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.
