Toroidal Pore Formation Research Articles

Energetics of toroidal pore formation in lipid membranes using molecular dynamics simulations

Energetics of toroidal pore formation in lipid membranes using molecular dynamics simulations

Molecular dynamics simulations of human α-defensin 5 (HD5) crossing gram-negative bacterial membrane.

Human α-defensin 5 (HD5) is a cationic antimicrobial peptide exhibiting a wide range of antimicrobial activities. It plays an important role in mucosal immunity of the small intestine. HD5 exerts its bactericidal activities through multiple mechanisms, one of which involves HD5 inducing the formation of pores in the bacterial membrane, subsequently allowing the peptide to enter the bacterial cytoplasm. Nevertheless, the precise molecular intricacies underlying its bactericidal mechanisms remain inadequately understood. In this work, the Potential of Mean Force (PMF) was computed to delve into the energetic properties governing the movement of HD5 across the lipopolysaccharide (LPS) membrane, which is a representative model of the gram-negative bacterial membrane. Our findings indicate that the most favorable free energy is attained when HD5 binds to the surface of the LPS membrane. This favorable interaction is primarily driven by the strong interactions between arginine residues in HD5 and the charged head groups of LPS, serving as the predominant forces facilitating the adhesion of HD5 to the membrane. Our analysis reveals that a dimeric form of HD5 alone is sufficient to create a water-filled channel in the membrane; however, achieving the complete lysis of the gram-negative bacterial membrane requires higher-order oligomerization of HD5. Our results suggest that HD5 employs the toroidal pore formation mechanism to disrupt the integrity of the LPS membrane. Furthermore, we identified that the primary energy barrier obstructing HD5 from traversing the membrane is localized within the hydrophobic core of the membrane, which is also observed for other defensins. Additionally, our study demonstrates that a mixture of HD5-LPS leads to a thinning of the membrane. Taken together, this work provides a deeper insight into the molecular intricacies governing the behavior of HD5 as it translocates through the gram-negative bacterial membrane.

Mechanistic Insight into the Early Stages of Toroidal Pore Formation by the Antimicrobial Peptide Smp24.

The antimicrobial peptide Smp24, originally derived from the venom of Scorpio maurus palmatus, is a promising candidate for further drug development. However, before doing so, greater insight into the mechanism of action is needed to construct a reliable structure-activity relationship. The aim of this study was to specifically investigate the critical early stages of peptide-induced membrane disruption. Single-channel current traces were obtained via planar patch-clamp electrophysiology, with multiple types of pore-forming events observed, unlike those expected from the traditional, more rigid mechanistic models. To better understand the molecular-level structures of the peptide-pore assemblies underlying these observed conductance events, molecular dynamics simulations were used to investigate the peptide structure and orientation both before and during pore formation. The transition of the peptides to transmembrane-like states within disordered toroidal pores occurred due to a peptide-induced bilayer-leaflet asymmetry, explaining why pore stabilization does not always follow pore nucleation in the experimental observations. To fully grasp the structure-activity relationship of antimicrobial peptides, a more nuanced view of the complex and dynamic mechanistic behaviour must be adopted.

Leaflet by Leaflet Synergistic Effects of Antimicrobial Peptides on Bacterial and Mammalian Membrane Models.

Antimicrobial peptides (AMPs) offer significant hope in the fight against antibiotic resistance. Operating via a mechanism different from that of antibiotics, they target the microbial membrane and ideally should damage it without impacting mammalian cells. Here, the interactions of two AMPs, magainin 2 and PGLa, and their synergistic effects on bacterial and mammalian membrane models were studied using electrochemical impedance spectroscopy, atomic force microscopy (AFM), and fluorescence correlation spectroscopy. Toroidal pore formation was observed by AFM when the two AMPs were combined, while individually AMP effects were confined to the exterior leaflet of the bacterial membrane analogue. Using microcavity-supported lipid bilayers, the diffusivity of each bilayer leaflet could be studied independently, and we observed that combined, the AMPs penetrate both leaflets of the bacterial model but individually each peptide had a limited impact on the proximal leaflet of the bacterial model. The impact of AMPs on a ternary, mammalian mimetic membrane was much weaker.

Temporin-GHb-Derived Peptides Exhibit Potent Antibacterial and Antibiofilm Activities against Staphylococcus aureus In Vitro and Protect Mice from Acute Infectious Pneumonia.

