Formation Of Transient Pores Research Articles

High Transmembrane Voltage Raised by Close Contact Initiates Fusion Pore.

Membrane fusion lies at the heart of neuronal communication but the detailed mechanism of a critical step, fusion pore initiation, remains poorly understood. Here, through atomistic molecular dynamics simulations, a transient pore formation induced by a close contact of two apposed bilayers is firstly reported. Such a close contact gives rise to a high local transmembrane voltage that induces the transient pore formation. Through simulations on two apposed bilayers fixed at a series of given distances, the process in which two bilayers approaching to each other under the pulling force from fusion proteins for membrane fusion was mimicked. Of note, this close contact induced fusion pore formation is contrasted with previous reported electroporation under ad hoc applied external electric field or ionic charge in-balance. We show that the transmembrane voltage increases with the decrease of the distance between the bilayers. Below a critical distance, depending on the lipid composition, the local transmembrane voltage can be sufficiently high to induce the transient pores. The size of these pores is approximately 1~2 nm in diameter, which is large enough to allow passing of neurotransmitters. A resealing of the membrane pores resulting from the neutralization of the transmembrane voltage by ions through the pores was then observed. We also found that the membrane tension can either prolong the lifetime of transient pores or cause them to dilate for full collapse. This result provides a possible mechanism for fusion pore formation and regulation of pathway of fusion process.

TEMPERATURE DEPENDENCE OF BILAYER PROPERTIES IN LIPOSOMES AND THE USE OF FLUORESCENT PROBES AS A TOOL TO ELUCIDATE THE PERMEATION MECHANISM OF HYDROPHILIC SOLUTES

Solute transport across lipidic membranes is a fundamental process for both living organisms and drug delivery. In order to establish the mechanism of solute passive transport through lipidic membranes, we determined the effect of temperature upon the rate of glucose transport (as hydrophilic solute model) through dipalmitoylphosphatidylcholine large unilamellar liposomes and compare the results to those obtained for a variety of fluorescent probes (pyrene, PRODAN, diphenylhexatriene, diphenylhexatriene-TMA). All these probes, independent of their localization in the liposomes, report a monotonous change in the micro-properties sensed with temperature, with a maximum rate of change at the main transition temperature of the bilayer. These results contrast with those obtained for the rate of glucose influx, were it is observed a clear maximum rate of intake at the transition temperature. These contrasting results imply that the microviscosity (common factor for used probes) is not the property of the bilayer that controls the rate off solute transport which, at least in the vicinity of the main transition temperature, is regulated by formation of transient pores, particularly at the transition temperature (Tm). This would indicate that, at least near Tm, glucose and other hydrophilic solutes diffuses through transient pores formed in the bilayer. Interestingly, the presence of these pores does not affect the fluorescence characteristics of the probes. Abbreviations LUV Large unilamellar vesicles GOx Glucose Oxidase DPPC 1,2-Dipalmitoyl-sn-glycero-3-phosphatydylcholine Tm Main phase transition temperatura

Fundamental study on a gene transfection methodology for mammalian cells using water-in-oil droplet deformation in a DC electric field

We have developed a gene transfection method called water-in-oil droplet electroporation (EP) that uses a dielectric oil and a liquid droplet containing live cells and exogenous DNA. When a cell suspension droplet is placed between a pair of electrodes, an intense DC electric field can induce droplet deformation, resulting in an instantaneous short circuit caused by the droplet elongating and contacting the two electrodes simultaneously. Small transient pores are generated in the cell membrane during the short, allowing the introduction of exogenous DNA into the cells. The droplet EP was characterized by varying the following experimental parameters: applied voltage, number of short circuits, type of medium (electric conductivity), concentration of exogenous DNA, and size of the droplet. In addition, the formation of transient pores in the cell membrane during droplet EP and the transfection efficiency were evaluated.

Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms.

Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.

Revealing the mechanism of passive transport in lipid bilayers via phonon-mediated nanometre-scale density fluctuations.

The passive transport of molecules through a cell membrane relies on thermal motions of the lipids. However, the nature of transmembrane transport and the precise mechanism remain elusive and call for a comprehensive study of phonon excitations. Here we report a high resolution inelastic X-ray scattering study of the in-plane phonon excitations in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine above and below the main transition temperature. In the gel phase, for the first time, we observe low-frequency transverse modes, which exhibit a phonon gap when the lipid transitions into the fluid phase. We argue that the phonon gap signifies the formation of short-lived nanometre-scale lipid clusters and transient pores, which facilitate the passive molecular transport across the bilayer plane. Our findings suggest that the phononic motion of the hydrocarbon tails provides an effective mechanism of passive transport, and illustrate the importance of the collective dynamics of biomembranes.

