The essential role of SepF in mycobacterial division

Mycobacteria lack several of the components that are essential in model systems as Escherichia coli or Bacillus subtilis for the formation of the divisome, a ring‐like structure assembling at the division site to initiate bacterial cytokinesis. Divisome assembly depends on the correct placement of the FtsZ protein into a structure called the Z ring. Notably, early division proteins that assist in the localisation of the Z ring to the cytoplasmic membrane and modulate its structure are missing in the so far known mycobacterial cell division machinery. To find mycobacterium‐relevant components of the divisome that might act at the level of FtsZ, a yeast two‐hybrid screening was performed with FtsZ from Mycobacterium tuberculosis. We identified the SepF homolog as a new interaction partner of mycobacterial FtsZ. Depending on the presence of FtsZ, SepF‐GFP fusions localised in ring‐like structures at potential division sites. Alteration of SepF levels in Mycobacterium smegmatis led to filamentous cells, indicating a division defect. Depletion of SepF resulted in a complete block of division. The sepF gene is highly conserved in the M. tuberculosis complex members. We therefore propose that SepF is an essential part of the core division machinery in the genus Mycobacterium.     

Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli

How bacteria coordinate cell growth with division is not well understood. Bacterial cell elongation is controlled by actin–MreB while cell division is governed by tubulin–FtsZ. A ring‐like structure containing FtsZ (the Z ring) at mid‐cell attracts other cell division proteins to form the divisome, an essential protein assembly required for septum synthesis and cell separation. The Z ring exists at mid‐cell during a major part of the cell cycle without contracting. Here, we show that MreB and FtsZ of Escherichia coli interact directly and that this interaction is required for Z ring contraction. We further show that the MreB–FtsZ interaction is required for transfer of cell‐wall biosynthetic enzymes from the lateral to the mature divisome, allowing cells to synthesise the septum. Our observations show that bacterial cell division is coupled to cell elongation via a direct and essential interaction between FtsZ and MreB.     

Divisome‐dependent subcellular localization of cell–cell joining protein SepJ in the filamentous cyanobacterium Anabaena

Heterocyst‐forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA‐dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two‐hybrid system. We found SepJ self‐interaction and a specific interaction with FtsQ, confirmed by co‐purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity.     

Macromolecular interactions of the bacterial division FtsZ protein: from quantitative biochemistry and crowding to reconstructing minimal divisomes in the test tube

The division of Escherichia coli is an essential process strictly regulated in time and space. It requires the association of FtsZ with other proteins to assemble a dynamic ring during septation, forming part of the functionally active division machinery, the divisome. FtsZ reversibly interacts with FtsA and ZipA at the cytoplasmic membrane to form a proto-ring, the first molecular assembly of the divisome, which is ultimately joined by the rest of the division-specific proteins. In this review we summarize the quantitative approaches used to study the activity, interactions, and assembly properties of FtsZ under well-defined solution conditions, with the aim of furthering our understanding of how the behavior of FtsZ is controlled by nucleotides and physiological ligands. The modulation of the association and assembly properties of FtsZ by excluded-volume effects, reproducing in part the natural crowded environment in which this protein has evolved to function, will be described. The subsequent studies on the reactivity of FtsZ in membrane-like systems using biochemical, biophysical, and imaging technologies are reported. Finally, we discuss the experimental challenges to be met to achieve construction of the minimum protein set needed to initiate bacterial division, without cells, in a cell-like compartment. This integrated approach, combining quantitative and synthetic strategies, will help to support (or dismiss) conclusions already derived from cellular and molecular analysis and to complete our understanding on how bacterial division works.     

A Multi-layered Protein Network Stabilizes the Escherichia coli FtsZ-ring and Modulates Constriction Dynamics

The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. The FtsZ-ring establishes the division plane and enables the assembly of the macromolecular division machinery (divisome). Although many molecular components of the divisome have been identified and their interactions extensively characterized, the spatial organization of these proteins within the divisome is unclear. Consequently, the physical mechanisms that drive divisome assembly, maintenance, and constriction remain elusive. Here we applied single-molecule based superresolution imaging, combined with genetic and biophysical investigations, to reveal the spatial organization of cellular structures formed by four important divisome proteins in E. coli: FtsZ, ZapA, ZapB and MatP. We show that these interacting proteins are arranged into a multi-layered protein network extending from the cell membrane to the chromosome, each with unique structural and dynamic properties. Further, we find that this protein network stabilizes the FtsZ-ring, and unexpectedly, slows down cell constriction, suggesting a new, unrecognized role for this network in bacterial cell division. Our results provide new insight into the structure and function of the divisome, and highlight the importance of coordinated cell constriction and chromosome segregation.     

