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

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

Molecular mechanisms of membrane targeting antibiotics.

The bacterial membrane provides a target for antimicrobial peptides. There are two groups of bacteria that have characteristically different surface membranes. One is the Gram-negative bacteria that have an outer membrane rich in lipopolysaccharide. Several antimicrobials have been found to inhibit the synthesis of this lipid, and it is expected that more will be developed. In addition, antimicrobial peptides can bind to the outer membrane of Gram-negative bacteria and block passage of solutes between the periplasm and the cell exterior, resulting in bacterial toxicity. In Gram-positive bacteria, the major bacterial lipid component, phosphatidylglycerol can be chemically modified by bacterial enzymes to convert the lipid from anionic to cationic or zwitterionic form. This process leads to increased levels of resistance of the bacteria against polycationic antimicrobial agents. Inhibitors of this enzyme would provide protection against the development of bacterial resistance. There are antimicrobial agents that directly target a component of bacterial cytoplasmic membranes that can act on both Gram-negative as well as Gram-positive bacteria. Many of these are cyclic peptides with a rigid binding site capable of binding a lipid component. This binding targets antimicrobial agents to bacteria, rather than being toxic to host cells. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

High-quality 3D structures shine light on antibacterial, anti-biofilm and antiviral activities of human cathelicidin LL-37 and its fragments.

Host defense antimicrobial peptides are key components of human innate immunity that plays an indispensible role in human health. While there are multiple copies of cathelicidin genes in horses, cattle, pigs, and sheep, only one cathelicidin gene is found in humans. Interestingly, this single cathelicidin gene can be processed into different forms of antimicrobial peptides. LL-37, the most commonly studied form, is not only antimicrobial but also possesses other functional roles such as chemotaxis, apoptosis, wound healing, immune modulation, and cancer metastasis. This article reviews recent advances made in structural and biophysical studies of human LL-37 and its fragments, which serve as a basis to understand their antibacterial, anti-biofilm and antiviral activities. High-quality structures were made possible by using improved 2D NMR methods for peptide fragments and 3D NMR spectroscopy for intact LL-37. The two hydrophobic domains in the long amphipathic helix (residues 2-31) of LL-37 separated by a hydrophilic residue serine 9 explain its cooperative binding to bacterial lipopolysaccharides (LPS). Both aromatic rings (F5, F6, F17, and F27) and interfacial basic amino acids of LL-37 directly interact with anionic phosphatidylglycerols (PG). Although the peptide sequences reported in the literature vary slightly, there is a consensus that the central helix of LL-37 is essential for disrupting superbugs (e.g., MRSA), bacterial biofilms, and viruses such as human immunodeficiency virus 1 (HIV-1) and respiratory syncytial virus (RSV). In the central helix, the central arginine R23 is of particular importance in binding to bacterial membranes or DNA. Mapping the functional roles of the cationic amino acids of the major antimicrobial region of LL-37 provides a basis for designing antimicrobial peptides with desired properties. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition/amino acid consensus site for membrane association.

Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its ß2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC(336) ((333)LEEYSKR(339)) is highly conserved among RTX toxins, whereas CRAC(503) ((501)VDYLK(505)) is unique to LtxA. A peptide corresponding to CRAC(336) inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC(336) and CRAC(503) bind cholesterol, only CRAC(336) competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC(336) site was essential for LtxA cytotoxicity. The conservation of CRAC(336) among RTX toxins suggests that this mechanism may be conserved among RTX toxins.

Dual function of mitochondrial Nm23-H4 protein in phosphotransfer and intermembrane lipid transfer: a cardiolipin-dependent switch.

The nucleoside diphosphate kinase Nm23-H4/NDPK-D forms symmetrical hexameric complexes in the mitochondrial intermembrane space with phosphotransfer activity using mitochondrial ATP to regenerate nucleoside triphosphates. We demonstrate the complex formation between Nm23-H4 and mitochondrial GTPase OPA1 in rat liver, suggesting its involvement in local and direct GTP delivery. Similar to OPA1, Nm23-H4 is further known to strongly bind in vitro to anionic phospholipids, mainly cardiolipin, and in vivo to the inner mitochondrial membrane. We show here that such protein-lipid complexes inhibit nucleoside diphosphate kinase activity but are necessary for another function of Nm23-H4, selective intermembrane lipid transfer. Mitochondrial lipid distribution was analyzed by liquid chromatography-mass spectrometry using HeLa cells expressing either wild-type Nm23-H4 or a membrane binding-deficient mutant at a site predicted based on molecular modeling to be crucial for cardiolipin binding and transfer mechanism. We found that wild type, but not the mutant enzyme, selectively increased the content of cardiolipin in the outer mitochondrial membrane, but the distribution of other more abundant phospholipids (e.g. phosphatidylcholine) remained unchanged. HeLa cells expressing the wild-type enzyme showed increased accumulation of Bax in mitochondria and were sensitized to rotenone-induced apoptosis as revealed by stimulated release of cytochrome c into the cytosol, elevated caspase 3/7 activity, and increased annexin V binding. Based on these data and molecular modeling, we propose that Nm23-H4 acts as a lipid-dependent mitochondrial switch with dual function in phosphotransfer serving local GTP supply and cardiolipin transfer for apoptotic signaling and putative other functions.