Straining soft colloids in aqueous nematic liquid crystals

Liquid crystals (LCs), because of their long-range molecular ordering, are anisotropic, elastic fluids. Herein, we report that elastic stresses imparted by nematic LCs can dynamically shape soft colloids and tune their physical properties. Specifically, we use giant unilamellar vesicles (GUVs) as soft colloids and explore the interplay of mechanical strain when the GUVs are confined within aqueous chromonic LC phases. Accompanying thermal quenching from isotropic to LC phases, we observe the elasticity of the LC phases to transform initially spherical GUVs (diameters of 2-50 µm) into two distinct populations of GUVs with spindle-like shapes and aspect ratios as large as 10. Large GUVs are strained to a small extent (R/r < 1.54, where R and r are the major and minor radii, respectively), consistent with an LC elasticity-induced expansion of lipid membrane surface area of up to 3% and conservation of the internal GUV volume. Small GUVs, in contrast, form highly elongated spindles (1.54 < R/r < 10) that arise from an efflux of LCs from the GUVs during the shape transformation, consistent with LC-induced straining of the membrane leading to transient membrane pore formation. A thermodynamic analysis of both populations of GUVs reveals that the final shapes adopted by these soft colloids are dominated by a competition between the LC elasticity and an energy (∼0.01 mN/m) associated with the GUV-LC interface. Overall, these results provide insight into the coupling of strain in soft materials and suggest previously unidentified designs of LC-based responsive and reconfigurable materials.

PSM Peptides of Staphylococcus aureus Activate the p38-CREB Pathway in Dendritic Cells, Thereby Modulating Cytokine Production and T Cell Priming.

The challenging human pathogen Staphylococcus aureus has highly efficient immune evasion strategies for causing a wide range of diseases, from skin and soft tissue to life-threatening infections. Phenol-soluble modulin (PSM) peptides are major pathogenicity factors of community-associated methicillin-resistant S. aureus strains. In previous work, we demonstrated that PSMs in combination with TLR2 ligand from S. aureus induce tolerogenic dendritic cells (DCs) characterized by the production of high amounts of IL-10, but no proinflammatory cytokines. This in turn promotes the activation of regulatory T cells while impairing Th1 response; however, the signaling pathways modulated by PSMs remain elusive. In this study, we analyzed the effects of PSMs on signaling pathway modulation downstream of TLR2. TLR2 stimulation in combination with PSMα3 led to increased and prolonged phosphorylation of NF-κB, ERK, p38, and CREB in mouse bone marrow-derived DCs compared with single TLR2 activation. Furthermore, inhibition of p38 and downstream MSK1 prevented IL-10 production, which in turn reduced the capacity of DCs to activate regulatory T cells. Interestingly, the modulation of the signaling pathways by PSMs was independent of the known receptor for PSMs, as shown by experiments with DCs lacking the formyl peptide receptor 2. Instead, PSMs penetrate the cell membrane most likely by transient pore formation. Moreover, colocalization of PSMs and p38 was observed near the plasma membrane in the cytosol, indicating a direct interaction. Thus, PSMs from S. aureus directly modulate the signaling pathway p38-CREB in DCs, thereby impairing cytokine production and in consequence T cell priming to increase the tolerance toward the pathogen.

The structure and behavior of the NA-CATH antimicrobial peptide with liposomes

Naja atra cathelicidin (NA-CATH) is a 34-amino acid highly cationic peptide identified in Chinese cobras to possess potent toxicity against gram-negative and gram-positive bacteria and low toxicity against host cells. Here, we report the NMR solution structure of the full-length NA-CATH peptide and its interaction with liposomes. The structure shows a well-defined α-helix between residues Phe3 to Lys23, on which one surface is lined by the side-chains of one arginine and 11 lysine residues, while the other side is populated by hydrophobic residues. The last eleven amino acids, which are predominately aromatic and hydrophobic in nature, have no defined structure. NMR data reveal that these residues do not interact with the hydrophobic residues of the helix, indicating that the C-terminal residues have random conformations. Fluorescence requenching experiments, in which liposomes serve as a mimic of the bacterial membranes, result in fluorophore leakage that is consistent with a membrane thinning or transient pore formation mechanism. NMR titration studies of the peptide–liposome interaction reveal that the peptide is in fast exchange with the liposome, consistent with the fluorescent studies. These data indicate that full length NA-CATH possesses a helical segment and unstructured C-terminal tail that disrupts the bilayer to induce leakage and lysing.

