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 solution A (20 mM MgCl2, 0.7 M sucrose, 10 mM Tris-Cl, pH 7.8) by dissolving 0.10 g MgCl2 (95.2 g/mol) and 11.98 g sucrose (342.3 g/mol) in 25 mL dH2O in a 125 mL flask.Add 500 &#181;L 1 M Tris-Cl (pH 7.8) and adjust the volume to 50 mL. 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 solution B (10 mM MgCl2, 0.8 M sucrose, 10 mM Tris-Cl, pH 7.8) by dissolving 0.05 g MgCl2 (95.2 g/mol) and 13.69 g sucrose (342.3 g/mol) in 25 mL dH2O in a 125 mL flask. Add 500 &#181;L 1 M Tris-Cl (pH 7.8) and adjust the volume to 50 mL. 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 0.8 M sucrose by dissolving 13.69 g sucrose (342.3 g/mol) in 50 mL 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 5 mg/mL deoxyribonuclease I (DNase I) by dissolving 0.015 g DNase I in 3 mL of dH2O in a 50 mL flask. Sterilize by filtering through a 25 mm syringe filter with a 0.2 &#181;m membrane. Aliquot solution into microfuge tubes and store at -20 &#176;C.</li>
	<li>Prepare 0.125 M ethylenediaminetetraacetic acid (EDTA), pH 8.0 by dissolving 0.698 g EDTA disodium dehydrate (372.2 g/mol) into 15 mL 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 600 &#181;g/mL cephalexin by dissolving 0.03 g of cephalexin hydrate (365.404 g/mol) 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 4 &#176;C.</li>
	<li>Prepare 5 mg/mL lysozyme by dissolving 0.015 g lysozyme in 3 mL of dH2O in a 50 mL flask. Sterilize by filtering through a 25 mm syringe filter with a 0.2 &#181;m membrane. Aliquot solution into microfuge tubes and store at -20 &#176;C.</li>
	<li>Prepare 3% w/v Trypticase Soy Broth (TSB) by dissolving 30 g of TSB in 1 L of dH2O in a 2 L flask. Aliquot the solution into flasks containing 25 mL or 100 mL TSB to be used in the preparation of E. coli spheroplasts or B. megaterium protoplasts, respectively. Autoclave flasks to sterilize the liquid medium. Sterile TSB can be stored at either room temperature or 4 &#176;C.</li>
	<li>Prepare solution C (1 M sucrose, 0.04 M maleate, 0.04 M MgCl2, pH 6.5) by dissolving 34.23 g sucrose (342.3 g/mol), 0.46 g maleic acid (116.07 g/mol), and 0.38 g MgCl2 (95.21 g/mol) in 100 mL of dH2O in a 250 mL flask. Adjust pH to 6.5. 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 200 mL protoplast medium by mixing 100 mL 3% w/v TSB and 100 mL solution C in a 1 L glass bottle. Autoclave to sterilize the solution and store at room temperature.</li>
</ol>
2. Preparation of Overnight Culture
NOTE: Perform sections 2-4 using appropriate sterile techniques. If desired, a bacterial strain can contain a plasmid for antibiotic resistance to reduce the potential contamination. If using a strain with antibiotic resistance, add the necessary antibiotics in steps 2.1, 3.1&#8211;3.2, and 4.1&#8211;4.4.
<ol>
	<li>Prepare an overnight culture by picking a single colony of bacteria using a sterile pipette tip and placing it into a 14 mL culture tube containing 2&#8211;3 mL of 3% w/v TSB. Incubate at 37 &#176;C, while shaking for 16&#8211;21 h.</li>
</ol>
3. Preparation of Gram-negative E. coli Spheroplasts
<ol>
	<li>In a 250 mL flask, dilute the overnight culture 1:100 in 25 mL volume of 3% w/v TSB and incubate the bacterial solution at 37 &#176;C, while shaking for approximately 2.5 h until the solution has reached an optical density of 0.5&#8211;0.8 at 600 nm. Measure the optical density using a spectrophotometer.</li>
	<li>In a 250 mL flask, dilute this culture 1:10 in 30 mL of 3% w/v TSB and incubate at 37 &#176;C, while shaking for 2.5 h in the presence of 60 &#181;g/mL cephalexin (347.4 g/mol) to produce single cell filaments of about 50&#8211;150 &#181;m in length, which are observable under 1,000X magnification using a light microscope (Figure 1B).</li>
	<li>Harvest the filaments by centrifuging the bacterial solution at 1,500 x g, 4 &#176;C for 4 min. Decant and discard the supernatant, reserving the pellet.</li>
	<li>Wash the filaments by gently adding 1 mL of 0.8 M sucrose, being careful not to disturb the pellet. Incubate for 1 min, and then discard the supernatant without disturbing the pellet.</li>
	<li>Add 150 &#181;L of 1 M Tris-Cl (pH 7.8), 120 &#181;L of 5 mg/mL lysozyme, 30 &#181;L of 5 mg/mL DNase I, and 120 &#181;L of 0.125 M EDTA in their respective order to the pellet and incubate the solution at room temperature for 10 min.</li>
	<li>Add 1 mL of solution A gradually over 1 min to the solution prepared in 3.5 using a micropipette while gently swirling the solution by hand. Incubate the solution for 4 min at room temperature.</li>
	<li>Put 7 mL of 4 &#176;C solution B into two 15 mL conical tubes. Add equal amounts of the solution prepared in 3.6 to each of these two tubes. Centrifuge the solution at 1,500 x g, 4 &#176;C for 4 min.</li>
	<li>Using a serological pipette, carefully remove all but 1-2 mL of supernatant without disturbing the pellet. Resuspend the pellet by gently pipetting up and down using a P1000 micropipette. Visually check to see spheroplasts formation by observing the sample at 1,000X magnification using a light microscope (Figure 1C).</li>