With the continuous development of drug resistance in bacteria to traditional antibiotics, the demand for novel antibacterial agents is urgent. Antimicrobial peptides (AMPs) are promising candidates because of their unique mechanism of action and low tendency to induce drug resistance. Previously, we cloned temporin-GHb (hereafter referred to simply as "GHb") from Hylarana guentheri. In this study, a series of derived peptides were designed, namely, GHbR, GHbK, GHb3K, GHb11K, and GHbK4R. The five derived peptides had stronger antibacterial activities against Staphylococcus aureus than the parent peptide GHb and could effectively inhibit the formation of biofilms and eradicate mature biofilms in vitro. GHbR, GHbK, GHb3K, and GHbK4R exerted bactericidal effects by disrupting membrane integrity. However, GHb11K exhibited bacteriostatic efficacy with toroidal pore formation on the cell membrane. In comparison to GHbK4R, GHb3K showed much lower cytotoxicity against A549 alveolar epithelial cells, with an IC50 > 200 μM, which was much higher than its minimal inhibitory concentration (MIC = 3.1 μM) against S. aureus. The anti-infection potential of GHbK4R and GHb3K was investigated in vivo. Compared with vancomycin, the two peptides displayed significant efficacy in a mouse model of acute pneumonia infected with S. aureus. Both GHbK4R and GHb3K also had no obvious toxicity to normal mice after intraperitoneal administration (15 mg/kg) for 8 days. Our results indicate that GHb3K and GHbK4R might be promising candidates for the treatment of bacterial pneumonia infected with S. aureus.

Cooperative antimicrobial action of melittin on lipid membranes: A coarse-grained molecular dynamics study

We conducted a series of coarse-grained molecular dynamics (CG-MD) simulations to investigate the complicated actions of melittin, which is an antimicrobial peptide (AMP) derived from honey bee venom, on a lipid membrane. To accurately simulate the AMP action, we developed and used a protein CG model as an extension of the pSPICA force field (FF), which was designed to reproduce several thermodynamic quantities and structural properties. At a low peptide-to-lipid (P/L) ratio (1/102), no defect was detected. At P/L = 1/51, toroidal pore formation was observed due to collective insertion of multiple melittin peptides from the N-termini. The pore formation was initiated by a local increase in membrane curvature in the vicinity of the peptide aggregate. At a higher P/L ratio (1/26), two more modes were detected, seemingly not controlled by the P/L ratio but by a local arrangement of melittin peptides: 1. Pore formation accompanied by lipid extraction by melittin peptides:a detergent-like mechanism. 2. A rapidly formed large pore in a significantly curved membrane: bursting. Thus, we observed three pore formation modes (toroidal pore formation, lipid extraction, and bursting) depending on the peptide concentration and local arrangement. These observations were consistent with experimental observations and hypothesized melittin modes. Through this study, we found that the local arrangements and population of melittin peptides and the area expansion rate by membrane deformation were key to the initiation of and competition among the multiple pore formation mechanisms.

Hydrophobic mismatch is a key factor in protein transport across lipid bilayer membranes via the Tat pathway

The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoids, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits: TatA, TatB, and TatC. TatA and TatB possess short transmembrane alpha helices (TMHs), both of which are only 15 residues long in Escherichia coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer, although the functional significance of this mismatch is unclear. Here, we sought to address the functional importance of the hydrophobic mismatch in the Tat transport mechanism in E. coli. We conducted three different assays to evaluate the effect of TMH length mutants on Tat activity and observed that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off following any modification of this length. Surprisingly, TatA and TatB with as few as 11 residues in their TMHs can still insert into the membrane bilayer, albeit with a decline in membrane integrity. These findings support a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, we conclude that the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes.

Antimicrobial Peptides with pH-Dependent Activity and Alkaline Optima: Their Origins, Mechanisms of Action and Potential Applications.