The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains.

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.

5-(Perylen-3-yl)ethynyl-arabino-uridine (aUY11), an arabino-based rigid amphipathic fusion inhibitor, targets virion envelope lipids to inhibit fusion of influenza virus, hepatitis C virus, and other enveloped viruses.

Entry of enveloped viruses requires fusion of viral and cellular membranes. Fusion requires the formation of an intermediate stalk structure, in which only the outer leaflets are fused. The stalk structure, in turn, requires the lipid bilayer of the envelope to bend into negative curvature. This process is inhibited by enrichment in the outer leaflet of lipids with larger polar headgroups, which favor positive curvature. Accordingly, phospholipids with such shape inhibit viral fusion. We previously identified a compound, 5-(perylen-3-yl)ethynyl-2'-deoxy-uridine (dUY11), with overall shape and amphipathicity similar to those of these phospholipids. dUY11 inhibited the formation of the negative curvature necessary for stalk formation and the fusion of a model enveloped virus, vesicular stomatitis virus (VSV). We proposed that dUY11 acted by biophysical mechanisms as a result of its shape and amphipathicity. To test this model, we have now characterized the mechanisms against influenza virus and HCV of 5-(perylen-3-yl)ethynyl-arabino-uridine (aUY11), which has shape and amphipathicity similar to those of dUY11 but contains an arabino-nucleoside. aUY11 interacted with envelope lipids to inhibit the infectivity of influenza virus, hepatitis C virus (HCV), herpes simplex virus 1 and 2 (HSV-1/2), and other enveloped viruses. It specifically inhibited the fusion of influenza virus, HCV, VSV, and even protein-free liposomes to cells. Furthermore, aUY11 inhibited the formation of negative curvature in model lipid bilayers. In summary, the arabino-derived aUY11 and the deoxy-derived dUY11 act by the same antiviral mechanisms against several enveloped but otherwise unrelated viruses. Therefore, chemically unrelated compounds of appropriate shape and amphipathicity target virion envelope lipids to inhibit formation of the negative curvature required for fusion, inhibiting infectivity by biophysical, not biochemical, mechanisms.

Sensitization of gram-negative bacteria by targeting the membrane potential.

Toward generating new tools for fighting multidrug-resistant (MDR) bacteria, we assessed the ability of a membrane-active peptide to sensitize gram-negative bacteria to various antibiotics. The mechanism for affecting inner and/or outer membrane functions was assessed by complementary biophysical methods (SPR, DSC, ITC). The implication of efflux pumps was examined using Acr-AB mutants, as tested with representative antibiotics, host defense peptides, and synthetic mimics. The ability to affect disease course systemically was compared for a single therapy and combination therapy, using the mouse thigh-infection model. The data show that potent antibiotic action can be provoked in vitro and in vivo, by a treatment combining two antibacterial compounds whose individual inefficiency against gram-negative bacteria stems from their efflux. Thus, at subminimal inhibitory concentrations, the lipopeptide-like sequence, N(α)(ω7)dodecenoyl-lysyl-[lysyl-aminododecanoyl-lysyl]-amide (designated C12(ω7)K-ß12), has, nonetheless, rapidly achieved a transient membrane depolarization, which deprived bacteria of the proton-motive force required for active efflux. Consequently, bacteria became significantly sensitive to intracellular targeting antibiotics. Collectively, these findings suggest a potentially useful approach for expanding the antibiotics sensitivity spectrum of MDR gram-negative bacteria to include efflux substrates.

Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization.

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while (31) P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization.

Rigid amphipathic fusion inhibitors, small molecule antiviral compounds against enveloped viruses.

