Membrane-disrupting Activity Research Articles

In vitro anticancer activity of Nε-acyl lysine methyl ester through folate receptor targeted liposomes

Delivering anticancer drugs to tumor tissues with high specificity to realize minimal adverse effects is currently the main research focus of cancer treatment. Nε-acyl lysine methyl esters (Nε-lys) have the potential to serve as novel and safe anticancer agents owing to their pH-responsive membrane-disruptive activity and non-toxic metabolic products. In this study, we synthesized three Nε-lys with different hydrophobic chain lengths and evaluated their cytotoxicity to cancer and normal cells. One of the compounds with an acyl chain of 14 carbon atoms (MKM) showed moderate cytotoxicity and was delivered to cancer cells encapsulated in liposomes (MKM-LP). We additionally modified the liposomes with folate and PEG as a cancer-targeting drug delivery system (FA-PEG-MKM-LP) to enhance the cell selectivity. We tested the difference in the cellular uptake of liposomes between a folate receptor-positive human ovarian cancer cell line (SKOV3) and a healthy human ovary epithelial cell line (IOSE). The experimental results showed that FA-PEG-MKM-LP had low cytotoxicity to IOSE and higher cancer-targeting ability than other tested liposomes. Fluorescence imaging of dead cells and a LDH release assay further demonstrated that the cytotoxicity of FA-PEG-MKM-LP possessed time- and dose-dependent activity and specificity to cancer cells. These results lay a foundation for the development of Nε-lys as a promising anticancer drug.

Engineered lipid bicelle nanostructures for membrane-disruptive antibacterial applications

Lipid bicelles are cell membrane-mimicking nanostructures that self-assemble from mixtures of long- and short-chain lipidic components and are widely used in various applications related to structural biology, drug delivery, and interfacial science. To date, most research efforts have focused on bicelles as passive structural carriers to host membrane proteins or hydrophobic drugs, while there remains untapped potential to engineer functionally active lipid bicelles that contain biologically important lipidic components for targeted applications. Herein, we developed antibiotic-free, antibacterial bicellar nanostructures composed of a long-chain phospholipid and glycerol monolaurate, which is a monoglyceride that exhibits membrane-disruptive inhibitory activity against various bacteria and membrane-enveloped viruses. Quartz crystal microbalance-dissipation and time-lapse fluorescence microscopy imaging experiments were conducted to identify fusogenic bicellar compositions with optimal levels of pore-like, membrane-disruptive activity that was distinct from the activity of the monoglyceride alone. Cryogenic transmission electron microscopy was performed to characterize the lamellar-phase nanostructure properties of the lead bicelle composition along with in vitro antibacterial assays, which identified that the bicelles inhibited Staphylococcus aureus bacteria via a killing mechanism. Collectively, these findings demonstrate the potential of applying molecular-level engineering strategies to fabricate lipid bicelles with membrane-disruptive properties relevant to anti-infective applications.

Stereorandomization as a Method to Probe Peptide Bioactivity

Solid-phase peptide synthesis (SPPS) is usually performed with optically pure building blocks to prepare peptides as single enantiomers. Herein we report that SPPS using racemic amino acids provides stereorandomized (sr) peptides, containing up to billions of different stereoisomers, as well-defined single HPLC peaks, single mass products with high yield, which can be used to investigate peptide bioactivity. To exemplify our method, we show that stereorandomization abolishes the membrane-disruptive effect of α-helical amphiphilic antimicrobial peptides but preserves their antibiofilm effect, implying different mechanisms involving folded versus disordered conformations. For antimicrobial peptide dendrimers by contrast, stereorandomization preserves antibacterial, membrane-disruptive, and antibiofilm effects but reduces hemolysis and cytotoxicity, thereby increasing their therapeutic index. Finally, we identify partially stereorandomized analogues of the last resort cyclic peptide antibiotic polymyxin B with preserved antibacterial activity but lacking membrane-disruptive and lipopolysaccharide-neutralizing activity, pointing to the existence of additional targets.