A number of disorders and diseases are associated with conditions of high pH, and many conventional antibiotics lose their efficacy under these pH conditions, generating a need for novel antimicrobials. A potential solution to fulfill this need is Antimicrobial Peptides (AMPs) with high pH optima. This review shows that a variety of anionic and cationic AMPs with this pH dependency are produced by creatures across the eukaryotic kingdom, including rabbits, cattle, sheep, fish, crabs and frog. These AMPs exhibit activity against viruses, bacteria, and fungi that involve membrane interactions and appear to be facilitated by a variety of mechanisms that generally promote passage across membranes to attack intracellular targets, such as DNA or protein synthesisand/or membrane lysis. Some of these mechanisms are unknown, but those elucidated include the use of bacterial pores and transporters, the self-promoted uptake pathway, and established models of membrane interaction, such as the carpet mechanism, toroidal pore formation, the adoption of tilted peptide, and the SHM model. A variety of potential roles have been proposed for these AMPs, including use as antivirals, antibacterials, antifungals, adjuvants to antimicrobial therapy, biomarkers of disease, and probes for pathogenic microbes. In this review, these properties are described and discussed, emphasizing the antimicrobial mechanisms used by these AMPs and the pH dependency of these mechanisms.

Advances in Molecular Understanding of α-Helical Membrane-Active Peptides.

Biological membranes separate the interior of cells or cellular compartments from their outer environments. This barrier function of membranes can be disrupted by membrane-active peptides, some of which can spontaneously penetrate through the membranes or open leaky transmembrane pores. However, the origin of their activity/toxicity is not sufficiently understood for the development of more potent peptides. To this day, there are no design rules that would be generally valid, and the role of individual amino acids tends to be sequence-specific.In this Account, we describe recent progress in understanding the design principles that govern the activity of membrane-active peptides. We focus on α-helical amphiphilic peptides and their ability to (1) translocate across phospholipid bilayers, (2) form transmembrane pores, or (3) act synergistically, i.e., to produce a significantly more potent effect in a mixture than the individual components.We refined the description of peptide translocation using computer simulations and demonstrated the effect of selected residues. Our simulations showed the necessity to explicitly include charged residues in the translocation description to correctly sample the membrane perturbations they can cause. Using this description, we calculated the translocation of helical peptides with and without the kink induced by the proline/glycine residue. The presence of the kink had no effect on the translocation barrier, but it decreased the peptide affinity to the membrane and reduced the peptide stability inside the membrane. Interestingly, the effects were mainly caused by the peptide's increased polarity, not the higher flexibility of the kink.Flexibility plays a crucial role in pore formation and affects distinct pore structures in different ways. The presence of a kink destabilizes barrel-stave pores, because the kink prevents the tight packing of peptides in the bundle, which is characteristic of the barrel-stave structure. In contrast, the kink facilitates the formation of toroidal pores, where the peptides are only loosely arranged and do not need to closely assemble. The exact position of the kink in the sequence further determines the preferred arrangement of peptides in the pore, i.e., an hourglass or U-shaped structure. In addition, we demonstrated that two self-associated (via termini) helical peptides could mimic the behavior of peptides with a helix-kink-helix motif.Finally, we review the recent findings on the peptide synergism of the archetypal mixture of Magainin 2 and PGLa peptides. We focused on a bacterial plasma membrane mimic that contains negatively charged lipids and lipids with negative intrinsic curvature. We showed that the synergistic action of peptides was highly dependent on the lipid composition. When the lipid composition and peptide/lipid ratios were changed, the systems exhibited more complex behavior than just the previously reported pore formation. We observed membrane adhesion, fusion, and even the formation of the sponge phase in this regime. Furthermore, enhanced adhesion/partitioning to the membrane was reported to be caused by lipid-induced peptide aggregation.In conclusion, the provided molecular insight into the complex behavior of membrane-active peptides provides clues for the design and modification of antimicrobial peptides or toxins.

Introduction of cell‐selectivity in bovine cathelicidin BMAP‐28 by exchanging heptadic isoleucine with the adjacent proline at a non‐heptadic position