Antiviral drugs targeting viral proteins often result in prompt selection for resistance. Moreover, the number of viral targets is limited. Novel antiviral targets are therefore needed. The unique characteristics of fusion between virion envelopes and cell membranes may provide such targets. Like all fusing bilayers, viral envelopes locally adopt hourglass-shaped stalks during the initial stages of fusion, a process that requires local negative membrane curvature. Unlike cellular vesicles, however, viral envelopes do not redistribute lipids between leaflets, can only use the energy released by virion proteins, and fuse to the extracellular leaflets of cell membranes. Enrichment in phospholipids with hydrophilic heads larger than their hydrophobic tails in the convex outer leaflet of vesicles favors positive curvature, therefore increasing the activation energy barrier for fusion. Such phospholipids can increase the activation barrier beyond the energy provided by virion proteins, thereby inhibiting viral fusion. However, phospholipids are not pharmacologically useful. We show here that a family of synthetic rigid amphiphiles of shape similar to such phospholipids, RAFIs (rigid amphipathic fusion inhibitors), inhibit the infectivity of several otherwise unrelated enveloped viruses, including hepatitis C and HSV-1 and -2 (lowest apparent IC(50) 48 nM), with no cytotoxic or cytostatic effects (selectivity index > 3,000) by inhibiting the increased negative curvature required for the initial stages of fusion.

The final conformation of the complete ectodomain of the HA2 subunit of influenza hemagglutinin can by itself drive low pH-dependent fusion.

One of the best characterized fusion proteins, the influenza virus hemagglutinin (HA), mediates fusion between the viral envelope and the endosomal membrane during viral entry into the cell. In the initial conformation of HA, its fusogenic subunit, the transmembrane protein HA2, is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. Acidification in the endosome triggers HA2 refolding toward the final lowest energy conformation. Is the fusion process driven by this final conformation or, as often suggested, by the energy released by protein restructuring? Here we explored structural properties as well as the fusogenic activity of the full sized trimeric HA2(1-185) (here called HA2*) that presents the final conformation of the HA2 ectodomain. We found HA2* to mediate fusion between lipid bilayers and between biological membranes in a low pH-dependent manner. Two mutations known to inhibit HA-mediated fusion strongly inhibited the fusogenic activity of HA2*. At surface densities similar to those of HA in the influenza virus particle, HA2* formed small fusion pores but did not expand them. Our results confirm that the HA1 subunit responsible for receptor binding as well as the transmembrane and cytosolic domains of HA2 is not required for fusion pore opening and substantiate the hypothesis that the final form of HA2 is more important for fusion than the conformational change that generates this form.

Phosphatidylethanolamines modified by γ-ketoaldehyde (γKA) induce endoplasmic reticulum stress and endothelial activation.

Peroxidation of plasma lipoproteins has been implicated in the endothelial cell activation and monocyte adhesion that initiate atherosclerosis, but the exact mechanisms underlying this activation remain unclear. Lipid peroxidation generates lipid aldehydes, including the γ-ketoaldehydes (γKA), also termed isoketals or isolevuglandins, that readily modify the amine headgroup of phosphatidylethanolamine (PE). We hypothesized that aldehyde modification of PE could mediate some of the proinflammatory effects of lipid peroxidation. We found that PE modified by γKA (γKA-PE) induced THP-1 monocyte adhesion to human umbilical cord endothelial cells. γKA-PE also induced expression of adhesion molecules and increased MCP-1 and IL-8 mRNA in human umbilical cord endothelial cells. To determine the structural requirements for γKA-PE activity, we tested several related compounds. PE modified by 4-oxo-pentanal induced THP-1 adhesion, but N-glutaroyl-PE and C(18:0)N-acyl-PE did not, suggesting that an N-pyrrole moiety was essential for cellular activity. As the N-pyrrole headgroup might distort the membrane, we tested the effect of the pyrrole-PEs on membrane parameters. γKA-PE and 4-oxo-pentanal significantly reduced the temperature for the liquid crystalline to hexagonal phase transition in artificial bilayers, suggesting that these pyrrole-PE markedly altered membrane curvature. Additionally, fluorescently labeled γKA-PE rapidly internalized to the endoplasmic reticulum (ER); γKA-PE induced C/EBP homologous protein CHOP and BiP expression and p38 MAPK activity, and inhibitors of ER stress reduced γKA-PE-induced C/EBP homologous protein CHOP and BiP expression as well as EC activation, consistent with γKA-PE inducing ER stress responses that have been previously linked to inflammatory chemokine expression. Thus, γKA-PE is a potential mediator of the inflammation induced by lipid peroxidation.

