our recent studies18,21. However, working with these radioactive ions has several drawbacks. They can be expensive, time-consuming, and messy, and can also expose the individual performing the experiment to harm, depending on the isotope of interest. Additionally, it is difficult to monitor rapid changes over time. Thus, we turned towards an alternative method of measurement that employs acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. By itself, the indicator dye is charged and does not pass through the membrane easily, but with the addition of the lipophilic ester group, the now uncharged molecule can pass through the membrane of the bacterium. Upon entering the interior, the bacterial esterases cleave the ester group, leaving the dye free inside the cell and charged again, significantly slowing its ability to exit the cell and allowing the dye to accumulate inside over time. However, use of these ester dyes has only been described in a few bacterial species to detect changes in intracellular Ca2+ 22-24 and H+ 16, with varying methods of loading, detection, and success.
With a desire to monitor changes in intracellular Ca2+ and also K+ and H+ levels in S. pneumoniae upon treatment with HAMLET and other compounds, we successfully created protocols to efficiently load fluorescent indicator dyes into bacterial cells. Effective loading into bacteria required both probenecid that increases dye retention by blocking anion-transporters and PowerLoad, a proprietary compound from Life Technologies that increases loading efficiency. Fura-2/AM (detecting Ca2+), PBFI/AM (detecting K+), and BCECF/AM (detecting H+) were successfully loaded into both unencapsulated and encapsulated pneumococcal strains enabling &#160;measurement of the resulting fluorescence patterns after addition of ionophores, such as ionomycin (Ca2+ uncoupler), valinomycin (K+ uncoupler) and CCCP (H+ uncoupler) using a fluorescence detection plate reader18,21.

<table border="0" cellpadding="0" cellspacing="0" width="864">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Todd Hewitt</td>
			<td style="width:139px;">Bacto, BD Diagnostics</td>
			<td style="width:143px;">249240</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Yeast Extract</td>
			<td style="width:139px;">Bacto, BD Diagnostics</td>
			<td style="width:143px;">212750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Phosphate Buffered Saline (PBS; pH 7.2)</td>
			<td style="width:139px;">Invitrogen (GIBCO)</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Dimethyl sulfoxide</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;">D5879</td>
			<td>DMSO</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:359px;">DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol)</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">B-438</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Propidium Iodide</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4170</td>
			<td>Make up in deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">D-(+)-Glucose</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">PowerLoad</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">P10020</td>
			<td>100X concentrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Probenecid</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">P36400</td>
			<td>Make 100X stock by adding 1 ml of PBS to one 77 mg vial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Fura-2/AM</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">F1221</td>
			<td>Special packaging (50 &#181;g aliquots)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">PBFI/AM</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">P1267</td>
			<td>Special packaging (50 &#181;g aliquots)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Nigericin</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;">N7143</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">KH2PO4</td>
			<td style="width:139px;">JT Baker</td>
			<td style="width:143px;">3246-01</td>
			<td>monobasic</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">K2HPO4</td>
			<td style="width:139px;">JT Baker</td>
			<td style="width:143px;">4012-01</td>
			<td>dibasic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">NaOH</td>
			<td style="width:139px;">JT Baker</td>
			<td style="width:143px;">5565-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">BCECF/AM</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">B1170</td>
			<td>Special packaging (50 &#181;g aliquots)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:359px;">CCCP</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;"></td>
			<td>Protonophore that causes an influx of H+ into the cytoplasm,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td>&#160;dissipating the electrical potential and the H+ gradient.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Culture tube&#160;</td>
			<td style="width:139px;">VWR</td>
			<td style="width:143px;">53283-802</td>
			<td>Fits the Spectronic spectrophotometer; borosilicate glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Spectrophotometer</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:143px;"></td>
			<td>Spectronic 20D+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">15 ml plastic conical tube</td>
			<td style="width:139px;">Corning</td>
			<td style="width:143px;">430790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Clear 96-well polystyrene microtiter plate</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">12-565-501</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Plate reader</td>
			<td style="width:139px;">BioTek</td>
			<td style="width:143px;">Synergy 2 Multi-Mode&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;"></td>
			<td style="width:139px;"></td>
			<td style="width:143px;">Microplate Reader</td>
			<td></td>
		</tr>
		<tr height="24">
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monitoring changes in membrane polarity, membrane integrity, and intracellular ion concentrations in streptococcus pneumoniae using fluorescent dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

For all experiments, there is one sample and set of conditions present in each well. Thus, each tracing represents the fluorescence intensity of an entire population of bacteria over time. The results should be easily interpretable, with a clear distinction between the fluorescence of the treated samples and that of the untreated controls. The kinetics and degree of an observed change in fluorescence could provide information about the possible mechanism and extent of the event being monitored.
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Watch this Scientific Journal Video about Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes at JoVE.c

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes (Video) | JoVE

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

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