ClpXP and ClpAP proteolytic activity on divisome substrates is differentially regulated following the Caulobacter asymmetric cell division

Proteolytic control of Caulobacter cell cycle proteins is primarily executed by ClpXP, a dynamically localized protease implicated in turnover of several factors critical for faithful cell cycle progression. Here, we show that the transient midcell localization of ClpXP that precedes cytokinesis requires the FtsZ component of the divisome. Although ClpAP does not exhibit subcellular localization, FtsZ is a substrate of both ClpXP and ClpAP in vivo and in vitro. A peptide containing the C‐terminal portion of the FtsA divisome protein is a substrate of both ClpXP and ClpAP in vitro but is primarily degraded by ClpAP in vivo. Caulobacter carries out an asymmetric division in which FtsZ and FtsA are stable in stalked cells but degraded in the non‐replicative swarmer cell where ClpAP alone degrades FtsA and both ClpAP and ClpXP degrade FtsZ. While asymmetric division in Caulobacter normally yields larger stalked and smaller swarmer daughters, we observe a loss of asymmetric size distribution among daughter cells when clpA is depleted from a strain in which FtsZ is constitutively produced. Taken together, these results suggest that the activity of both ClpXP and ClpAP on divisome substrates is differentially regulated in daughter cells.     

3D-SIM Super Resolution Microscopy Reveals a Bead-Like Arrangement for FtsZ and the Division Machinery: Implications for Triggering Cytokinesis

FtsZ is a tubulin-like GTPase that is the major cytoskeletal protein in bacterial cell division. It polymerizes into a ring, called the Z ring, at the division site and acts as a scaffold to recruit other division proteins to this site as well as providing a contractile force for cytokinesis. To understand how FtsZ performs these functions, the in vivo architecture of the Z ring needs to be established, as well as how this structure constricts to enable cytokinesis. Conventional wide-field fluorescence microscopy depicts the Z ring as a continuous structure of uniform density. Here we use a form of super resolution microscopy, known as 3D-structured illumination microscopy (3D-SIM), to examine the architecture of the Z ring in cells of two Gram-positive organisms that have different cell shapes: the rod-shaped Bacillus subtilis and the coccoid Staphylococcus aureus. We show that in both organisms the Z ring is composed of a heterogeneous distribution of FtsZ. In addition, gaps of fluorescence were evident, which suggest that it is a discontinuous structure. Time-lapse studies using an advanced form of fast live 3D-SIM (Blaze) support a model of FtsZ localization within the Z ring that is dynamic and remains distributed in a heterogeneous manner. However, FtsZ dynamics alone do not trigger the constriction of the Z ring to allow cytokinesis. Lastly, we visualize other components of the divisome and show that they also adopt a bead-like localization pattern at the future division site. Our data lead us to propose that FtsZ guides the divisome to adopt a similar localization pattern to ensure Z ring constriction only proceeds following the assembly of a mature divisome.     