Antibacterial properties of an oligo-acyl-lysyl hexamer targeting Gram-negative species.

Toward developing new tools for fighting resistance to antibiotics, we investigated the antibacterial properties of a new decanoyl-based oligo-acyl-lysyl (OAK) hexamer, aminododecanoyl-lysyl-[aminodecanoyl-lysyl](5) (α(12)-5α(10)). The OAK exhibited preferential activity against Gram-negative bacteria (GNB), as determined using 36 strains, including diverse species, with an MIC(90) of 6.2 µM. The OAK's bactericidal mode of action was associated with rapid membrane depolarization and cell permeabilization, suggesting that the inner membrane was the primary target, whereas the observed binding affinity to lipoteichoic acid suggested that inefficacy against Gram-positive species resulted from a cell wall interaction preventing α(12)-5α(10) from reaching internal targets. Interestingly, perturbation of the inner membrane structure and function was preserved at sub-MIC values. This prompted us to assess the OAK's effect on the proton motive force-dependent efflux pump AcrAB-TolC, implicated in the low sensitivity of GNB to various antibiotics, including erythromycin. We found that under sub-MIC conditions, wild-type Escherichia coli was significantly more sensitive to erythromycin (the MIC dropped by >10-fold), unlike its acr-deletion mutant. Collectively, the data suggest a useful approach for treating GNB infections through overcoming antibiotic efflux.

Decoding the functional roles of cationic side chains of the major antimicrobial region of human cathelicidin LL-37.

Human cathelicidin LL-37 is a critical cationic antimicrobial peptide for host defense against infection, immune modulation, and wound healing. This article elucidates the functional roles of the cationic side chains of the major antimicrobial region of LL-37, corresponding to residues 17 to 32 (designated GF-17). Antimicrobial assays, killing kinetics studies, and vesicle leakage experiments all indicate that a conversion of lysines to arginines affected the ability of the peptide to kill the Gram-positive Staphylococcus aureus strain USA300. Alanine scanning experiments show that S. aureus is less sensitive than Escherichia coli to a single cationic residue mutation of GF-17. Among the five cationic residues, R23 appears to be somewhat important in killing S. aureus. However, R23 and K25 of GF-17 are of prime importance in killing the Gram-negative organism E. coli. In particular, R23 is essential for (i) rapid recognition, (ii) permeation of the E. coli outer membrane, (iii) clustering of anionic lipids in a membrane system mimicking the E. coli inner membrane, and (iv) membrane disruption. Bacterial aggregation (i.e., rapid recognition via charge neutralization) is the first step of the peptide action. Structurally, R23 is located in the interface (i.e., the first action layer), a situation ideal for the interactions listed above. In contrast, residues K18, R19, and R29 are on the hydrophilic surface of the amphipathic helix and play only a secondary role. Mapping of the functional spectrum of cationic residues of GF-17 provides a solid basis for engineering bacterium-specific antimicrobials using this highly potent template.

Soluble respiratory syncytial virus fusion protein in the fully cleaved, pretriggered state is triggered by exposure to low-molarity buffer.

The paramyxovirus fusion (F) glycoprotein is anchored in the virion membrane in a metastable, pretriggered form. Once triggered, the F protein undergoes a dramatic conformational extension that inserts its hydrophobic fusion peptide into the target cell membrane, then folds back on itself to bring the membranes together and initiate fusion. Unlike most other paramyxoviruses, the respiratory syncytial virus (RSV) F protein alone is sufficient to mediate membrane fusion and virus infection. To study the triggering mechanism of the RSV F protein, we have generated a soluble F (sF) protein by replacing the transmembrane and cytoplasmic tail domains with a 6His tag. The sF protein is secreted efficiently from 293T cells in a fully cleaved form. It is recognized by neutralizing monoclonal antibodies, appears spherical by electron microscopic analysis, and is not aggregated, all consistent with a native, pretriggered trimer. The sF protein was purified on a Ni(+2) column and eluted with 50 mM phosphate buffer containing 500 mM NaCl and 250 mM imidazole. Dialysis against 10 mM buffer caused the sF protein to trigger, forming "hat pin"-shaped molecules that aggregated as rosettes, characteristic of the posttriggered form. Further dialysis experiments indicated that the efficiency of triggering correlated well with the reduction of buffer molarity. Reduction of buffer molarity by dilution also resulted in exposure of the fusion peptide, as detected by liposome association, confirming sF protein triggering. Mutation of the furin cleavage site adjacent to the fusion peptide prevented liposome association, further confirming that association is via the fusion peptide.

