Name: production and visualization of bacterial spheroplasts and protoplasts to characterize antimicrobial peptide localization
Uploaded: 2018-08-11T15:00:00
Description: Watch this Scientific Journal Video about Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization at JoVE.com

Research was supported by National Institute of Allergy and Infectious Diseases (NIH-NIAID) award R15AI079685.

1. Solution Preparation
NOTE: Prepare solutions described in steps 1.1&#8211;1.9 and 1.8&#8211;1.11 in order to produce E. coli spheroplasts and B. megaterium protoplasts, respectively.
<ol>
	<li>Prepare 1 M Tris-Cl, pH 7.8 by dissolving 10.34 g Tris HCl and 4.17 g of Tris OH in 50 mL of dH2O in a 125 mL flask. Sterilize by filtering through a 25 mm syringe filter with a 0.2 &#181;m membrane and store in a conical tube at room temperature.</li>
	<li>Prepare solut...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+antimicrobial+agents.">Peptide antimicrobial agents.</a> Clinical Microbiology Reviews. 19 (3), 491-511 (2006).</li><li>Toke, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides:+new+candidates+in+the+fight+against+bacterial+infections.">Antimicrobial peptides: new candidates in the fight against bacterial infections.</a> Biopolymers. 80 (6), 717-735 (2005).</li><li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+in+2014.">Antimicrobial peptides in 2014.</a> Pharmaceuticals. 8 (1), 123-150 (2015).</li><li>Epand, R. M., Vogel, H. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+analysis+of+buforin+II,+a+histone+H2A-derived+antimicrobial+peptide:+The+proline+hinge+is+responsible+for+the+cell-penetrating+ability+of+buforin+II.">Structure-activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: The proline hinge is responsible for the cell-penetrating ability of buforin II.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (15), 8245-8250 (2000).</li><li>Pavia, K. E., Spinella, S. A., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+histone-derived+antimicrobial+peptides+use+different+antimicrobial+mechanisms.">Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms.</a> Biochim Biophys Acta. 1818 (3), 869-876 (2012).</li><li>Wei, L., LaBouyer, M. A., Darling, L. E., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Spheroplasts+as+a+Model+for+Visualizing+Membrane+Translocation+of+Antimicrobial+Peptides.">Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides.</a> Antimicrobial Agents and Chemotherapy. 60 (10), 6350-6352 (2016).</li><li>Chassy, B. M., Giuffrida, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+Lysis+of+Gram-Positive,+Asporogenous+Bacteria+with+Lysozyme.">Method for the Lysis of Gram-Positive, Asporogenous Bacteria with Lysozyme.</a> Appl. Environ. Microbiol. 39 (1), 153-158 (1980).</li><li>Fitz-James, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+and+Chemical+Studies+of+the+Browth+of+Protoplasts+of+Bacillus+megaterium.">Cytological and Chemical Studies of the Browth of Protoplasts of Bacillus megaterium.</a> J. Biophysic. and Biochem. Cytol. 4 (3), 257-266 (1958).</li><li>Martinac, B., Buechner, M., Delcour, A. H., Adler, J., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-sensitive+ion+channel+in+Escherichia+coli.">Pressure-sensitive ion channel in Escherichia coli.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 2297-2301 (1986).</li><li>Martinac, B., Rohde, P. R., Cranfield, C. G., Nomura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+electrophysiology+for+the+study+of+bacterial+ion+channels+in+giant+spheroplasts+of+E.+coli.">Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli.</a> Methods Mol Biol. 966, 367-380 (2013).