Antimicrobial Peptides Grafted onto a Plasma Polymer Interlayer Platform: Performance upon Extended Bacterial Challenge

To combat infections on biomedical devices, antimicrobial coatings have attracted considerable attention, including coatings comprising naturally occurring antimicrobial peptides (AMPs). In this study the aim was to explore performance upon extended challenge by bacteria growing in media above samples. The AMPs LL37, Magainin 2, and Parasin 1 were selected on the basis of well-known membrane disruption activity in solution and were covalently grafted onto a plasma polymer platform, which enables application of this multilayer coating strategy to a wide range of biomaterials. Detailed surface analyses were performed to verify the intended outcomes of the coating sequence. Samples were challenged by incubation in bacterial growth media for 5 and 20 h. Compared with the control plasma polymer surface, all three grafted AMP coatings showed considerable reductions in bacterial colonization even at the high bacterial challenge of initial seeding at 1 × 107 CFU, but there were increasing numbers of dead bacteria attached to the surface. All three grafted AMP coatings were found to be non-toxic to primary fibroblasts. These coatings thus could be useful to produce antibacterial surface coatings for biomaterials, though possible consequences arising from the presence of dead bacteria need to be studied further, and compared to non-fouling coatings that avoid attachment of dead bacteria.

Spatially regulated activation of membrane fusogenic peptides with chaperone-like ionic copolymers

Controlled or targeted membrane lysis induced by cascades of assembly and activation of biomolecules on membrane surfaces is important in programmed cell death and host defense systems. In a previous study, we reported that an ionic graft copolymer with a polycation backbone and water-soluble graft chains, poly(allylamine)-graft-dextran (PAA-g-Dex) chaperoned folding and assembly of E5, a membrane-destructive peptide derived from influenza hemagglutinin, to its increase membrane-disruptive activity. In this study, we modified the copolymer with long acyl chains, which resulted in delivery of the copolymer to membrane surfaces of liposomes and living cells. The liposomes with PAA-g-Dex functionalized with stearic acid (PAA-g-Dex-SA) on their surfaces underwent vesicle-to-sheet conversion upon addition of E5, whereas control liposomes did not. E5 also induced selective lysis of cells incubated with PAA-g-Dex-SA. The spatially specific activation of E5 on target membrane surfaces driven by self-assembly of copolymer and activation of E5 should find application in lipid-based delivery devices and cell-based therapeutics.

Modification of carboxyl groups converts α-lactalbumin into an active molten globule state with membrane-perturbing activity and cytotoxicity

We investigated whether the modification of the negatively charged carboxyl groups with semicarbazide could confer membrane-disrupting and cytotoxic properties to bovine α-lactalbumin (LA). MALDI-TOF analysis revealed that eighteen of the twenty-one carboxyl groups in LA were coupled with semicarbazide molecules. Measurement of circular dichroism spectra and Trp fluorescence quenching studies showed that semicarbazide-modified LA (SEM-LA) had a molten globule-like conformation that retained the α-helix secondary structure but lost the tertiary structure of LA. Compared to LA, SEM-LA had a higher structural flexibility in response to trifluoroethanol- and temperature-induced structural transitions. In sharp contrast to LA, SEM-LA exhibited membrane-damaging activity and cytotoxicity. Furthermore, SEM-LA-induced membrane permeability promoted the uptake of daunorubicin and thereby its cytotoxicity. The microenvironment surrounding the Trp residues of SEM-LA was enriched in positive charges, as revealed by iodide quenching studies. The binding of SEM-LA with lipid vesicles altered the positively charged cluster around Trp residues. Although LA and SEM-LA displayed similar lipid-binding affinities, the membrane interaction modes of SEM-LA and LA differed. Collectively, these results suggest that blocking of negatively charged residues enables the formation of a molten-globule conformation of LA with structural flexibility and increased positive charge, thereby generating functional LA with membrane-disrupting activity and cytotoxicity.