AbstractBovine cathelicidin, BMAP‐28 exhibits potent antimicrobial activity without any cell‐selectivity. Considering the role of its C‐terminus amino acid residues in its cytotoxic activities, we envisioned to rearrange the amino acids at its C‐terminus without altering its composition for designing new cell‐selective BMAP‐28‐analog. Thus an isoleucine residue at 20th position, also an “a” position of its previously identified isoleucine zipper sequence, was exchanged with a proline residue at 19th position. As a result, a remarkable reduction in the hemolytic activity of BMAP‐28 toward human red blood cells (hRBCs) was observed without hampering its antibacterial activity in liquid media, and in presence of salts and serum. BMAP‐28‐analog, BMAP‐28M depolarized S. aureus membrane and bacterial membrane‐mimetic, PE/PG lipid vesicles similar to BMAP‐28 but showed weaker efficacy to depolarize hRBC membrane and mammalian membrane‐mimetic, PC/Chol vesicles. Differences in the localization of tryptophan residues of BMAP‐28 and BMAP‐28M were observed only in PC/Chol vesicles. BMAP‐28 induced pores of ~3.4 nm diameter onto hRBCs whereas BMAP‐28M showed minor perturbation onto these cells. BMAP‐28 and BMAP‐28M induced the entry of calcein (size, ~1 nm) and 4.4 kDa FITC‐dextran (size, ~2.8 nm) into B. megaterium but not of higher sized fluorescent‐dextrans indicating toroidal pore formation onto these bacterial membranes.

Correlation between Single-Molecule Dynamics and Biological Functions of Antimicrobial Peptide Melittin.

Many fundamental biological processes occur on cell membranes, and a typical example is the membrane permeabilization by peptides for an antimicrobial purpose. Previous studies of the underlying mechanism mostly focus on structural changes of membranes and peptides during their interactions. Herein, from a new perspective of single-molecule dynamics, the real-time three-dimensional motions of individual phospholipid and peptide molecules were monitored, and specifically, their correlation with the membrane poration function of melittin, a most representative natural antimicrobial peptide, was studied. We found that the adsorption and accumulation of melittin on the membrane surface significantly sped up the lateral diffusion of lipids surrounding the peptides, which in turn facilitated the peptide insertion at such heterogeneous regions. A unique "U"-bending pathway of melittin during membrane insertion and the ultimate formation of toroidal pores with dynamical translocations of peptides and lipids with several metastable states between the two leaflets of bilayer were observed.

Curved or linear? Predicting the 3-dimensional structure of α-helical antimicrobial peptides in an amphipathic environment.

α-Helical membrane-active antimicrobial peptides (AMPs) are known to act via a range of mechanisms, including the formation of barrel-stave and toroidal pores and the micellisation of the membrane (carpet mechanism). Different mechanisms imply that the peptides adopt different 3D structures when bound at the water-membrane interface, a highly amphipathic environment. Here, an evolutionary algorithm is used to predict the 3D structure of a range of α-helical membrane-active AMPs at the water-membrane interface by optimising amphipathicity. This amphipathic structure prediction (ASP) is capable of distinguishing between curved and linear peptides solved experimentally, potentially allowing the activity and mechanism of action of different membrane-active AMPs to be predicted. The ASP algorithm is accessible via a web interface at http://atb.uq.edu.au/asp/.

Membrane Remodeling by the Lytic Fragment of SticholysinII: Implications for the Toroidal Pore Model

Sticholysins are pore-forming toxins of biomedical interest and represent a prototype of proteins acting through the formation of protein-lipid or toroidal pores. Peptides spanning the N-terminus of sticholysins can mimic their permeabilizing activity and, together with the full-length toxins, have been used as a tool to understand the mechanism of pore formation in membranes. However, the lytic mechanism of these peptides and the lipid shape modulating their activity are not completely clear. In this article, we combine molecular dynamics simulations and experimental biophysical tools to dissect different aspects of the pore-forming mechanism of StII1–30, a peptide derived from the N-terminus of sticholysin II (StII). With this combined approach, membrane curvature induction and flip-flop movement of the lipids were identified as two important membrane remodeling steps mediated by StII1–30. Pore formation by this peptide was enhanced by the presence of the negatively curved lipid phosphatidylethanolamine in membranes. This lipid emerged not only as a facilitator of membrane interactions but also as a structural element of the StII1–30 pore that is recruited to the ring upon its assembly. Collectively, these, to our knowledge, new findings support a toroidal model for the architecture of the pore formed by StII1–30 and provide new molecular insight into the role of phosphatidylethanolamine as a membrane component that can easily integrate into the ring of toroidal pores, thus probably aiding in their stabilization. This study contributes to a better understanding of the molecular mechanism underlying the permeabilizing activity of StII1–30 and peptides or proteins acting via a toroidal pore mechanism and offers an informative framework for the optimization of the biomedical application of this and similar molecules.