</li><li>Nadeau, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Experimental Biophysics: Biological Methods for Physical Scientists. , (2016).</li><li>Sun, Y., Sun, T. L., Huang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+properties+of+Escherichia+coli+spheroplast+membranes.">Physical properties of Escherichia coli spheroplast membranes.</a> Biophysical Journal. 107 (9), 2082-2090 (2014).</li><li>Branco, P., Viana, T., Albergaria, H., Arneborg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+(AMPs)+produced+by+Saccharomyces+cerevisiae+induce+alterations+in+the+intracellular+pH,+membrane+permeability+and+culturability+of+Hanseniaspora+guilliermondii+cells.">Antimicrobial peptides (AMPs) produced by Saccharomyces cerevisiae induce alterations in the intracellular pH, membrane permeability and culturability of Hanseniaspora guilliermondii cells.</a> Int J Food Microbiol. 205, 112-118 (2015).</li><li>Kobayashi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Translocation+Mechanism+of+the+Antimicrobial+Peptide+Buforin+2.">Membrane Translocation Mechanism of the Antimicrobial Peptide Buforin 2.</a> Biochemistry. 43 (49), 15610-15616 (2004).</li><li>Spinella, S. A., Nelson, R. B., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+peptide+translocation+into+large+unilamellar+vesicles.">Measuring peptide translocation into large unilamellar vesicles.</a> J Vis Exp. (59), e3571 (2012).</li><li>van der Kraan, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactoferrampin:+a+novel+antimicrobial+peptide+in+the+N1-domain+of+bovine+lactoferrin.">Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin.</a> Peptides. 25 (2), 177-183 (2004).</li><li>Sun, Y., Sun, T. L., Huang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+electrophysiology+for+the+study+of+bacterial+ion+channels+in+giant+spheroplasts+of+E.+coli.">Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli.</a> Biophys J. 111 (1), 132-139 (2016).</li><li>Xie, Y., Fleming, E., Chen, J. L., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+proline+position+on+the+antimicrobial+mechanism+of+buforin+II.">Effect of proline position on the antimicrobial mechanism of buforin II.</a> Peptides. 32 (4), 677-682 (2011).</li><li>Kobayashi, S., Takeshima, K., Park, C. B., Kim, S. C., Matsuzaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+Novel+Antimicrobial+Peptide+Buforin+2+with+Lipid+Bilayers:+Proline+as+a+Translocation+Promoting+Factor.">Interactions of the Novel Antimicrobial Peptide Buforin 2 with Lipid Bilayers: Proline as a Translocation Promoting Factor.</a> Biochem. 39 (29), 8648-8654 (2000).</li><li>Decad, G. M., Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+Membrane+of+Gram-Negative+Bacteria+XII.+Molecular-Sieving+Function+of+Cell+Wall.">Outer Membrane of Gram-Negative Bacteria XII. Molecular-Sieving Function of Cell Wall.</a> J. Bacteriol. 128 (1), 325-336 (1976).</li><li>Choi, H., Yang, Z., Weisshaar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell,+real-time+detection+of+oxidative+stress+induced+in+Escherichia+coli+by+the+antimicrobial+peptide+CM15.">Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15.</a> Proc Natl Acad Sci U S A. 112 (3), E303-E310 (2015).</li><li>Yang, Z., Choi, H., Weisshaar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melittin-Induced+Permeabilization,+Re-sealing,+and+Re-permeabilization+of+E.+coli+Membranes.">Melittin-Induced Permeabilization, Re-sealing, and Re-permeabilization of E. coli Membranes.</a> Biophys J. 114 (2), 368-379 (2018).</li><li>Ruthe, H. J., Adler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+of+bacterial+spheroplasts+by+electric+fields.">Fusion of bacterial spheroplasts by electric fields.</a> Biochim. Biophys. Acta. 819 (1), (1985).</li></ol>