Functional studies of cochleate assemblies of an oligo-acyl-lysyl with lipid mixtures for combating bacterial multidrug resistance.

The cationic antimicrobial oligo-acyl-lysyls (OAKs) interact with lipid mixtures mimicking the composition of bacterial cytoplasmic membranes. We have reported the ability of one such OAK, C(12)K-7α(8), to cluster anionic lipids and to promote a structural change with lipid bilayers to form rolled cylindrical structures or cochleates, without requiring divalent cations for their assembly. These assemblies can be exploited for drug delivery, permitting their synergistic use with antibiotics in systemic therapy to increase efficacy and reduce toxicity. Our previous studies of the biophysical properties of these systems led us to select mixtures with the goal of optimizing their potential for enhancing effectiveness in combating bacterial multidrug resistance. Here, we further investigate the properties of such mixtures that result in enhanced in vivo activity. The role of erythromycin in the assembly of cochleates with OAK in the gel and the liquid crystalline states were assessed, as well as the encapsulation efficiency of the systems chosen. In addition, we found that erythromycin did not undermine the ability of OAKs to induce fusion of vesicles, fusion being an essential component of cochleate formation. The in vivo activity of the new assemblies tested resulted in higher survival rates of animals infected with multidrug resistant bacteria.

Lipid complexes with cationic peptides and OAKs; their role in antimicrobial action and in the delivery of antimicrobial agents.

Antimicrobial agents are toxic to bacteria by a variety of mechanisms. One mechanism that is very dependent on the lipid composition of the bacterial membrane is the clustering of anionic lipid by cationic antimicrobial agents. Certain species of oligo-acyl-lysine (OAK) antimicrobial agents are particularly effective in clustering anionic lipids in mixtures mimicking the composition of bacterial membranes. The clustering of anionic lipids by certain cationic antimicrobial agents contributes to the anti-bacterial action of these agents. Bacterial membrane lipids are a determining factor, resulting in some species of bacteria being more susceptible than others. In addition, lipids can be used to increase the effectiveness of antimicrobial agents when administered in vivo. Therefore, we review some of the structures in which lipid mixtures can assemble, to more effectively be utilized as antimicrobial delivery systems. We describe in more detail the complexes formed between mixtures of lipids mimicking bacterial membranes and an OAK and their usefulness in synergizing with antibiotics to overcome bacterial multidrug resistance.

Lipid clustering by three homologous arginine-rich antimicrobial peptides is insensitive to amino acid arrangement and induced secondary structure.

Three Arg-rich nonapeptides, containing the same amino acid composition but different sequences, PFWRIRIRR-amide (PR-9), RRPFWIIRR-amide (RR-9) and PRFRWRIRI-amide (PI-9), are able to induce segregation of anionic lipids from zwitterionic lipids, as shown by changes in the phase transition properties of lipid mixtures detected by differential scanning calorimetry and freeze fracture electron microscopy. The relative Minimal Inhibitory Concentration (MIC) of these three peptides against several strains of Gram positive bacteria correlated well with the extent to which the lipid composition of the bacterial membrane facilitated peptide-induced clustering of anionic lipids. The lower activity of these three peptides against Gram negative bacteria could be explained by the retention of these peptides in the LPS layer. The membrane morphologies produced by PR-9 as well as by a cathelicidin fragment, KR-12 that had previously been shown to induce anionic lipid clustering, was directly visualized using freeze fracture electron microscopy. This work shows the insensitivity of phase segregation to the specific arrangement of the cationic charges in the peptide sequence as well as to their tendency to form different secondary structures. It also establishes the role of anionic lipid clustering in the presence of zwitterionic lipids in determining antimicrobial selectivity.

Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion.

BACKGROUND: We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear. RESULTS: We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization. CONCLUSIONS: Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.

Bacterial membrane lipids in the action of antimicrobial agents.

Many antimicrobial agents that target bacteria are cationic and can interact with the anionic lipid components that are exposed on the bacterial membrane. Bacteria vary widely in the nature of the major lipid components that are in the cell membrane. Those bacteria with both anionic as well as zwitterionic or neutral lipids can be induced to form domains in the presence of antimicrobial peptides possessing several cationic charges. This segregation of anionic and zwitterionic lipids into domains can result in the arrest of cell growth or in cell death. Such agents are generally more toxic to Gram-negative bacteria, than to Gram-positive ones. These findings emphasize the importance of the lipid composition of bacterial membranes in determining the susceptibility of the organism to the action of certain antimicrobial agents.