Shared PKS Module in Biosynthesis of Synergistic Laxaphycins

Cyanobacteria produce a wide range of lipopeptides that exhibit potent membrane-disrupting activities. Laxaphycins consist of two families of structurally distinct macrocyclic lipopeptides that act in a synergistic manner to produce antifungal and antiproliferative activities. Laxaphycins are produced by range of cyanobacteria but their biosynthetic origins remain unclear. Here, we identified the biosynthetic pathways responsible for the biosynthesis of the laxaphycins produced by Scytonema hofmannii PCC 7110. We show that these laxaphycins, called scytocyclamides, are produced by this cyanobacterium and are encoded in a single biosynthetic gene cluster with shared polyketide synthase enzymes initiating two distinct non-ribosomal peptide synthetase pathways. The unusual mechanism of shared enzymes synthesizing two distinct types of products may aid future research in identifying and expressing natural product biosynthetic pathways and in expanding the known biosynthetic logic of this important family of natural products.

Supramolecular Mechanism of Viral Envelope Disruption by Molecular Tweezers.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the “phosphate tweezer” CLR01, a “carboxylate tweezer” CLR05, and a “phosphate clip” PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.

Myofiber necroptosis promotes muscle stem cell proliferation via releasing Tenascin-C during regeneration

Necroptosis, a form of programmed cell death, is characterized by the loss of membrane integrity and release of intracellular contents, the execution of which depends on the membrane-disrupting activity of the Mixed Lineage Kinase Domain-Like protein (MLKL) upon its phosphorylation. Here we found myofibers committed MLKL-dependent necroptosis after muscle injury. Either pharmacological inhibition of the necroptosis upstream kinase Receptor Interacting Protein Kinases 1 (RIPK1) or genetic ablation of MLKL expression in myofibers led to significant muscle regeneration defects. By releasing factors into the muscle stem cell (MuSC) microenvironment, necroptotic myofibers facilitated muscle regeneration. Tenascin-C (TNC), released by necroptotic myofibers, was found to be critical for MuSC proliferation. The temporary expression of TNC in myofibers is tightly controlled by necroptosis; the extracellular release of TNC depends on necroptotic membrane rupture. TNC directly activated EGF receptor (EGFR) signaling pathway in MuSCs through its N-terminus assembly domain together with the EGF-like domain. These findings indicate that necroptosis plays a key role in promoting MuSC proliferation to facilitate muscle regeneration.

Surface Curvature and Aminated Side-Chain Partitioning Affect Structure of Poly(oxonorbornenes) Attached to Planar Surfaces and Nanoparticles of Gold.

Cationic amphiphilic polymers are often used to coat nanoparticles as they increase chemical stability in solution and exhibit membrane disruption activities. Among these, poly(oxonorbornenes) (PONs) are tunable membrane disruptors. They can be constructed with either one amine-terminated side chain and one hydrophobic alkyl side chain (PON-50) or two amine-terminated side chains (PON-100) on each repeat unit and can then be conjugated to gold nanoparticles using O-(2-carboxyethyl)-O'-(2-mercaptoethyl) heptaethylene glycol (HEG) spacers. While the amine content and membrane disruption activity of PONs can be controlled, the detailed structural properties of PONs conjugated to gold nanoparticles remain less understood. To address this, we performed molecular dynamics simulations of PON-50 and PON-100 to determine the nonbonded energies of PON structures as a function of amine composition. We found increasing energetic stabilization with decreasing amine composition. These results were consistent with experimental observations obtained with X-ray photoelectron spectroscopy (XPS) in which PON-100 was found to have the lowest conjugation efficiency to gold surfaces out of a range of PON amination ratios. Computationally obtained energetics suggest that replacing the aliphatic amine groups with aromatic amine groups can reverse this behavior and lead to more stable PON structures with increasing amine content. We also found that the curvature of the gold nanoparticle surface affects interactions between the surface and the amine groups of PON-50. Increasing curvature decreased these interactions, resulting in a smaller effective footprint of the HEG-PON-50 structure.