The protocols presented here make it feasible for researchers to more rapidly obtain larger sample sizes of bacterial images because the enlarged, spherical bacteria are much easier to locate, orient, and image. This enhanced ability to collect data is valuable in several respects. First, it enables a more systematic quantitative analysis of peptide localization patterns. While qualitative trends can be demonstrated from smaller sets of images, only a large sample set of high-quality images reveal more nuanced trends in ...

Antimicrobial peptides (AMPs) have gained attention due to their potential use as alternatives to conventional antibiotics1,2,3,4,5. AMPs kill bacteria by either translocating across the cell membrane and interacting with intracellular components such as nucleic acids or by permeabilizing the membrane causing leakage of cell contents6. In addition to their use as antibiotics, translocating AMPs may be adapted for drug delivery applications because they can non-disruptively cross the impermeable cell membrane7,8. We, therefore, seek to understand fundamental AMP mechanisms of action to lay the foundation for their use in drug design.
Confocal microscopy offers a way to assess localization patterns of fluorescently labeled AMPs in bacterial cells providing insights into their mechanism of action9,10,11,12,13,14. By labeling the membrane of the bacteria, one can determine if a fluorescently labeled peptide localizes to the membrane or the intracellular space of a bacterial cell. However, this technique is limited by the small size and rod shape of bacteria, which can make imaging challenging due to the resolution limits of conventional light microscopes and the variable orientation of the bacteria on the slide15.
The goal of the presented method is to enable enhanced visualization of the fluorescently labeled peptide localization patterns using confocal microscopy. Visualization is enhanced by turning the small, thin, rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) bacteria into enlarged, spherical forms referred to as spheroplasts (for gram-negative strains) and protoplasts (for gram-positive strains)16,17,18,19,20,21. Spheroplasts and protoplasts are easier to image because of both their increased size and their symmetric shape, which makes the orientation of a bacterium on a slide irrelevant for its imaging. In addition, we present a systematic approach to quantitatively analyze confocal microscopy data in order to characterize AMPs as either membrane localizing or translocating. Applying these methods makes it easier to distinguish fluorescently labeled peptide localization patterns. The protocols presented here can be used to assess the localization of a variety of membrane-active agents other than AMPs, including cell-penetrating peptides.
One distinct advantage of this technique is that it provides insights into the mechanism of action of AMPs on a single cell level, which may reveal cell-to-cell heterogeneity15, as opposed to other fluorescence assays commonly used to identify the mechanisms of action of AMPs, which only provide bulk estimates9,22,23,24,25. The use of spheroplasts and protoplasts in order to assess AMP cell entry is particular useful26&#160;because they are more physiologically relevant15&#160;than other models used for assessing cell entry, such as lipid vesicles24.

<table>
	<tbody>
		<tr>
			<td>Trizma hydrocloride (Tris HCl)</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base (Tris OH)</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>D4527</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma</td>
			<td>106361</td>
			<td>Used Sigma 106361 in original protocol development; 106361 discontinued with ED2SS as replacement</td>
		</tr>
		<tr>
			<td>Cephalexin hydrate</td>
			<td>Sigma</td>
			<td>C4895</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Trypticase soy broth</td>
			<td>Fisher Scientific</td>
			<td>B11768</td>
			<td></td>
		</tr>
		<tr>
			<td>BF2 P11A FITC</td>
			<td>NeoScientific</td>
			<td>Custom ordered</td>
			<td></td>
		</tr>
		<tr>
			<td>di-8-ANEPPS</td>
			<td>Biotium</td>
			<td>61012</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>34869</td>
			<td>Used Sigma D8779 in original protocol development; D8779 discontinued with 34869 as replacement</td>
		</tr>
		<tr>
			<td>Maleic acid</td>
			<td>Sigma</td>
			<td>M0375</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrodisc 25 mm Syringe Filter w/ 0.2 &#956;m HT Tuffryn Membrane</td>
			<td>Pall Corporation</td>
			<td>4192</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Leica Microsystems</td>
			<td>TCS SP5 II</td>
			<td>For image acquisition</td>
		</tr>
		<tr>
			<td>Leica Application Suite, Advanced Fluorescence</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>For image processing</td>
		</tr>
	</tbody>
</table>

production and visualization of bacterial spheroplasts and protoplasts to characterize antimicrobial peptide localization

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

By enlarging bacteria and making them spherical, we can easily distinguish whether peptides localize to the bacterial membrane or readily translocate across the bacterial membrane. The resolution limits of conventional light microscopes make it challenging to distinguish whether peptide signals arise from the membrane or intracellular space in normal bacteria because signals localized to the membrane will appear to overlap with the intracellular space (Figure 3A</stro...

