Modular analysis of hipposin, a histone-derived antimicrobial peptide consisting of membrane translocating and membrane permeabilizing fragments

Abstract

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.

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

“…However, with spheroplasts we can consider the percentage of images showing translocation or membrane localization, providing more systematic data (Table 1). Again, these percentages support the previously observed trends for membrane entry (5,6,16), with BF2 and HipC entering significantly more spheroplasts than P11A BF2 and magainin. Interestingly, none of these peptides exclusively exhibit membrane localization or membrane translocation behavior.…”

“…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)(5)(6)(7)(8)(9)(10)(11). However, bacterial cells are so small that effective imaging is limited by the resolution of conventional light microscopes.…”

“…As in previous studies, BF2 and magainin peptides included F10W and F5W variations, respectively, which allow for straightforward quantification without significantly altering the peptide activity or mechanism. We also considered HipC, a cell-penetrating peptide without antibacterial activity that was previously observed to enter E. coli (5).…”

Role of arginine and lysine in the antimicrobial mechanism of histone‐derived antimicrobial peptides

“…27, 37-39 To form experimental vesicles, lipids dissolved in chloroform at a 75:20:5 ratio of POPC:POPG:DNS-PE were dried and reconsituted in a 1:1 volume mixture by volume of 10 mM HEPES buffer and 0.4 mM trypsin in 10 mM HEPES buffer solution for a final concentration of 0.2 mM trypsin. For control vesicles, Bowman-Birk Inhibitor was added to the reconstitution mixture for a final concentration of 2.0 mM.…”

Histones as Mediators of Host Defense, Inflammation and Thrombosis

“…A slightly larger N-terminal cleavage product (51 aa) of histone H2A, known as hipposin I, was discovered in Atlantic halibut [34]. A recent study found that the N-terminal domain of hipposin is responsible for bacterial membrane permeabilization and killing, while a C-terminal fragment (termed HipC) can enter cells without killing the bacteria [59]. Such studies raise interest in the potential of selective use of fragments of histones, or novel combinations of histone fragments, for antimicrobial therapy as proposed by Kawasaki et al [55].…”