Antimicrobial properties of membrane-active dodecapeptides derived from MSI-78

Antimicrobial peptides (AMPs) are a class of broad-spectrum antibiotics known by their ability to disrupt bacterial membranes and their low tendency to induce bacterial resistance, arising as excellent candidates to fight bacterial infections. In this study we aimed at designing short 12-mer AMPs, derived from a highly effective and broad spectrum synthetic AMP, MSI-78 (22 residues), by truncating this peptide at the N- and/or C-termini while spanning its entire sequence with 1 amino acid (aa) shifts. These designed peptides were evaluated regarding antimicrobial activity against selected gram-positive Staphylococcus strains and the gram-negative Pseudomonas aeruginosa (P. aeruginosa).The short 12-mer peptide CEM1 (GIGKFLKKAKKF) was identified as an excellent candidate to fight P. aeruginosa infections as it displays antimicrobial activity against this strain and selectivity, with negligible toxicity to mammalian cells even at high concentrations. However, in general most of the short 12-mer peptides tested showed a reduction in antimicrobial activity, an effect that was more pronounced for gram-positive Staphylococcus strains. Interestingly, CEM1 and a highly similar peptide differing by only one aa-shift (CEM2: IGKFLKKAKKFG), showed a remarkably contrasting AMP activity. These two peptides were chosen for a more detailed study regarding their mechanism of action, using several biophysical assays and simple membrane models that mimic the mammalian and bacterial lipid composition.We confirmed the correlation between peptide helicity and antimicrobial activity and propose a mechanism of action based on the disruption of the bacterial membrane permeability barrier.     

Investigation of the antimicrobial activity of soy peptides by developing a high throughput drug screening assay

BackgroundAntimicrobial resistance is a great concern in the medical community, as well as food industry. Soy peptides were tested against bacterial biofilms for their antimicrobial activity. A high throughput drug screening assay was developed using microfluidic technology, RAMAN spectroscopy, and optical microscopy for rapid screening of antimicrobials and rapid identification of pathogens.MethodsSynthesized PGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides were tested against Pseudomonas aeruginosa and Listeria monocytogenes using a microdilution assay. Microfluidic technology in combination with Surface Enhanced RAMAN Spectroscopy (SERS) and optical microscopy was used for rapid screening of soy peptides, pathogen identification, and to visualize the impact of selected peptides.ResultsThe PGTAVFK peptide did not significantly affect P. aeruginosa, although it had an inhibitory effect on L. monocytogenes above a concentration of 625 µM. IKAFKEATKVDKVVVLWTA was effective against both P. aeruginosa and L. monocytogenes above a concentration of 37.2 µM. High throughput drug screening assays were able to reduce the screening and bacterial detection time to 4 h. SERS spectra was used to distinguish the two bacterial species.ConclusionsPGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides showed antimicrobial activity against P. aeruginosa and L. monocytogenes. Development of high throughput assays could streamline the drug screening and bacterial detection process.General significanceThe results of this study show that the antimicrobial properties, biocompatibility, and biodegradability of soy peptides could possibly make them an alternative to the ineffective antimicrobials and antibiotics currently used in the food and medical fields. High throughput drug screening assays could help hasten pre-clinical trials in the medical field.     

Differential interactions of the antimicrobial peptide,RQ18, with phospholipids and cholesterol modulate its selectivity for microorganism membranes

BackgroundAntimicrobial peptides (AMPs) are molecules with potential application for the treatment of microorganism infections. We, herein, describe the structure, activity, and mechanism of action of RQ18, an α-helical AMP that displays antimicrobial activity against Gram-positive and Gram-negative bacteria, and yeasts from the Candida genus.MethodsA physicochemical-guided design assisted by computer tools was used to obtain our lead peptide candidate, named RQ18. This peptide was assayed against Gram-positive and Gram-negative bacteria, yeasts, and mammalian cells to determine its selectivity index. The secondary structure and the mechanism of action of RQ18 were investigated using circular dichroism, large unilamellar vesicles, and molecular dynamic simulations.ResultsRQ18 was not cytotoxic to human lung fibroblasts, peripheral blood mononuclear cells, red blood cells, or Vero cells at MIC values, exhibiting a high selectivity index. Circular dichroism analysis and molecular dynamic simulations revealed that RQ18 presents varying structural profiles in aqueous solution, TFE/water mixtures, SDS micelles, and lipid bilayers. The peptide was virtually unable to release carboxyfluorescein from large unilamellar vesicles composed of POPC/cholesterol, model that mimics the eukaryotic membrane, indicating that vesicles' net charges and the presence of cholesterol may be related with RQ18 selectivity for bacterial and fungal cell surfaces.ConclusionsRQ18 was characterized as a membrane-active peptide with dual antibacterial and antifungal activities, without compromising mammalian cells viability, thus reinforcing its therapeutic application.General significanceThese results provide further insight into the complex process of AMPs interaction with biological membranes, in special with systems that mimic prokaryotic and eukaryotic cell surfaces.     