Watch this Scientific Journal Video about Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization at JoVE.com

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

This method can help answer key questions in the antimicrobial peptide field about whether peptides localize to or trans locate across the cell membrane. Using spheroplasts and protoplasts allows the visualization and analysis of peptide localization in a physiologically relevant model system without the resolution challenges encountered with bacterial cell imaging. Though this method can provide insights into antimicrobial peptides, it can also be applied to other membrane active agents such as cell penetrating peptides.Gram-negative E.coli spheroplasts and Gram-positive B.megaterium protoplast should be prepared in a biological safety cabinet or laminar flow hood using appropriate sterile technique. If desired, bacterial strains can contain an antibiotic resistant plasmid, and spheroplasts and protoplasts can be prepared at an open lab bench. In this video spheroplasts and protoplasts are prepared at an open lab bench using antibiotic resistant bacterial strains.To set up an overnight culture, use a sterile micropipette tip to transfer a single bacterial colony from a solid agar culture plate to a 14 milliliter culture tube, containing two to three milliliters of three percent trypticase soy broth, TSB, for a 16 to 21 hour incubation at 37 degrees celsius with shaking. To prepare Gram-negative E.coli spheroplasts, dilute the overnight culture at one to 100 ratio, and 25 milliliter of TSB in a 250 milliliter flask, and return the bacteria to the shaking incubator for another 2.5 hours. When the solution has reached an optical density of 0.5 to 0.8 at 600 nanometers, dilute the culture at a one to ten ratio in fresh TSB in a new 250 milliliter flask for a second 2.5 hour incubation with a final effective concentration of 60 micrograms per milliliter of cephalexin.To produce single cell filaments of about 50 to 150 micrometers in length, as visualized under a light microscope at a 1000x magnification. Centrifuge the bacterial solution to harvest the filaments and decant the supernatant. Wash the filaments with the gentle addition of one milliliter of 0.8 molar sucrose, taking care not to disturb the pellet.After one minute discard the supernatant without disturbing the pellet, and add the indicated reagents in their respective order to the filaments. After ten minutes at room temperature, gradually add one milliliter of solution A over a period of one minute, while gently swirling the solution by hand. Incubate the solution for four minutes at room temperature, then split the filament suspension into to two equal aliquots between two 15 milliliter conical tubes containing seven milliliters of four degree Celsius solution B.Next, pellet the filaments by centrifugalization, and use assyriological pipet to carefully remove all but one to two milliliters of the supernatant without disturbing the pellets.Use a p1000 micropipette to gently re suspend the pellets. Check a small aliquots of the sample for spheroplast formation by light microscopy. The spheroplast can then be stored at minus 20 degrees Celsius for up to a week or until they have gone through three freeze thaw cycles.To prepare Gram-positive B.megaterium protoplasts, dilute the overnight bacterial culture at a one to 1000 ratio in 100 milliliters of three percent TSB in a 250 milliliter flask for a 4.5 hour incubation at 37 degrees Celsius with shaking. When the liquid culture reaches an optical density of 0.9 to 1.0 at 600 nanometers, split the bacteria between two 50 milliliter conical tubes for their centrifugation, and use assyriological pipet to asperate the supernatants. Re suspend the pellets in 2.5 milliliters of protoplast medium per tube, and pull the suspension into a single conical tube for culture in a 125 milliliter flask.Add one milliliter of five milligrams per milliliter lysozyme to the culture for a one hour incubation at 37 degrees Celsius with shaking. Monitoring the growth of the protoplast under the light microscope and noting any irregularities, such as bacteria that appear as swollen rods instead of spheres. At the end of the incubation, transfer the suspension to a 15 milliliter conical tube for centrifugation, and re suspend the pellet in five milliliters of protoplast medium.The protoplast can then be stored at minus 20 degrees Celsius for up to a week or until they have gone through three freeze thaw cycles. For visualization of the spheroplast or protoplast transfer five microliters of the suspension of interest onto a Poly L Lysine coated glass slide, followed by two microliters of fitc labeled 100 to 200 micromolar antimicrobial