Chemical Characterization and Suppressor Potent of Juglans regia Essential Oil on Tramadol-Induced Cell Death

Tramadol is an opioid pain reliever. Its abuse causes neurotoxicity in the brain cells. In high concentration, it increases the production of NO, and decreases the mitochondrial membrane potential in the central nervous system. Cellular toxicity in brain cells leads to death, by activating caspase. In this study, we aimed to investigate of chemical characterization and suppressor effects of Juglans regia essential oil on tramadol-induced cell death. PC12, rat neuron-like cell line, was used for the best simulation. The Gas Chromatography-Mass Spectroscopy (GC/MS) indicated that α-pinene and β-pinene were the most frequently found chemical constituents in J. regia essential oil. The viability and proliferation percent of all the J. regia essential oil treatments were higher than tramadol-treated cells significantly. The cell cytotoxicity and cell death index of all the J. regia essential oil treatments were lower than tramadol-treated cells. All treatment increased mitochondrial membrane potential and decreased NO production, IL-1β, IL-6, INFγ, and TNFα release, and Caspase-3 activity. We concluded that J. regia essential oil suppressed the tramadol-induced cell death in neuron-like, PC12 cells through inhibition of NO production, mitochondrial membrane disruption, DNA fragmentation, and caspase-3 activity.

It Takes Two: Dimerization Is Essential for the Broad-Spectrum Predatory and Defensive Activities of the Venom Peptide Mp1a from the Jack Jumper Ant Myrmecia pilosula.

Ant venoms have recently attracted increased attention due to their chemical complexity, novel molecular frameworks, and diverse biological activities. The heterodimeric peptide ∆-myrtoxin-Mp1a (Mp1a) from the venom of the Australian jack jumper ant, Myrmecia pilosula, exhibits antimicrobial, membrane-disrupting, and pain-inducing activities. In the present study, we examined the activity of Mp1a and a panel of synthetic analogues against the gastrointestinal parasitic nematode Haemonchus contortus, the fruit fly Drosophila melanogaster, and for their ability to stimulate pain-sensing neurons. Mp1a was found to be both insecticidal and anthelmintic, and it robustly activated mammalian sensory neurons at concentrations similar to those reported to elicit antimicrobial and cytotoxic activity. The native antiparallel Mp1a heterodimer was more potent than heterodimers with alternative disulfide connectivity, as well as monomeric analogues. We conclude that the membrane-disrupting effects of Mp1a confer broad-spectrum biological activities that facilitate both predation and defense for the ant. Our structure–activity data also provide a foundation for the rational engineering of analogues with selectivity for particular cell types.

Conjugates of Ciprofloxacin and Levofloxacin with Cell-Penetrating Peptide Exhibit Antifungal Activity and Mammalian Cytotoxicity.

Seven conjugates composed of well-known fluoroquinolone antibacterial agents, ciprofloxacin (CIP) or levofloxacin (LVX), and a cell-penetrating peptide transportan 10 (TP10-NH2) were synthesised. The drugs were covalently bound to the peptide via an amide bond, methylenecarbonyl moiety, or a disulfide bridge. Conjugation of fluoroquinolones to TP10-NH2 resulted in congeners demonstrating antifungal in vitro activity against human pathogenic yeasts of the Candida genus (MICs in the 6.25–100 µM range), whereas the components were poorly active. The antibacterial in vitro activity of most of the conjugates was lower than the activity of CIP or LVX, but the antibacterial effect of CIP-S-S-TP10-NH2 was similar to the mother fluoroquinolone. Additionally, for two representative CIP and LVX conjugates, a rapid bactericidal effect was shown. Compared to fluoroquinolones, TP10-NH2 and the majority of its conjugates generated a relatively low level of reactive oxygen species (ROS) in human embryonic kidney cells (HEK293) and human myeloid leukemia cells (HL-60). The conjugates exhibited cytotoxicity against three cell lines, HEK293, HepG2 (human liver cancer cell line), and LLC-PK1 (old male pig kidney cells), with IC50 values in the 10–100 µM range and hemolytic activity. The mammalian toxicity was due to the intrinsic cytoplasmic membrane disruption activity of TP10-NH2 since fluoroquinolones themselves were not cytotoxic. Nevertheless, the selectivity index values of the conjugates, both for the bacteria and human pathogenic yeasts, remained favourable.