A mutation in Escherichia coli ftsZ bypasses the requirement for the essential division gene zipA and confers resistance to FtsZ assembly inhibitors by stabilizing protofilament bundling

The earliest step in Escherichia coli cell division consists of the assembly of FtsZ protein into a proto‐ring structure, tethered to the cytoplasmic membrane by FtsA and ZipA. The proto‐ring then recruits additional cell division proteins to form the divisome. Previously we described an ftsZ allele, ftsZ_L169R, which maps to the side of the FtsZ subunit and confers resistance to FtsZ assembly inhibitory factors including Kil of bacteriophage λ. Here we further characterize this allele and its mechanism of resistance. We found that FtsZ_L169R permits the bypass of the normally essential ZipA, a property previously observed for FtsA gain‐of‐function mutants such as FtsA* or increased levels of the FtsA‐interacting protein FtsN. Similar to FtsA*, FtsZ_L169R also can partially suppress thermosensitive mutants of ftsQ or ftsK, which encode additional divisome proteins, and confers strong resistance to excess levels of FtsA, which normally inhibit FtsZ ring function. Additional genetic and biochemical assays provide further evidence that FtsZ_L169R enhances FtsZ protofilament bundling, thereby conferring resistance to assembly inhibitors and bypassing the normal requirement for ZipA. This work highlights the importance of FtsZ protofilament bundling during cell division and its likely role in regulating additional divisome activities.     

Interaction of a novel antimicrobial peptide isolated from the venom of solitary bee Colletes daviesanus with phospholipid vesicles and Escherichia coli cells

The peptide named codesane (COD), consisting of 18 amino acid residues and isolated from the venom of wild bee Colletes daviesanus (Hymenoptera : Colletidae), falls into the category of cationic α‐helical amphipathic antimicrobial peptides. In our investigations, synthetic COD exhibited antimicrobial activity against Gram‐positive and Gram‐negative bacteria and Candida albicans but also noticeable hemolytic activity. COD and its analogs (collectively referred to as CODs) were studied for the mechanism of their action. The interaction of CODs with liposomes led to significant leakage of calcein entrapped in bacterial membrane‐mimicking large unilamellar vesicles made preferentially from anionic phospholipids while no calcein leakage was observed from zwitterionic liposomes mimicking membranes of erythrocytes. The preference of CODs for anionic phospholipids was also established by the blue shift in the tryptophan emission spectra maxima when the interactions of tryptophan‐containing COD analogs with liposomes were examined. Those results were in agreement with the antimicrobial and hemolytic activities of CODs. Moreover, we found that the studied peptides permeated both the outer and inner cytoplasmic membranes of Escherichia coli. This was determined by measuring changes in the fluorescence of probe N‐phenyl‐1‐naphthylamine and detecting cytoplasmic β‐galactosidase released during the interaction of peptides with E. coli cells. Transmission electron microscopy revealed that treatment of E. coli with one of the COD analogs caused leakage of bacterial content mainly from the septal areas of the cells. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of dimerization of the cell‐penetrating peptide Tat analog on antimicrobial activity and mechanism of bactericidal action

The cell‐penetrating peptide Tat (48–60) (GRKKRRQRRRPPQ) derived from HIV‐1 Tat protein showed potent antibacterial activity (MIC: 2–8 µM ). To investigate the effect of dimerization of Tat (48–60) analog, [Tat(W): GRKKRRQRRRPWQ‐NH₂], on antimicrobial activity and mechanism of bactericidal action, its dimeric peptides, di‐Tat(W)‐C and di‐Tat(W)‐K, were synthesized by a disulfide bond linkage and lysine linkage of monomeric Tat(W), respectively. From the viewpoint of a weight basis and the monomer concentration, these dimeric peptides displayed almost similar antimicrobial activity against six bacterial strains tested but acted more rapidly against Staphylococcus aureus on kinetics of bactericidal activity, compared with monomeric Tat(W). Unlike monomeric Tat(W), these dimeric peptides significantly depolarized the cytoplasmic membrane of intact S. aureus cells at MIC and induced dye leakage from bacterial‐membrane‐mimicking egg yolk L ‐α‐phosphatidylethanolamine/egg yolk L ‐α‐phosphatidyl‐DL ‐glycerol (7:3, w/w) vesicles. Furthermore, these dimeric peptides were less effective to translocate across lipid bilayers than monomeric Tat(W). These results indicated that the dimerization of Tat analog induces a partial change in the mode of its bactericidal action from intracellular target mechanism to membrane‐targeting mechanism. Collectively, our designed dimeric Tat peptides with high antimicrobial activity and rapid bactericidal activity appear to be excellent candidates for future development as novel antimicrobial agents. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Atomic force microscopy study of the antimicrobial action of Sushi peptides on Gram negative bacteria