Biogenesis and structure of a type VI secretion membrane core complex.

Bacteria share their ecological niches with other microbes. The bacterial type VI secretion system is one of the key players in microbial competition, as well as being an important virulence determinant during bacterial infections. It assembles a nano-crossbow-like structure in the cytoplasm of the attacker cell that propels an arrow made of a haemolysin co-regulated protein (Hcp) tube and a valine-glycine repeat protein G (VgrG) spike and punctures the prey's cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here we show that this complex is assembled by the sequential addition of three type VI subunits (Tss)-TssJ, TssM and TssL-and present a structure of the fully assembled complex at 11.6 Å resolution, determined by negative-stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via ten arches to form a double-ring structure containing the carboxy-terminal domain of TssM (TssMct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssMct-TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane, allowing passage of the attacking Hcp tube/VgrG spike.

Recruitment of endocytosis in sonopermeabilization-mediated drug delivery: a real-time study

Microbubbles (MBs) in combination with ultrasound (US) can enhance cell membrane permeability, and have the potential to facilitate the cellular uptake of hydrophilic molecules. However, the exact mechanism behind US- and MB-mediated intracellular delivery still remains to be fully understood. Among the proposed mechanisms are formation of transient pores and endocytosis stimulation. In our study, we investigated whether endocytosis is involved in US- and MB-mediated delivery of small molecules. Dynamic fluorescence microscopy was used to investigate the effects of endocytosis inhibitors on the pharmacokinetic parameters of US- and MB-mediated uptake of SYTOX Green, a 600 Da hydrophilic model drug. C6 rat glioma cells, together with SonoVue® MBs, were exposed to 1.4 MHz US waves at 0.2 MPa peak-negative pressure. Collection of the signal intensity in each individual nucleus was monitored during and after US exposure by a fibered confocal fluorescence microscope designed for real-time imaging. Exposed to US waves, C6 cells pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, showed up to a 2.5-fold significant increase of the uptake time constant, and a 1.1-fold increase with genistein, an inhibitor of caveolae-mediated endocytosis. Both inhibitors slowed down the US-mediated uptake of SYTOX Green. With C6 cells and our experimental settings, these quantitative data indicate that endocytosis plays a role in sonopermeabilization-mediated delivery of small molecules with a more predominant contribution of clathrin-mediated endocytosis.

Theoretical evaluation of wall teichoic acids in the cavitation-mediated pores formation in Gram-positive bacteria subjected to an electric field

BackgroundElectroporation is a method of choice to transform living cells. The ability of electroporation to transfer small or large chemicals across the lipid bilayer membrane of eukaryotic cells or Gram-negative bacteria relies on the formation of transient pores across the membrane. To exist, these pores rely on an insulator (the bilayer membrane) and the presence of a potential difference on either side of the membrane mediated by an external electric field. In Gram-positive bacteria, however, the wall is not an insulator but pores can still form when an electric field is applied. Past works have shown that the electrostatic charge of teichoic acids, a major wall component; sensitizes the wall to pore formation when an external electric field is applied. These results suggest that teichoic acids mediate the formation of defects in the wall of Gram-positive bacteria. MethodsWe model the electrostatic repulsion between teichoic acids embedded in the bacterial wall composed of peptidoglycan when an electric field is applied. The repulsion between teichoic acids gives rise to a stress pressure that is able to rupture the wall when a threshold value has been reached. The size of such small defects can diverge leading to the formation of pores. ResultsIt is demonstrated herein that for a bonding energy of about ~1−10kBT between peptidoglycan monomers an intra-wall pressure of about ~5−120kBT/nm3 generates spherical defects of radius ~0.1−1nm diverging in size to create pores. ConclusionThe electrostatic cavitation of the bacterial wall theory has the potential to highlight the role of teichoic acids in the formation pores, providing a new step in the understanding of electroporation in Gram-positive bacteria without requiring the use of an insulator.

Nanoscale phase behavior on flat and curved membranes

The diverse physical properties of membranes play a critical role in many membrane associated biological processes. Proteins responsible for membrane transport can be affected by the lateral membrane order and lateral segregation of proteins is often controlled by the preference of certain membrane anchors for membrane phases having a physically ordered state. The dynamic properties of coexisting membrane phases are often studied by investigating their thermal behavior. Optical trapping of gold nanoparticles is a useful tool to generate local phase transitions in membranes. The high local temperatures surrounding an irradiated gold nanoparticle can be used to melt a part of a giant unilamellar lipid vesicle (GUV) which is then imaged using phase sensitive fluorophores embedded within the bilayer. By local melting of GUVs we reveal how a protein-free, one component lipid bilayer can mediate passive transport of fluorescent molecules by localized and transient pore formation. Also, we show how tubular membrane curvatures can be generated by optical pulling from the melted region on the GUV. This will allow us to measure the effect of membrane curvature on the phase transition temperature.

