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>
	<li>Baltzer, S. A., Brown, M. H. <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:+promising+alternatives+to+conventional+antibiotics.">Antimicrobial peptides: promising alternatives to conventional antibiotics.</a> Journal of Molecular Microbiology Biotechnology. 20 (4), 228-235 (2011).</li><li>Hancock, R. E., Sahl, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+and+host-defense+peptides+as+new+anti-infective+therapeutic+strategies.">Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies.</a> Nature Biotechnology. 24 (12), 1551-1557 (2006).</li><li>Jenssen, H., Hamill, P., Hancock, R. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+antimicrobial+peptides+and+their+mechanisms+of+action.">Diversity of antimicrobial peptides and their mechanisms of action.</a> Biochim Biophys Acta. 1462, 11-28 (1999).</li><li>Drin, G., Rousselle, C., Scherrmann, J. -. M., Rees, A. R., Temsamani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+Delivery+to+the+Brain+via+Adsorptive-Mediated+Endocytosis:+Advances+With+SynB+Vectors.">Peptide Delivery to the Brain via Adsorptive-Mediated Endocytosis: Advances With SynB Vectors.</a> AAPS PharmSciTech. 4 (4), 61-67 (2002).</li><li>Splith, K., Neundorf, I. <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+with+cell-penetrating+peptide+properties+and+vice+versa.">Antimicrobial peptides with cell-penetrating peptide properties and vice versa.</a> European Biophysics Journal. 40 (4), 387-397 (2011).</li><li>Bustillo, M. E., 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=Modular+analysis+of+hipposin,+a+histone-derived+antimicrobial+peptide+consisting+of+membrane+translocating+and+membrane+permeabilizing+fragments.">Modular analysis of hipposin, a histone-derived antimicrobial peptide consisting of membrane translocating and membrane permeabilizing fragments.</a> Biochim Biophys Acta. 1838 (9), 2228-2233 (2014).</li><li>Koo, Y. 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=Structure-activity+relations+of+parasin+I,+a+histone+H2A-derived+antimicrobial+peptide.">Structure-activity relations of parasin I, a histone H2A-derived antimicrobial peptide.</a> Peptides. 29 (7), 1102-1108 (2008).</li><li>Libardo, M. D., Cervantes, J. L., Salazar, J. C., Angeles-Boza, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+bioactivity+of+antimicrobial+peptides+by+addition+of+amino-terminal+copper+and+nickel+(ATCUN)+binding+motifs.">Improved bioactivity of antimicrobial peptides by addition of amino-terminal copper and nickel (ATCUN) binding motifs.</a> ChemMedChem. 9 (8), 1892-1901 (2014).</li><li>Park, C. B., Kim, H. S., Kim, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Action+of+the+Antimicrobial+Peptide+Buforin+II:+Buforin+II+Kills+Microorganisms+by+Penetrating+the+Cell+Membrane+and+Inhibiting+Cellular+Functions.">Mechanism of Action of the Antimicrobial Peptide Buforin II: Buforin II Kills Microorganisms by Penetrating the Cell Membrane and Inhibiting Cellular Functions.</a> Biochemical and Biophysical Research Communications. , 253-257 (1998).</li><li>Park, C. B., Yi, K. -. S., Matsuzaki, K., Kim, M. S., Kim, S. 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>

No conflicts of interest are declared.

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

11.3K visualizações.  Wellesley College. 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...