peptide or AMP. After a three minute incubation protected from light add one microliter of an appropriate membrane die to the sample for another three minute incubation in the dark.At the end of the incubation cover the labeled spheroplast or protoplast with a glass cover slip, and seal the cover slip with the nail polish. To image the cells, turn on the confocal microscope and appropriate laser and select the 63x objective, then use the appropriate imaging software to obtain eight bit 512 by 512 composite Z stack images composed of 0.5 micrometer slices of the entirety of each bacterial cell of interest. To visualize localization of the fluorescently labeled AMP open the composite Z stack images in an appropriate imaging analysis program and locate the center most slice of the first spheroplast or protoplast of interest.Place one circular 0.3 micrometer diameter region of interest on the membrane, one at the center of the cell, and one away from the cell to measure the background fluorescence, taking care to avoid saturated pixels. The mean fluorescence intensity in each region of interest can then be calculated by the software. The resolution limits of conventional light microscopes make it challenging to distinguish whether fluorescently labeled peptide signals arise from the membrane or the intracellular space in normal bacteria.As the signals localize to the membrane will appear to overlap with the intracellular space. In contrast the enlarged size of the spheroplast and protoplast compared to normal bacteria, results in a clear resolution between the membrane marker and the intracellular space. Making it easier to distinguish peptide localization.For example, the localization to the membrane and translocation across the cell membrane of fluorescently labeled peptide can be visualized in both E.coli spheroplast and B.megaterium protoplast, by confocal fluorescence microscopy as just demonstrated. The placement of regions of interest on the membrane and intracellular space of the spheroplast or protoplast and in the background, allows calculation of the ratio of intracellular peptide fluorescence intensity to peptide fluorescence intensity on the membrane of each spheroplast or protoplast interacting with AMP. For example, this method can be used to visualize the interactions of a histone derived microbial peptide mutant with E.coli spheroplast and B.megaterium protoplasts.In this experiment the AMP demonstrated a similar localization pattern in both strains of bacteria. Highlighting it's reduced translocation relative to the wild type peptide. Ultimately this technique enables researchers to explore peptide localization patterns on a single cell level.Offering insights into the mechanism of action of a peptide of interest. In addition to allowing researchers to collect larger numbers of images for the systematic characterization of peptide mechanisms, images of larger spheroplasts and protoplasts are more manageable for quantitative analysis than those of smaller bacterial cells.

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Research

JoVE Journal

Biology

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for generation of transgenics for in vivo imaging. These unique capabilities make the zebrafish an ideal platform to study ocular lens development and physiology. Our recent findings that an Aquaporin-0, Aqp0a, is required for stability of the anterior lens suture, as well as for the shift of the lens nucleus to the lens center with age led us to develop tools especially suited to analyzing the properties of zebrafish lenses. Here we outline detailed methods for lens dissection that can be applied to both larval and adult lenses, to prepare them for histological analysis, immunohistochemistry and imaging. We focus on analysis of lens suture integrity and cortical cell morphology and compare data generated from dissected lenses with data obtained from in vivo imaging of lens morphology made possible by a novel transgenic zebrafish line with a genetically encoded fluorescent marker. Analysis of dissected lenses perpendicular to their optical axis allows quantification of the relative position of the lens nucleus along the anterior-posterior axis. Movement of the lens nucleus from an initial anterior position to the center is required for normal lens optics in adult zebrafish. Thus, a quantitative measure of lens nuclear position directly correlates with its optical properties.

These protocols were developed to analyze cortical lens morphology, structural integrity of the zebrafish lens sutures in fixed and live lenses and to measure the position of the zebrafish lens nucleus along the anterior-posterior axis.

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for ...