	<li>Store the spheroplasts at -20 &#176;C for up to a week or until they have gone through 3 freeze-thaw cycles.</li>
</ol>
4. Preparation of Gram-positive B. megaterium Protoplasts
<ol>
	<li>In a 250 mL flask, dilute the overnight culture 1:1,000 in 100 mL of 3% w/v TSB and incubate the bacterial solution at 37 &#176;C, while shaking for approximately 4.5 h until the solution has reached an optical density of 0.9-1.0 at 600 nm. Measure the optical density using a spectrophotometer.</li>
	<li>Pour the liquid culture into two 50 mL conical tubes and centrifuge at 2,000 x g, 4 &#176;C for 10 min.</li>
	<li>Using a serological pipette, discard the supernatant from both conical tubes. Resuspend each pellet in 2.5 mL protoplast medium and combine the resuspended solutions into a single conical tube. Pipette the combined resuspended solution in a 125 mL flask.</li>
	<li>Add 1 mL of 5 mg/mL lysozyme and incubate for 1 h at 37 &#176;C, while shaking.</li>
	<li>Monitor the growth of protoplasts under 1,000x magnification using a light microscope, noting any irregularities, such as bacteria that appear as swollen rods instead of spheres (Figure 2). Using a serological pipette, transfer the solution to a 15 mL conical tube and centrifuge at 2,000 x g, 4 &#176;C for 10 min.</li>
	<li>Decant the supernatant and re-suspend the pellet in 5 mL protoplast media. Store protoplasts at -20 &#176;C for up to a week or until they have gone through 3 freeze-thaw cycles.</li>
</ol>
5. Preparation of Peptide Solution and Membrane Dye for Imaging
<ol>
	<li>Peptide solution
	<ol>
		<li>In a microfuge tube wrapped in aluminum foil, dissolve 2 mg of FITC labeled BF2 P11A in 800 &#181;L dH2O. Use a peptide containing at least one tryptophan residue in order to conduct protein concentration measurements.</li>
		<li>Spectrophotometrically, measure the absorbance of the peptide solution at 280 nm in triplicate. Calculate the peptide concentration using the molar extinction coefficient for tryptophan (5,700/Mcm).</li>
		<li>Dilute the peptide concentration in dH2O to a final concentration of 100&#8211;200 &#181;M. Store in a microfuge tube at -20 &#176;C wrapped in aluminum foil to protect from light.</li>
	</ol>
	</li>
	<li>Membrane dye
	<ol>
		<li>In a microfuge tube, prepare a 10 mM stock of di-8-ANEPPS (592.9 g/mol) by dissolving 5 mg of di-8-ANEPPS in 843.3 &#181;L of DMSO. Store at 4 &#176;C wrapped in aluminum foil to protect from light.</li>
		<li>In a microfuge tube, prepare 1 mL of 0.03 mM di-8-ANEPPS by adding 3 &#181;L of 10 mM di-8-ANEPPS to 997 &#181;L DMSO. Store in a microfuge tube at 4 &#176;C wrapped in aluminum foil to protect from light.</li>
	</ol>
	</li>
</ol>
6. Visualization of E. coli Spheroplasts and B. megaterium Protoplasts Using Confocal Microscopy
<ol>
	<li>Pipette 5 &#181;L of spheroplasts or protoplasts onto a poly-L-lysine coated glass slide. Add 2 &#181;L of FITC labeled AMP (100-200 &#181;M) to the slide and incubate for 3 min protected from light.</li>
	<li>Add 1 &#181;L of the membrane dye di-8-ANEPPS (0.03 mM) to the slide and incubate for 3 min protected from light. Cover with a glass coverslip and seal with the nail polish.</li>
	<li>Turn on the confocal microscope and argon laser. Adjust the argon laser to 20% power output and 20% transmission.</li>
	<li>Set emission wavelength ranges of 499&#8211;532 nm and 670&#8211;745 nm for the FITC-labeled peptide emission channel and di-8-ANEPPS-labeled membrane dye emission channel, respectively.</li>
	<li>Image protoplasts or spheroplasts (Figure 1 and Figure 2) with a 63X objective. Use imaging software to obtain 8-bit, 512 x 512 composite z-stack images composed of 0.5 &#181;m slices of the entirety of the spheroplast or protoplast. 
	NOTE: Take careful consideration to reduce the emission bleed-through while imaging spheroplasts and protoplasts. See discussion for details.</li>
</ol>
7. Characterization of AMP Localization
<ol>
	<li>Open the composite z-stack image in imaging software. Locate the center-most slice of the spheroplast or protoplast and place one circular Region of Interest (ROI) (0.3 &#181;m in diameter) on the membrane (ROI 1), one at the center of the spheroplast or protoplast (ROI 2), and one away from spheroplast or protoplast to measure the background fluorescence (ROI 3) (Figure 5). Avoid the inclusion of saturated pixels. The fluorescence intensity in each ROI can be calculated by imaging software. 
	NOTE: In instances when peptide does not localize to the entirety of the membrane, draw ROI 1 on an area of the membrane where the peptide is localized.</li>
	<li>Use the following equation to determine the ratio of intracellular peptide fluorescence intensity to membrane peptide fluorescence intensity: 
	<img alt="Equation" src="/files/ftp_upload/57904/57904eq1.jpg" /> 
	NOTE: The fluorescence intensity in ROI 3 is subtracted from fluorescence intensities in ROI 2 and ROI 1 in order to account for background fluorescence.</li>
</ol>