Combination of hydrophobically modified γ-poly(glutamic acid) and trehalose achieving high cryosurvival of RBCs

Trehalose is expected to be an alternative for toxic glycerol as a biocompatible cryoprotectant of red blood cells (RBCs). In this work, γ-poly(glutamic acid) (PGA) is modified by grafting hydrophobic phenethylamine, 3,4-dimethoxyphenylethylamine and dodecylamine, respectively. The graft-modified PGA can significantly enhance cryosurvival of RBCs in combination with trehalose. Analyses of dynamic light scattering, hemolysis assay, atomic force microscope and confocal laser scanning microscope suggest that the modified PGA polymers can self-assemble into nanoparticles in phosphate buffer saline solutions at the pH range of 6.0-7.4, and exhibit membrane-disruptive activity due to hydrogen bond, conjugation and hydrophobic interactions with cell membranes. It is assumed that the modified PGA polymers can improve the cryosurvival of RBCs by promoting membrane permeability of trehalose. Among the three graft-modified polymers, phenethylamine-grafted PGA (PGA-g-PEA) can significantly increase the intracellular trehalose-loading content to 11.3±2.4 mM at pH 7.4, much higher than the value 0.17±0.66 mM when trehalose is used without any polymers. In view of the aforementioned merit, the cryosurvival rate of sheep RBCs is increased to about 90% by incubation with 1.0 mg mL-1 PGA-g-PEA and 0.36 M trehalose. In vitro cell culture of L929 fibroblasts demonstrates low cytotoxicity of PGA-g-PEA. Therefore, hydrophobic PEA-modified PGA with enhanced intracellular trehalose-loading ability can be potentially applied in glycerol-free RBC cryopreservation or other related biomacromolecule delivery systems.

Serum amyloid A exhibits pH dependent antibacterial action and contributes to host defense against Staphylococcus aureus cutaneous infection

Serum amyloid A (SAA), one of the major highly conserved acute-phase proteins in most mammals, is predominantly produced by hepatocytes and also by a variety of cells in extrahepatic tissues. It is well-known that the expression of SAA is sharply increased in bacterial infections. However, the exact physiological function of SAA during bacterial infection remains unclear. Herein, we showed that SAA expression significantly increased in abscesses of Staphylococcus aureus cutaneous infected mice, which exert direct antibacterial effects by binding to the bacterial cell surface and disrupting the cell membrane in acidic conditions. Mechanically, SAA disrupts anionic liposomes by spontaneously forming small vesicles or micelles under acidic conditions. Especially, the N-terminal region of SAA is necessary for membrane disruption and bactericidal activity. Furthermore, we found that mice deficient in SAA1/2 were more susceptible to infection by S. aureus In addition, the expression of SAA in infected skin was regulated by interleukin-6. Taken together, these findings support a key role of the SAA in host defense and may provide a novel therapeutic strategy for cutaneous bacterial infection.

Revisiting the Interaction of Melittin with Phospholipid Bilayers: The Effects of Concentration and Ionic Strength.