The antibacterial effect of the endotoxin-binding Sushi peptides against Gram-negative bacteria (GNB) is investigated in this study. Similar characteristics observed for Atomic force microscopy (AFM) images of peptide-treated Escherichia coli and Pseudomonas aeruginosa suggest that the Sushi peptides (S3) evoke comparable mechanism of action against different strains of GNB. The results also indicate that the Sushi peptides appear to act in three stages: damage of the bacterial outer membrane, permeabilization of the inner membrane and disintegration of both membranes. The AFM approach has provided vivid and detailed close-up images of the GNB undergoing various stages of antimicrobial peptide actions at the nanometer scale. The AFM results support our hypothesis that the S3 peptide perturbs the GNB membrane via the “carpet-model” and thus, provide important insights into their antimicrobial mechanisms.     

Interaction of an artificial antimicrobial peptide with lipid membranes

Antimicrobial peptides constitute an important part of the innate immune defense and are promising new candidates for antibiotics. Naturally occurring antimicrobial peptides often possess hemolytic activity and are not suitable as drugs. Therefore, a range of new synthetic antimicrobial peptides have been developed in recent years with promising properties. But their mechanism of action is in most cases not fully understood. One of these peptides, called V4, is a cyclized 19 amino acid peptide whose amino acid sequence has been modeled upon the hydrophobic/cationic binding pattern found in Factor C of the horseshoe crab (Carcinoscorpius rotundicauda). In this work we used a combination of biophysical techniques to elucidate the mechanism of action of V4. Langmuir-Blodgett trough, atomic force microscopy, Fluorescence Correlation Spectroscopy, Dual Polarization Interference, and confocal microscopy experiments show how the hydrophobic and cationic properties of V4 lead to a) selective binding of the peptide to anionic lipids (POPG) versus zwitterionic lipids (POPC), b) aggregation of vesicles, and above a certain concentration threshold to c) integration of the peptide into the bilayer and finally d) to the disruption of the bilayer structure. The understanding of the mechanism of action of this peptide in relation to the properties of its constituent amino acids is a first step in designing better peptides in the future.     

FtsZ and bacterial cell division

In this review we describe proteins and supermolecular structures which take part in the division of bacterial cells. FtsZ, a eukaryotic tubulin homolog is a key cell division protein in most prokaryotes. FtsZ, as well as tubulin, is capable of binding and hydrolyzing GTP. The division of a bacterial cell begins with the forming of a so-called divisome. The basis of such a divisome is a contractile ring (Z ring) which encircles the cell about midcell. The Z-ring consists of a bundle of laterally bound protofilaments formed in result of FtsZ polymerization. Z-ring is rigidly bounded to the cytosolic side of the inner membrane with the participation of FtsA, ZipA, FtsW and many other divisome cell division proteins. The ring directs the process of cytokinesis transmitting constriction power to the membrane. The primary structures of the prokaryotic FtsZ family members significantly differ from eukaryotic tubulins except for the sites of GTP binding. There is a high degree of structural homology between these proteins in the region. FtsZ is one of the most conserved proteins in prokaryotes. However, ftsZ genes have not been found in several species of microorganisms with completely sequenced genomes. They include two species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea. Consequently, these organisms divide without FtsZ participation. The genomes of U. parvum and M. mobile have many open reading frames which encode proteins with unknown functions. A comparison of the primary structures of these hypothetical proteins did not identify any known cell division proteins. We hypothesize that the process of cell division in these organisms should involve proteins similar to FtsZ in function and homologous to FtsZ or other cell division proteins in structure.     

