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 ca....

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 dH

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

List of materials used in this article

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