Melittin is an anti-microbial peptide (AMP) and one of the most studied membrane-disrupting peptides. There is, however, a lack of accurate measurements of the concentration-dependent kinetics and affinity of binding of melittin to phospholipid membranes. In this study, we used surface plasmon resonance spectroscopy to determine the concentration-dependent effect on the binding of melittin to 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayers in vesicles. Three concentration ranges were considered, and when combined, covered two orders of magnitudes (0.04 µM to 8 µM), corresponding to concentrations relevant to the membrane-disrupting and anti-microbial activities of melittin. Binding kinetics data were analysed using a 1:1 Langmuir-binding model and a two-state reaction model. Using in-depth quantitative analysis, we characterised the effect of peptide concentration, the addition of NaCl at physiological ionic strength and the choice of kinetic binding model on the reliability of the calculated kinetics and affinity of binding parameters. The apparent binding affinity of melittin for POPC bilayers was observed to decrease with increasing peptide/lipid (P/L) ratio, primarily due to the marked decrease in the association rate. At all concentration ranges, the two-state reaction model provided a better fit to the data and, thus, a more reliable estimate of binding affinity. Addition of NaCl significantly reduced the signal response during the association phase; however, no substantial effect on the binding affinity of melittin to the POPC bilayers was observed. These findings based on POPC bilayers could have important implications for our understanding of the mechanism of action of melittin on more complex model cell membranes of higher physiological relevance.

Bioinspired Membrane-Disruptive Macromolecules as Drug-Free Therapeutics.

Membrane-disruptive, drug-free macromolecular therapeutics may help overcome cancer drug-resistance. Their inability to distinguish cancerous from normal cells, however, results in significant off-target toxicity. Note that the tumor has a slightly acidic microenvironment (pH 6.5-6.8) in contrast to the alkaline microenvironment in normal tissues (pH 7.4) and that host-defense peptides (HDPs) and their synthetic mimetics need to be net cationic to be membrane-disruptive. We herein endow polymer mimetics of HDPs with acid-triggered cationicity, to make them membrane-disruptive at only tumor pH. For these polymer mimetics, there exists a maximal threshold of chain length that determines whether the micelle of a mimetic inherits its pH-sensitive activity. Using the most and least active micelles as representatives, we find that their distinct potency in disrupting membranes arises because of their striking tendency to dissociate upon exposure to tumor pH. As expected, these micelles exhibit in vitro cytotoxicity profiles that correlate with their membrane-disruptive activity profiles. When administered intravenously, these micelles-irrespective of their distinct activity profiles-unanimously exhibit long systemic circulation as do PEGylated micelle nanoparticles, despite of their lacking stealth materials, owing to the zwitterionic nature of their surfaces at blood pH. Nevertheless, the pH-sensitive micelle achieves significantly higher tumor uptake and strikingly better therapeutic efficacy than its completely inactive analogue. More important, the pH-sensitive micelle exhibits undetectable off-target toxicity, owing to its pH-sensitivity. Clearly, making HDPs and their mimetics sensitive to tumor-characteristic cues (e.g., acidic pH) is efficient in minimizing their off-target toxicity, thereby offering membrane-disruptive, drug-free macromolecular therapeutics for fighting against cancer drug-resistance.

Antibacterial, anti-biofilm and in vivo activities of the antimicrobial peptides P5 and P6.2