The bacterial divisome: ready for its close-up

Bacterial cells divide by targeting a transmembrane protein machine to the division site and regulating its assembly and disassembly so that cytokinesis occurs at the correct time in the cell cycle. The structure and dynamics of this machine (divisome) in bacterial model systems are coming more clearly into focus, thanks to incisive cell biology methods in combination with biochemical and genetic approaches. The main conserved structural element of the machine is the tubulin homologue FtsZ, which assembles into a circumferential ring at the division site that is stabilized and anchored to the inner surface of the cytoplasmic membrane by FtsZ-binding proteins. Once this ring is in place, it recruits a series of transmembrane proteins that ultimately trigger cytokinesis. This review will survey the methods used to characterize the structure of the bacterial divisome, focusing mainly on the Escherichia coli model system, as well as the challenges that remain. These methods include recent super-resolution microscopy, cryo-electron tomography and synthetic reconstitution.     

Modulating short tryptophan- and arginine-rich peptides activity by substitution with histidine

BackgroundHigh antimicrobial efficacy of short tryptophan-and arginine-rich peptides makes them good candidates in the fight against pathogens. Substitution of tryptophan and arginine by histidine could be used to modulate the peptides efficacy by optimizing their structures.MethodsThe peptide (RRWWRWWRR), reported to showed good antimicrobial efficacy, was used as template, seven new analogs being designed substituting tryptophan or arginine with histidine. The peptides' efficacy was tested against E. coli, B. subtilis and S. aureus. The cytotoxicity and hemolytic effect were evaluated and the therapeutic index was inferred for each peptide. Atomic force microscopy and molecular simulation were used to analyze the effects of peptides on bacterial membrane.ResultsThe substitution of tryptophan by histidine proved to strongly modulate the antimicrobial activity, mainly by changing the peptide-to-membrane binding energy. The substitution of arginine has low effect on the antimicrobial efficacy. The presence of histidine residue reduced the cytotoxic and hemolytic activity of the peptides in some cases maintaining the same efficacy against bacteria. The peptides' antimicrobial activity was correlated to the 3D-hydrophobic moment and to a simple structure-based packing parameter.ConclusionThe results show that some of these peptides have the potential to become good candidates to fight against bacteria. The substitution by histidine proved to fine tune the therapeutic index allowing the optimization of the peptide structure mainly by changing its binding energy and 3D-hydrophobic moment.General significanceThe short tryptophan reach peptides therapeutic index can be maximized using the histidine substitution to optimize their structure.     

Antimicrobial activities and membrane‐active mechanism of CPF‐C1 against multidrug‐resistant bacteria,a novel antimicrobial peptide derived from skin secretions of the tetraploid frog Xenopus clivii

Hospital‐acquired infections caused by multidrug‐resistant bacteria pose significant challenges for treatment, which necessitate the development of new antibiotics. Antimicrobial peptides are considered potential alternatives to conventional antibiotics. The skin of Anurans (frogs and toads) amphibians is an extraordinarily rich source of antimicrobial peptides. CPF‐C1 is a typical cationic antimicrobial peptide that was originally isolated from the tetraploid frog Xenopus clivii. Our results showed that CPF‐C1 has potent antimicrobial activity against both sensitive and multidrug‐resistant bacteria. It disrupted the outer and inner membranes of bacterial cells. CPF‐C1 induced both propidium iodide uptake into the bacterial cell and the leakage of calcein from large liposome vesicles, which suggests a mode of action that involves membrane disturbance. Scanning electron microscopy and transmission electron microscopy verified the morphologic changes of CPF‐C1‐treated bacterial cells and large liposome vesicles. The membrane‐dependent mode of action signifies that the CPF‐C1 peptide functions freely and without regard to conventional resistant mechanisms. Additionally, it is difficult for bacteria to develop resistance against CPF‐C1 under this action mode. Other studies indicated that CPF‐C1 had low cytotoxicity against mammalian cell. In conclusion, considering the increase in multidrug‐resistant bacterial infections, CPF‐C1 may offer a new strategy that can be considered a potential therapeutic agent for the treatment of diseases caused by multidrug‐resistant bacteria. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.