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

Summary

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.

Abstract

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.

Introduction

Antimicrobial peptides (AMPs) have gained attention due to their potential use as alternatives to conventional antibiotics^1,2,3,4,5. 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 membrane^7,8. 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 action^{9,10,11,12,13,14}. 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 estimates^{9,22,23,24,25}. The use of spheroplasts and protoplasts in order to assess AMP cell entry is particular useful²⁶ because they are more physiologically relevant¹⁵ than other models used for assessing cell entry, such as lipid vesicles²⁴.

Protocol

1. Solution Preparation

NOTE: Prepare solutions described in steps 1.1–1.9 and 1.8–1.11 in order to produce E. coli spheroplasts and B. megaterium protoplasts, respectively.

1. 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 µm membrane and store in a conical tube at room temperature.
2. 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 µL 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 µm membrane and store in a conical tube at room temperature.
3. 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 µL 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 µm membrane and store in a conical tube at room temperature.
4. 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 µm membrane and store in a conical tube at room temperature.
5. 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 µm membrane. Aliquot solution into microfuge tubes and store at -20 °C.
6. 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 µm membrane and store in a conical tube at room temperature.
7. Prepare 600 µg/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 µm membrane and store in a conical tube at 4 °C.
8. 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 µm membrane. Aliquot solution into microfuge tubes and store at -20 °C.
9. 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 °C.
10. 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 µm membrane and store in a conical tube at room temperature.
11. 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.

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, 3.1–3.2, and 4.1–4.4.

1. 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 2–3 mL of 3% w/v TSB. Incubate at 37 °C, while shaking for 16–21 h.

3. Preparation of Gram-negative E. coli Spheroplasts

1. 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 °C, while shaking for approximately 2.5 h until the solution has reached an optical density of 0.5–0.8 at 600 nm. Measure the optical density using a spectrophotometer.
2. In a 250 mL flask, dilute this culture 1:10 in 30 mL of 3% w/v TSB and incubate at 37 °C, while shaking for 2.5 h in the presence of 60 µg/mL cephalexin (347.4 g/mol) to produce single cell filaments of about 50–150 µm in length, which are observable under 1,000X magnification using a light microscope (Figure 1B).
3. Harvest the filaments by centrifuging the bacterial solution at 1,500 x g, 4 °C for 4 min. Decant and discard the supernatant, reserving the pellet.
4. 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.
5. Add 150 µL of 1 M Tris-Cl (pH 7.8), 120 µL of 5 mg/mL lysozyme, 30 µL of 5 mg/mL DNase I, and 120 µL of 0.125 M EDTA in their respective order to the pellet and incubate the solution at room temperature for 10 min.
6. 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.
7. Put 7 mL of 4 °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 °C for 4 min.
8. 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).
9. Store the spheroplasts at -20 °C for up to a week or until they have gone through 3 freeze-thaw cycles.

4. Preparation of Gram-positive B. megaterium Protoplasts

1. 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 °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.
2. Pour the liquid culture into two 50 mL conical tubes and centrifuge at 2,000 x g, 4 °C for 10 min.
3. 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.
4. Add 1 mL of 5 mg/mL lysozyme and incubate for 1 h at 37 °C, while shaking.
5. 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 °C for 10 min.
6. Decant the supernatant and re-suspend the pellet in 5 mL protoplast media. Store protoplasts at -20 °C for up to a week or until they have gone through 3 freeze-thaw cycles.

5. Preparation of Peptide Solution and Membrane Dye for Imaging

1. Peptide solution
   1. In a microfuge tube wrapped in aluminum foil, dissolve 2 mg of FITC labeled BF2 P11A in 800 µL dH₂O. Use a peptide containing at least one tryptophan residue in order to conduct protein concentration measurements.
   2. 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).
   3. Dilute the peptide concentration in dH₂O to a final concentration of 100–200 µM. Store in a microfuge tube at -20 °C wrapped in aluminum foil to protect from light.
2. Membrane dye
   1. 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 µL of DMSO. Store at 4 °C wrapped in aluminum foil to protect from light.
   2. In a microfuge tube, prepare 1 mL of 0.03 mM di-8-ANEPPS by adding 3 µL of 10 mM di-8-ANEPPS to 997 µL DMSO. Store in a microfuge tube at 4 °C wrapped in aluminum foil to protect from light.

6. Visualization of E. coli Spheroplasts and B. megaterium Protoplasts Using Confocal Microscopy

1. Pipette 5 µL of spheroplasts or protoplasts onto a poly-L-lysine coated glass slide. Add 2 µL of FITC labeled AMP (100-200 µM) to the slide and incubate for 3 min protected from light.
2. Add 1 µL 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.
3. Turn on the confocal microscope and argon laser. Adjust the argon laser to 20% power output and 20% transmission.
4. Set emission wavelength ranges of 499–532 nm and 670–745 nm for the FITC-labeled peptide emission channel and di-8-ANEPPS-labeled membrane dye emission channel, respectively.
5. 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 µm 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.

7. Characterization of AMP Localization

1. 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 µm 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.
2. Use the following equation to determine the ratio of intracellular peptide fluorescence intensity to membrane peptide fluorescence intensity:
   
   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.

Results

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

Discussion

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 ...

Materials

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