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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 &amp;mu;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 ...

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Research

JoVE Journal

Biology

11.7K Views.  Wellesley College. Hier presenteren we een protocol voor de productie van gramnegatieve Escherichia coli (E. coli) spheroplasts en gram-positieve Bacillus megaterium (B. megaterium) protoplasten duidelijk visualiseren en snel karakteriseren Peptide-bacteriën interacties. Dit biedt een systematische methode om te definiëren membraan lokaliseren en translocating peptiden.

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

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

Biochemistry

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Immunology and Infection

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

Bioengineering

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Whole&#45;Organ Pathogen Tracking via Fluorescent Reporter Quantification

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

Author Spotlight: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In part, this reflects the availability of methods for the identification of break sites, and characterization of factors recruited to DSBs at those sequences. However, DSBs also appear as intermediates during the processing of DNA adducts formed by compounds that do not directly cause breaks, and do not react at specific sequence sites. Consequently, for most of these agents, technologies that permit the analysis of binding interactions with response factors and repair proteins are unknown. For example, DNA interstrand crosslinks (ICLs) can provoke breaks following replication fork encounters. Although formed by drugs widely used as cancer chemotherapeutics, there has been no methodology for monitoring their interactions with replication proteins.
Here, we describe our strategy for following the cellular response to fork collisions with these challenging adducts. We linked a steroid antigen to psoralen, which forms photoactivation dependent ICLs in nuclei of living cells. The ICLs were visualized by immunofluorescence against the antigen tag. The tag can also be a partner in the Proximity Ligation Assay (PLA) which reports the close association of two antigens. The PLA was exploited to distinguish proteins that were closely associated with the tagged ICLs from those that were not. It was possible to define replisome proteins that were retained after encounters with ICLs and identify others that were lost. This approach is applicable to any structure or DNA adduct that can be detected immunologically.

While replication fork collisions with DNA adducts can induce double strand breaks, less is known about the interaction between replisomes and blocking lesions. We have employed the proximity ligation assay to visualize these encounters and to characterize the consequences for replisome composition.

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In ...

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

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