Cationic antimicrobial peptides (AMPs) are short linear amino acid sequences, which display antimicrobial activity against a wide range of bacterial species. They are promising novel antimicrobials since they have shown bactericidal effects against multiresistant bacteria. Their amphiphilic structure with hydrophobic and cationic regions drives their interaction with anionic bacterial cytoplasmic membranes, which leads to their disruption. In this work two synthetic designed AMPs, P5 and P6.2, which have been previously analyzed in their ability to interact with bacterial or eukaryotic membranes, were evaluated in their anti-biofilm and in vivo antibacterial activity. In a first step, a time-kill kinetic assay against P. aeruginosa and S. aureus and a curve for hemolytic activity were performed in order to determine the killing rate and the possible undesirable toxic effect, respectively, for both peptides. The biofilm inhibitory activity was quantified at sub MIC concentrations of the peptides and the results showed that P5 displayed antibiofilm activity on both strains while P6.2 only on S. aureus. Scanning electron microscopy (SEM) of bacteria treated with peptides at their MIC revealed protruding blisters on Gam-negative P. aeruginosa strain, but almost no visible surface alteration on Gram-positive S. aureus. These micrographs highlighted different manifestations of the membrane-disrupting activity that these kinds of peptides possess. Finally, both peptides were analyzed in vivo, in the lungs of neutropenic mice previously instilled with P. aeruginosa. Mice lungs were surgically extracted and bacteria and pro-inflammatory cytokines (IL-β, IL-6 and TNF-α) were quantified by colony forming units and ELISA, respectively. Results showed that instillation of the peptides produced a significant decrease in the number of living bacteria in the lungs, concomitant with a decrease in pro-inflammatory cytokines. Overall, the results presented here suggest that these two new peptides could be good candidates for future drug development for anti-biofilm and anti-infective therapy.

A bactericidal permeability-increasing protein (BPI) from manila clam Ruditapes philippinarum: Investigation on the antibacterial activities and antibacterial action mode

Bactericidal permeability-increasing protein (BPI) is an antimicrobial protein with potent endotoxin-neutralising activity and plays a crucial role in innate immunity against bacterial infection. In the present study, a bpi (designed as rpbpi) was identified and characterized from manila clam Ruditapes philippinarum. Multiple alignments and phylogenetic analysis suggested that rpbpi was a new member of the bpis family. In non-stimulated clams, rpbpi transcripts were ubiquitously expressed in all tested tissues with the highest expression level in hemocytes. After Vibrio anguillarum challenge, the expression levels of rpbpi mRNA in hemocytes were up-regulated significantly at 3 h and 48 h compared with that in the control, which were 4.01- and 19.10-fold (P < 0.05), respectively. The recombinant RpBPI (rRpBPI) showed high antibacterial activities against Gram-negative bacteria Escherichia coli and V. anguillarum, but not Staphylococcus aureus. Moreover, membrane integrity analysis revealed that rRpBPI increased the membrane permeability of Gram-negative bacteria, and then resulted in cell death. Overall, our results suggested that RpBPI played an important role in the elimination of invaded bacteria through membrane-disruptive activity.

Structural analysis and mode of action of BMAP-27, a cathelicidin-derived antimicrobial peptide

BMAP-27, a member of cathelicidin family, plays an important role against microorganisms, including bacteria and fungi. BMAP-27 may exert antimicrobial effects through membrane integrity disruption, but the exact molecular mechanism remains unclear. To identify the structural features important for antimicrobial activity and propose a mechanism underlying antibacterial effects, we determined the nuclear magnetic resonance structure of BMAP-27 in a membrane-mimetic environment and investigated its interactions with lipid membranes. BMAP-27 exhibited a long N-terminal α-helix with faces patterned into aromatic and cationic regions, central kink, and short hydrophobic C-terminal helix. While the N-terminal 18-residue peptide (BMAP-18) exerted only antibacterial activity, BMAP-27 showed potent activity against bacteria and cancer cells. Both peptides inhibited bacterial growth, but BMAP-18 showed delayed bactericidal activity and BMAP-27 completely killed bacteria within 20 min. The differences in antimicrobial activities and microbicidal kinetics may be associated with membrane permeabilisation; BMAP-27 rapidly and largely disrupted membrane integrity, whereas BMAP-18 showed low membrane disruption activity. Thus, the N-terminal helix is sufficient to inhibit bacterial growth and the C-terminal helix is involved in membrane permeabilisation for rapid bactericidal and efficient anticancer activities. The structural and functional characterisation of BMAP-27 may encourage the development of novel antimicrobial/anticancer agents.