Antimicrobial peptides continue to garner attention as potential alternatives to conventional antibiotics. Hipposin is a histone-derived antimicrobial peptide (HDAP) that was previously isolated from Atlantic halibut. Though its potency against several bacterial strains has been documented, its antibacterial mechanism had not been characterized. The mechanism of this peptide is particularly interesting to consider since the full hipposin sequence contains the sequences of parasin and buforin II (BF2), two other known antimicrobial peptides that act via different antibacterial mechanisms. While parasin kills bacteria by inducing membrane permeabilization, buforin II enters cells without causing significant membrane disruption, harming bacteria through interactions with intracellular nucleic acids. In this study, we used a modular approach to characterize hipposin and determine the role of the parasin and buforin II fragments in the overall hipposin mechanism. Our results show that hipposin kills bacteria by inducing membrane permeabilization, and this membrane permeabilization is promoted by the presence of the N-terminal parasin domain. Portions of hipposin lacking the parasin sequence do not cause membrane permeabilization and function more similarly to buforin II. We also determined that the C-terminal portion of hipposin, HipC, is a cell-penetrating peptide that readily enters bacterial cells but has no measurable antimicrobial activity. HipC is the first membrane active histone fragment identified that does not kill bacterial or eukaryotic cells. Together, these results not only characterize hipposin but also provide a useful starting point for considering the activity of chimeric peptides made by combining peptides that operate via differing mechanisms.

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...

We have investigated the bactericidal activity, specificity, and mechanism of a several antimicrobial peptides (AMPs) derived from histone H2A alone and combined into chimeric peptides. Relative antibacterial efficacy of these AMPs was determined using radial diffusion assays, and eukaryotic cytotoxicity assays indicate that these peptides demonstrate bacterial specificity. Confocal microscopy was used to determine the mechanism of action of the peptides. Parasin, buforin II, and buforin I are segments of the larger hipposin AMP. Buforin II can translocate membranes while parasin is a permeabilizing peptide. Buforin I and hipposin are larger peptides containing both parasin and buforin II segments, and both are permeabilizing peptides relatively unable to translocate the membrane. These findings suggest that the addition of a lytic fragment to an otherwise‐translocating peptide confers a lytic property. We further explored this hypothesis by studying a chimeric peptide of parasin and DesHDAP1, another peptide known to translocate effectively. This chimera was found to be more strongly bactericidal than either parasin or DesHDAP1 alone.

The ability of antimicrobial peptides (AMPs) to target and lyse the harmful microbial membrane over that of a host's is a unique characteristic, making these innate immune effectors promising candidates to fill a growing therapeutic void resulting from antibiotic drug resistance. This selectivity is believed to depend on the chemical and structural properties of the lipids that comprise the cell membrane. The selectivity of AMPs can be based on the electrostatic attraction of these predominately cationic peptides for the bacterial membrane surface heavily populated with negatively charged lipid components. We have previously shown with atomic force microscopy that zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayers display concentration-dependent structural transformations induced by protegrin-1 (PG-1) that progress from finger-like instabilities at bilayer edges, to the formation of pores, and finally to a network of worm-like micelles. The increasing degree of membrane disruption in chargeneutral membranes demonstrates that a more complex interaction than that suggested by a simple electrostatic argument is needed to explain AMP selectivity. We propose that in addition to an electrostatic element, specific membrane compositional differences between host and pathogen tunes AMP activity to selectively disrupt microbial membranes. We have tailored our investigations to utilize membrane components which eukaryotes and prokaryotes contain in drastically different proportions, specifically the presence and absence of cholesterol. In these results we have employed a variety of biophysical techniques to elucidate how increasing cholesterol content in both phospholipid monolayers and bilayers attenuates the ability of PG-1 to induce membrane disruption. Atomic force microscopy and isothermal titration calorimetry were used to assess the propensity for peptide insertion and pore formation. X-ray and neutron reflectivity measurements were advantageous in providing molecular level detail on the location and orientation of PG-1 with respect to the membrane. 2793-Pos Board B223What Vesicle Leakage Reveals about Antimicrobial Activity (and What It Doesn't) The mode of action of antimicrobial peptides and their mimics is often assessed by vesicle leakage experiments, and most of these compounds are believed to act by membrane permeabilization. This work aims at improving the interpretation of vesicle leakage data in general, and at understanding and optimizing the fungicidal activity of nylon-3 polymers. We have studied the membrane permeabilizing properties of the cationic homopolymer poly-NM (Liu, R et al. JACS, 2013, 135, 5270), which displays significant antifungal activity, and two related cationic/hydrophobic binary copolymers using the lifetime-based leakage assay of calcein-loaded vesicles. We compared the results with biological activities against Candida albicans. Poly-NM induces all-or-none leakage of vesicles that are made from yeast polar lipid extract (YPLE), at the polymer's MIC against C. albicans (3 m...