Mechanistic insight into CM18-Tat11 peptide membrane-perturbing action by whole-cell patch-clamp recording.

The membrane-destabilization properties of the recently-introduced endosomolytic CM18-Tat11 hybrid peptide (KWKLFKKIGAVLKVLTTG-YGRKKRRQRRR, residues 1–7 of cecropin-A, 2–12 of melittin, and 47–57 of HIV-1 Tat protein) are investigated in CHO-K1 cells by using the whole-cell configuration of the patch-clamp technique. CM18-Tat11, CM18, and Tat11 peptides are administered to the cell membrane with a computer-controlled micro-perfusion system. CM18-Tat11 induces irreversible cell-membrane permeabilization at concentrations (≥4 µM) at which CM18 triggers transient pore formation, and Tat11 does not affect membrane integrity. We argue that the addition of the Tat11 module to CM18 is able to trigger a shift in the mechanism of membrane destabilization from “toroidal” to “carpet”, promoting a detergent-like membrane disruption. Collectively, these results rationalize previous observations on CM18-Tat11 delivery properties that we believe can guide the engineering of new modular peptides tailored to specific cargo-delivery applications.

Inhibitory effect of biosynthetic nanoscale peptide Melittin on hepatocellular carcinoma, driven by survivin promoter.

Hepatic resection and orthotopic liver transplantation are the only potentially curative treatments for hepatocellular carcinoma (HCC) but are indicated only in a minority of patients. Biosynthetic nanoscale peptide Melittin (Mel) is postulated to disrupt microbial phospholipid membranes by formation of stable or transient pores. Survivin, a member of the inhibitor of apoptosis family, is transcriptionally upregulated in most malignant tissues but not in normal tissues. It has been reported that the survivin promoter activity is tumor-specific and makes it a good candidate for construction of gene therapy vectors. In the present study, a non-viral vector (pSURV-Mel), encoding Mel gene, was developed to evaluate its anti-tumor effect in HCC cell lines and in vivo in a mouse model of human HCC xenograft tumor. Our results showed that the survivin promoter is specifically activated in tumor cells, and the pSURV-Mel plasmid expressed Mel selectively in tumor cells and also induced cytotoxicity. Moreover, intratumoral Injection of pSURV-Mel significantly suppressed the growth of xenograft tumors. Mechanistically, pSURV-Mel induced cell death by an apoptosis-dependent pathway. All taken together, this study elucidates a relatively safe, highly effective and cancer specific gene therapy strategy for HCC. The mechanisms of non-viral vector-induced cell death which were revealed by this work will shed light on the construction of more powerful vectors for cancer therapy.

Electroporation enhances antimicrobial photodynamic therapy mediated by the hydrophobic photosensitizer, hypericin

The effective transport of photosensitizers (PS) across the membrane and the intracellular accumulation of PS are the most crucial elements in antimicrobial photodynamic therapy (aPDT). However, due to the morphological complexity of Gram-negative bacteria the penetration of PS is limited, especially hydrophobic PS. Electroporation (EP) could increase the effectiveness of aPDT, by promoting the formation of transient pores that enhance the permeability of the bacterial membrane to PS. In this study we evaluated the combination of aPDT mediated by the hydrophobic PS, hypericin and EP (aPDT/EP) against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. These bacteria were exposed to light (590 nm) in the presence of hypericin (4 μM), following electroporation. The results showed that aPDT/EP inactivated 3.67 logs more E. coli and 2.65 logs more S. aureus than aPDT alone. Based on these results we suggest that EP can potentiate the aPDT effect.

Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers

Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.