Nano-viscosimetry analysis of the membrane disrupting action of the bee venom peptide melittin

Melittin is one of the most studied α-helical cationic membrane disrupting peptides. It is the main component of bee venom, however it is considered an antimicrobial peptide for its ability to kill bacteria. Melittin is believed to act by opening large toroidal pores in the plasma membrane of the targeted cells/bacteria, although this is questioned by some authors. Little is known, however, about the molecular mechanism leading to this activity. In this study the mechanism of action of melittin was studied by dye leakage and quartz crystal microbalance fingerprinting analysis in biomimetic model membranes. The results revealed the existence of multiple stages in the membrane disrupting action with characteristic differences between different membrane types. In bacterial-mimetic (charged) lipid mixtures the viscoelastic fingerprints suggest a surface-acting mechanism, whereas in mammalian-mimetic (neutral) membranes melittin appears to penetrate the bilayer already at low concentrations. In domain-forming mixed membranes melittin shows a preference for the domain containing predominantly zwitterionic lipids. The results confirm membrane poration but are inconsistent with the insertion-to-toroidal pore pathway. Therefore hypotheses of the two membrane disrupting pathways were developed, describing the membrane disruption as either surface tension modulation leading to toroidal pore formation, or linear aggregation leading to fissure formation in the membrane.

Membrane Permeabilization by Bordetella Adenylate Cyclase Toxin Involves Pores of Tunable Size.

RTX (Repeats in ToXin) pore-forming toxins constitute an expanding family of exoproteins secreted by many Gram-negative bacteria and involved in infectious diseases caused by said pathogens. Despite the relevance in the host/pathogen interactions, the structure and characteristics of the lesions formed by these toxins remain enigmatic. Here, we capture the first direct nanoscale pictures of lytic pores formed by an RTX toxin, the Adenylate cyclase (ACT), secreted by the whooping cough bacterium Bordetella pertussis. We reveal that ACT associates into growing-size oligomers of variable stoichiometry and heterogeneous architecture (lines, arcs, and rings) that pierce the membrane, and that, depending on the incubation time and the toxin concentration, evolve into large enough “holes” so as to allow the flux of large molecular mass solutes, while vesicle integrity is preserved. We also resolve ACT assemblies of similar variable stoichiometry in the cell membrane of permeabilized target macrophages, proving that our model system recapitulates the process of ACT permeabilization in natural membranes. Based on our data we propose a non-concerted monomer insertion and sequential mechanism of toroidal pore formation by ACT. A size-tunable pore adds a new regulatory element to ACT-mediated cytotoxicity, with different pore sizes being putatively involved in different physiological scenarios or cell types.

Correlation of an Antimicrobial Peptide's Potency and Its Influences on Membrane Elasticity

The killing power of membrane-active antimicrobial peptides (AMPs) lies in their ability to disrupt the structures of pathogenic membranes. While understanding the AMPs' mechanisms of action is key to realizing their therapeutic potentials, how the AMPs modulate membrane elasticity and thereby affect membrane structure remains an open question, even though the AMP-induced variations in membrane curvature are widely considered to be crucial. Here, we exploit the x-ray diffraction technique to examine how the dominant bacterial lipid, phosphatidylethanolamine, varies its monolayer elastic properties upon interacting with six artificial peptides mimicking the AMPs' common amino acid content. Remarkably, the monolayer spontaneous curvature ${C}_{0}$ is unaffected by any of the peptides. In contrast, the peptides designated as ${(\mathrm{K}2\mathrm{W})}^{2}$, K4W2, R6, and R9, mimicking the AMP mutants with microbicidal potency, are able to modify the monolayer bending moduli ${K}_{cp}$, while those derived from the impotent mutants cannot. The results are consistent with the scenario that the AMPs disrupt the structure of pathogenic monolayers by, additionally if not exclusively, modulating their ${K}_{cp}$'s, with stiffening and softening leading to two different modes of disruption, that is, toroidal pore formation and membrane micellation, respectively.

Styrene maleic acid copolymer induces pores in biomembranes.

We investigated the interactions between styrene-maleic acid (SMA) copolymers and phospholipid bilayers, using confocal microscopy and surface acoustic wave resonance (SAR) sensing. For the first time we experimentally observed and followed pore formation by SMA copolymers in intact supported bilayers and unilamellar vesicles, showing that fluorescein, a water-soluble organic compound with a mean diameter of 6.9 Å, can traverse the membrane. Our findings are in agreement with recent theoretical predictions, which suggested that SMA copolymers may insert along the plane of the bilayer to form stable toroidal pores.

Correlation of an antimicrobial peptide's potency and its influences on membrane elasticity

The killing power of membrane-active antimicrobial peptides (AMPs) lies in their ability to disrupt the structures of pathogenic membranes. While understanding the AMPs' mechanisms of action is key to realizing their therapeutic potentials, how the AMPs modulate membrane elasticity and thereby affect membrane structure remains an open question, even though the AMP-induced variations in membrane curvature are widely considered to be crucial. Here, we exploit the x-ray diffraction technique to examine how the dominant bacterial lipid, phosphatidylethanolamine, varies its monolayer elastic properties upon interacting with six artificial peptides mimicking the AMPs' common amino acid content. Remarkably, the monolayer spontaneous curvature ${C}_{0}$ is unaffected by any of the peptides. In contrast, the peptides designated as ${(\mathrm{K}2\mathrm{W})}^{2}$, K4W2, R6, and R9, mimicking the AMP mutants with microbicidal potency, are able to modify the monolayer bending moduli ${K}_{cp}$, while those derived from the impotent mutants cannot. The results are consistent with the scenario that the AMPs disrupt the structure of pathogenic monolayers by, additionally if not exclusively, modulating their ${K}_{cp}$'s, with stiffening and softening leading to two different modes of disruption, that is, toroidal pore formation and membrane micellation, respectively.

Dissecting the mechanism of action of actinoporins. Role of the N-terminal amphipathic α-helix in membrane binding and pore activity of sticholysins I and II.

Actinoporins sticholysin I and sticholysin II (St I, St II) are proposed to lyse model and biomembranes via toroidal pore formation by their N-terminal domain. Based on the hypothesis that peptide fragments can reproduce the structure and function of this domain, the behavior of peptides containing St I residues 12–31 (StI12-31), St II residues 11–30 (StII11-30), and its TOAC-labeled analogue (N-TOAC-StII11-30) was examined. Molecular modeling showed a good match with experimental structures, indicating amphipathic α-helices in the same regions as in the toxins. CD spectra revealed that the peptides were essentially unstructured in aqueous solution, acquiring α-helical conformation upon interaction with micelles and large unilamellar vesicles (LUV) of variable lipid composition. Fluorescence quenching studies with NBD-containing lipids indicated that N-TOAC-StII11-30’s nitroxide moiety is located in the membranes polar head group region. Pyrene-labeled phospholipid inter-leaflet redistribution suggested that the peptides form toroidal pores, according to the mechanism of action proposed for the toxins. Binding occurred only to negatively charged LUV, indicating the importance of electrostatic interactions; in contrast the peptides bound to both negatively charged and zwitterionic micelles, pointing to a lesser influence of these interactions. In addition, differences between bilayers and micelles in head group packing and in curvature led to differences in peptide-membrane interaction. We propose that the peptides topography in micelles resembles that of the toxins in the toroidal pore. The peptides mimicked the toxins permeabilizing activity, St II peptides being more effective than StI12-31. To our knowledge, this is the first demonstration that differences in the toxins N-terminal amphipathic α-helix play a role in the difference between St I and St II activities.

The novel cathelicidin of naked mole rats, Hg-CATH, showed potent antimicrobial activity and low cytotoxicity

We performed the in silico genome-wide identification of antimicrobial peptides against the available genome sequence of the naked mole rat Heterocephalus glaber (H. glaber). Our results showed the presence of Hg-CATH, the single cathelicidin containing the antimicrobial domain in H. glaber. We chemically synthesized a 25 amino-acid peptide (ΔHg-CATH) corresponding to the predicted antimicrobial-active core region of Hg-CATH, and evaluated its antibacterial activity against seven bacterial strains. The ΔHg-CATH peptide exhibited strong bactericidal activity against gram-negative bacteria, including a multi-drug resistant strain, while showing low toxicity towards mammalian cells, including erythrocytes. Scanning electron microscopy images of bacterial cells treated with ΔHg-CATH showed disruption of their membranes due to the formation of toroidal pores. Identifying novel antimicrobial peptides, such as Hg-CATH, may be important for identifying candidate peptides for the control of multi-drug resistant bacteria.