Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides

Abstract

Studies attempting to characterize the membrane translocation of antimicrobial and cell-penetrating peptides are frequently limited by the resolution of conventional light microscopy. This study shows that spheroplasts provide a valuable approach to overcome these limits. Spheroplasts produce less ambiguous images and allow for more systematic analyses of localization. Data collected with spheroplasts are consistent with studies using normal bacterial cells and imply that a particular peptide may not always follow the same mechanism of action.A ntimicrobial peptides (AMPs) represent a promising alternative to conventional therapeutics in the face of concerns about the rise of antibiotic-resistant bacteria in clinical settings (1). Traditionally, AMPs were believed to kill bacteria through membrane disruption. While many AMPs do induce membrane permeabilization, researchers have identified increasing numbers of peptides that function by translocating into bacterial cells and targeting intracellular components (2). Thus, it has become increasingly important for researchers to reliably determine whether AMPs are able to effectively translocate into bacterial cells (3). Many researchers have turned to confocal microscopy in order to assess cell entry (4-11). However, bacterial cells are so small that effective imaging is limited by the resolution of conventional light microscopes. For example, in order to distinguish whether any observed signal from peptides arises from inside the cell versus on the cell membrane, researchers ideally should examine individual focal plane images throughout cells. However, if signal on the membrane is sufficiently strong it can "contaminate" slices ostensibly taken "inside" the cell, as we have observed in measurements of the membrane-localized dye di-8-ANEPPS (Fig. 1).In order to overcome these resolution limits, we have employed bacterial spheroplasts (12)(13)(14). Spheroplasts are produced by culturing bacteria in the presence of an antibiotic, such as cephalexin, that prevents division while still allowing cells to grow. The resulting elongated bacterial "snakes" are then exposed to lysozyme, which digests the outer cell wall and produces spherical spheroplasts that are typically 2 to 5 m in diameter (see Fig. S1 in the supplemental material). Perhaps even more important than larger size, the spherical shape allows one to obtain consistent slices regardless of how a spheroplast is oriented during imaging.In order to test the validity of using spheroplasts to assess peptide translocation, we have measured the cellular localization of four previously characterized peptides (Table 1). To this end, we exposed Escherichia coli spheroplasts to peptides with an N-terminally conjugated fluorescein isothiocyanate (FITC) label for imaging; detailed methods for spheroplast preparation and peptide incubation are provided in the supplemental material. As one set of positive and negative controls, we chose buforin II (BF2), arguably the best-studied membrane-translocating AMP (15), and BF2 with...

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

“…Additionally, because we have not assessed the viability of spheroplast populations, one cannot distinguish between a peptide that is readily able to translocate across the cell membrane of a living cell versus a peptide that can only translocate across a dead or dying cell. Despite these differences, we have seen localization patterns in E. coli spheroplasts for many AMPs that are consistent with known mechanisms of action 15 , and our results for BF2 P11A with B. megaterium protoplasts presented here seem consistent with other observations for that peptide 13,27,28 . The membrane compositions of spheroplasts and protoplasts are also certainly more physiologically relevant than the typical mixtures used in other model systems, such as lipid vesicles.…”

“…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 slide 15 .…”

Investigating the nucleic acid interactions of histone‐derived antimicrobial peptides

“…For example, considering membrane translocation and DNA binding led to a more complete interpretation how proline mutants affect activity of BF2 [39]. Although AMPs are often characterized as primarily killing bacteria by either permeabilizing membranes or interacting with an intracellular component, several studies have proposed that at least some peptides may function through a combination of these different mechanisms [19, 23, 49]. Because of their well-characterized membrane and nucleic acid interactions, HDAPs may continue to provide a valuable model system for considering how different factors combine in AMP activity.…”

Analysis of the antimicrobial mechanism of porcine beta defensin 2 against E. coli by electron microscopy and differentially expressed genes

“…Wei et al . have reported that the antimicrobial peptide buforin II (BF2), and HipC (a cell-penetrating peptide) entered into the majority of spheroplasts, while BF2 with a P11A mutation and magainin localized with the membrane 40 . Nan et al .…”