Mechanism of Polyplex- and Lipoplex-Mediated Delivery of Nucleic Acids: Real-Time Visualization of Transient Membrane Destabilization without Endosomal Lysis

Lipoplexes and polyplexes are widely applied as nonviral gene delivery carriers. Although their efficiencies of transfection are comparable, their mechanisms of delivery, specifically at the level of nucleic acid release from endosomes, are different. Thus, lipoplex-mediated release is proposed to rely on lipid mixing, as occurs between lipoplex and endosomal target membrane, the ensuing membrane destabilization leading to nucleic acid delivery into the cytosol. By contrast, the mechanism by which polyplexes, particularly those displaying a high proton buffering capacity, release their nucleic acid cargo from the endosome, is thought to rely on a so-called "proton sponge effect", in essence an osmotically induced rupturing of the endosomal membrane. However, although a wealth of indirect insight supports both these mechanisms, direct evidence is still lacking. Therefore, to further clarify these mechanisms, we have investigated the interaction of lipo- and polyplexes with HeLa cells by live cell imaging. As monitored over an incubation period of 2 h, our data reveal that in contrast to the involvement of numerous nanocarriers in case of lipoplex-mediated delivery, only a very limited number of polyplexes, that is, as few as one up to four/five nanocarriers per cell, with an average of one/two per cell, contribute to the release of nucleic acids from endosomes and their subsequent accumulation into the nucleus. Notably, in neither case complete rupture of endosomes nor release of intact polyplexes or lipoplexes into the cytosol was observed. Rather, at the time of endosomal escape both the polymer and its genetic payload are separately squirted into the cytoplasm, presumably via (a) local pore(s) within the endosomal membrane. Specifically, an almost instantaneous and complete discharge of nucleic acids and carrier (remnants) from the endosomes is observed. In case of lipoplexes, the data suggest the formation of multiple transient pores over time within the same endosomal membrane, via which the cargo is more gradually transferred into the cytosol.

Deformation and poration of lipid bilayer membranes by cationic nanoparticles

Synthetic lipid bilayers in a giant unilamellar vesicle (GUV) format were used to study potentially harmful interactions between nanoparticles and biomembranes. Confocal fluorescence microscopy shows that 20 nm polystyrene nanoparticles with cationic surfaces adhere strongly to these lipid bilayer membranes. This adhesion is sensitive to the ionic strength of the surrounding medium and is independent of the lipid composition of the membrane, indicating that it is driven by nonspecific electrostatic interactions. Nanoparticles bound to the GUV membrane form extended protrusions that incorporate the membrane lipids. As the membrane material is pulled into these protrusions, the diameter of the GUV shrinks. This process is accompanied by the formation of transient pores in the membrane, as indicated by the leakage of high-molecular weight polymers from the GUV interior. There is an upper limit to the molecular weight of polymer that will leak through these pores; based on this size limit, the pore diameter can be estimated as 18–27 nm. These results suggest that nanoparticle adhesion imposes surface tension on biomembranes via a steric crowding mechanism, leading to poration. The phenomenon is potentially a physiologically relevant mode of interaction between nanoparticles and biomembranes, and may help explain observed plasma membrane permeabilization in the presence of broad classes of nanoparticles.

Microfluidic electro-sonoporation: a multi-modal cell poration methodology through simultaneous application of electric field and ultrasonic wave

A microfluidic device that simultaneously applies the conditions required for microelectroporation and microsonoporation in a flow-through scheme toward high-efficiency and high-throughput molecular delivery into mammalian cells is presented. This multi-modal poration microdevice using simultaneous application of electric field and ultrasonic wave was realized by a three-dimensional (3D) microelectrode scheme where the electrodes function as both electroporation electrodes and cell flow channel so that acoustic wave can be applied perpendicular to the electric field simultaneously to cells flowing through the microfluidic channel. This 3D microelectrode configuration also allows a uniform electric field to be applied while making the device compatible with fluorescent microscopy. It is hypothesized that the simultaneous application of two different fields (electric field and acoustic wave) in perpendicular directions allows formation of transient pores along two axes of the cell membrane at reduced poration intensities, hence maximizing the delivery efficiency while minimizing cell death. The microfluidic electro-sonoporation system was characterized by delivering small molecules into mammalian cells, and showed average poration efficiency of 95.6% and cell viability of 97.3%. This proof of concept result shows that by combining electroporation and sonoporation together, significant improvement in molecule delivery efficiency could be achieved while maintaining high cell viability compared to electroporation or sonoporation alone. The microfluidic electro-sonoporation device presented here is, to the best of our knowledge, the first multi-modal cell poration device using simultaneous application of electric field and ultrasonic wave. This new multi-modal cell poration strategy and system is expected to have broad applications in delivery of small molecule therapeutics and ultimately in large molecule delivery such as gene transfection applications where high delivery efficiency and high viability are crucial.

Antibacterial and leishmanicidal activities of temporin-SHd, a 17-residue long membrane-damaging peptide

Temporins are a family of short antimicrobial peptides (8-17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF(amide)), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins.