Henri G. Franquelim

Escherichia coli cell surface perturbation and disruption induced by antimicrobial peptides, BP100 and pepR

The potential of antimicrobial peptides (AMPs) as an alternative to conventional therapies is well recognized. Insights into the biological and biophysical properties of AMPs are thus key to understanding their mode of action. In this study, the mechanisms adopted by two AMPs in disrupting the Gram-negative Escherichia coli bacterial envelope were explored. BP100 is a short cecropin A-melittin hybrid peptide known to inhibit the growth of phytopathogenic Gram-negative bacteria. pepR, on the other hand, is a novel AMP derived from the dengue virus capsid protein. Both BP100 and pepR were found to inhibit the growth of E. coli at micromolar concentrations. Zeta potential measurements of E. coli incubated with increasing peptide concentrations allowed for the establishment of a correlation between the minimal inhibitory concentration (MIC) of each AMP and membrane surface charge neutralization. While a neutralization-mediated killing mechanism adopted by either AMP is not necessarily implied, the hypothesis that surface neutralization occurs close to MIC values was confirmed. Atomic force microscopy (AFM) was then employed to visualize the structural effect of the interaction of each AMP with the E. coli cell envelope. At their MICs, BP100 and pepR progressively destroyed the bacterial envelope, with extensive damage already occurring 2 h after peptide addition to the bacteria. A similar effect was observed for each AMP in the concentration-dependent studies. At peptide concentrations below MIC values, only minor disruptions of the bacterial surface occurred.

Extracellular Alpha-Synuclein Oligomers Modulate Synaptic Transmission and Impair LTP Via NMDA-Receptor Activation

Parkinsons disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of α-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.

Decoding the Membrane Activity of the Cyclotide Kalata B1 THE IMPORTANCE OF PHOSPHATIDYLETHANOLAMINE PHOSPHOLIPIDS AND LIPID ORGANIZATION ON HEMOLYTIC AND ANTI-HIV ACTIVITIES

Cyclotides, a large family of cyclic peptides from plants, have a broad range of biological activities, including insecticidal, cytotoxic, and anti-HIV activities. In all of these activities, cell membranes seem likely to be the primary target for cyclotides. However, the mechanistic role of lipid membranes in the activity of cyclotides remains unclear. To determine the role of lipid organization in the activity of the prototypic cyclotide, kalata B1 (kB1), and synthetic analogs, their bioactivities and affinities for model membranes were evaluated. We found that the bioactivity of kB1 is dependent on the lipid composition of target cell membranes. In particular, the activity of kB1 requires specific interactions with phospholipids containing phosphatidylethanolamine (PE) headgroups but is further modulated by nonspecific peptide-lipid hydrophobic interactions, which are favored in raft-like membranes. Negatively charged phospholipids do not favor high kB1 affinity. This lipid selectivity explains trends in antimicrobial and hemolytic activities of kB1; it does not target bacterial cell walls, which are negatively charged and lacking PE-phospholipids but can insert in the membranes of red blood cells, which have a low PE content and raft domains in their outer layer. We further show that the anti-HIV activity of kB1 is the result of its ability to target and disrupt the membranes of HIV particles, which are raft-like membranes very rich in PE-phospholipids.

Design and characterization of novel antimicrobial peptides, R-BP100 and RW-BP100, with activity against Gram-negative and Gram-positive bacteria.

BP100 is a short cationic antimicrobial peptide with a mechanism of action dependent on peptide-lipid interactions and microbial surface charge neutralization. Although active against Gram-negative bacteria, BP100 is inactive against Gram-positive bacteria. In this study we report two newly designed BP100 analogues, RW-BP100 and R-BP100 that have the Tyr residue replaced with a Trp and/or the Lys residues replaced with an Arg. The new analogues in addition to being active against Gram-negative bacteria, possess activity against all tested Gram-positive bacteria. Mechanistic studies using atomic force microscopy, surface plasmon resonance and fluorescence methodologies reveal that the antibacterial efficiency follows the affinity for bacterial membrane. The studies suggest that the activity of BP100 and its analogues against Gram-negative bacteria is mainly driven by electrostatic interactions with the lipopolysaccharide layer and is followed by binding to and disruption of the inner membrane, whereas activity against Gram-positive bacteria, in addition to electrostatic attraction to the exposed lipoteichoic acids, requires an ability to more deeply insert in the membrane environment, which is favoured with Arg residues and is facilitated in the presence of a Trp residue. Knowledge on the mechanism of action of these antimicrobial peptides provides information that assists in the design of antimicrobials with higher efficacy and broader spectra of action, but also on the design of peptides with higher specificity if required.

Quantitative assessment of peptide-lipid interactions. Ubiquitous fluorescence methodologies.

Peptide-membrane interactions have been gaining increased relevance, mainly in biomedical investigation, as the potential of the natural, nature-based and synthetic peptides as new drugs or drug candidates also expands. These peptides must face the cell membrane when they interfere with or participate in intracellular processes. Additionally, several peptide drugs and drug leads actions occur at the membrane level (e.g., antimicrobial peptides, cell-penetrating peptides and enveloped viruses membrane fusion inhibitors). Here we explore fluorescence spectroscopy methods that can be used to monitor such interactions. Two main approaches are considered, centered either on the peptide or on the membrane. On the first, we consider mainly the methodologies based on the intrinsic fluorescence of the aminoacid residues tryptophan and tyrosine. Regarding membrane-centric approaches, we review methods based on lipophilic probes sensitive to membrane potentials. The use of fluorescence constitutes a simple and sensitive method to measure these events. Unraveling the molecular mechanisms that govern these interactions can unlock the key to understand specific biological processes involving natural peptides or to optimize the action of a peptide drug.

Sifuvirtide screens rigid membrane surfaces. Establishment of a correlation between efficacy and membrane domain selectivity among HIV fusion inhibitor peptides

Sifuvirtide, a 36 amino acid negatively charged peptide, is a novel and promising HIV fusion inhibitor, presently in clinical trials. Because of the aromatic amino acid residues of the peptide, its behavior in aqueous solution and the interaction with lipid-membrane model systems (large unilammelar vesicles) were studied by using mainly fluorescence spectroscopy techniques (both steady-state and time-resolved). No significant aggregation of the peptide was observed with aqueous solution. Various biological and nonbiological lipid-membrane compositions were analyzed, and atomic force microscopy was used to visualize phase separation in several of those mixtures. Results showed no significant interaction of the peptide, neither with zwitterionic fluid lipid membranes (liquid-disordered phase), nor with cholesterol-rich membranes (liquid-ordered phase). However, significant partitioning was observed with the positively charged lipid models (K(p) = (2.2 +/- 0.3) x 10(3)), serving as a positive control. Fluorescence quenching using Förster resonance acrylamide and lipophilic probes was carried out to study the location of the peptide in the membrane models. In the gel-phase DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) membrane model, an adsorption of the peptide at the surface of these membranes was observed and confirmed by using Förster resonance energy-transfer experiments. These results indicate a targeting of the peptide to gel-phase domains relatively to liquid-disordered or liquid-ordered phase domains. This larger affinity and selectivity toward the more rigid areas of the membranes, where most of the receptors are found, or to viral membrane, may help explain the improved clinical efficiency of sifuvirtide, by providing a local increased concentration of the peptide at the fusion site.

Antimicrobial protein rBPI21-induced surface changes on Gram-negative and Gram-positive bacteria

UNLABELLED New classes of antibiotics, such as antimicrobial peptides or proteins (AMPs), are crucial to deal with threatening bacterial diseases. rBPI21 is an AMP based on the human neutrophil BPI protein, with potential clinical use against meningitis. We studied the membrane perturbations promoted by rBPI21 on Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Its interaction with bacteria was also studied in the presence of lipopolysaccharide (LPS), rBPI21 major ligand. Flow cytometry analysis of both bacteria shows that rBPI21 induces membrane depolarization. rBPI21 increases the negative surface charge of both bacteria toward positive values, as shown by zeta-potential measurements. This is followed by surface perturbations, culminating in cell lysis, as visualized by atomic force microscopy (AFM). Force spectroscopy measurements show that soluble LPS decreases the interaction of rBPI21 with bacteria, especially with S. aureus. This suggests that the rBPI21 LPS-binding pocket may also participate on the binding to Gram-positive bacteria. FROM THE CLINICAL EDITOR In this study, rBPI21, an antimicrobial protein based on the human neutrophil BPI protein, with potential clinical use against meningitis, is analyzed with multiple tools including zeta-potential measurements, clarifying its actions on E. coli and S. aureus. Since antimicrobial peptides are potentially important new additions to antibiotic regimens, studies like this represent important cornerstones in efficiency and mechanism of action testing of these new approaches.

The Antimicrobial Activity of Sub3 is Dependent on Membrane Binding and Cell-Penetrating Ability

Because of their high activity against microorganisms and low cytotoxicity, cationic antimicrobial peptides (AMPs) have been explored as the next generation of antibiotics. Although they have common structural features, the modes of action of AMPs are extensively debated, and a single mechanism does not explain the activity of all AMPs reported so far. Here we investigated the mechanism of action of Sub3, an AMP previously designed and optimised from high‐throughput screening with bactenecin as the template. Sub3 has potent activity against Gram‐negative and Gram‐positive bacteria as well as against fungi, but its mechanism of action has remained elusive. By using AFM imaging, ζ potential, flow cytometry and fluorescence methodologies with model membranes and bacterial cells, we found that, although the mechanism of action involves membrane targeting, Sub3 internalises inside bacteria at lethal concentrations without permeabilising the membrane, thus suggesting that its antimicrobial activity might involve both the membrane and intracellular targets. In addition, we found that Sub3 can be internalised into human cells without being toxic. As some bacteria are able to survive intracellularly and consequently evade host defences and antibiotic treatment, our findings suggest that Sub3 could be useful as an intracellular antimicrobial agent for infections that are notoriously difficult to treat.

Amphipathic DNA Origami Nanoparticles to Scaffold and Deform Lipid Membrane Vesicles

We report a synthetic biology-inspired approach for the engineering of amphipathic DNA origami structures as membrane-scaffolding tools. The structures have a flat membrane-binding interface decorated with cholesterol-derived anchors. Sticky oligonucleotide overhangs on their side facets enable lateral interactions leading to the formation of ordered arrays on the membrane. Such a tight and regular arrangement makes our DNA origami capable of deforming free-standing lipid membranes, mimicking the biological activity of coat-forming proteins, for example, from the I-/F-BAR family.

Anti-HIV-1 antibodies 2F5 and 4E10 interact differently with lipids to bind their epitopes

Objectives:2F5 and 4E10 are two broadly neutralizing monoclonal antibodies (mAbs) targeting the membrane proximal external region (MPER) of HIV-1 gp41 envelope protein. This region, which contacts the viral membrane, is highly conserved and has been regarded as a promising target for vaccine development. We aimed to clarify the basis of 2F5 and 4E10 molecular interactions with epitope cores in MPER and lipid bilayers. Design:Microscopy-based approaches were used to infer and quantify the effects of both mAbs on membranes, in the presence and absence of the epitope cores. Supported lipid bilayers (SLBs), with and without phase separation, were used as membrane models. Fluorescent-labeled and nonlabeled MPER-derived peptides containing both 2F5 and 4E10 epitopes were used. Methods:mAbs 2F5 and 4E10 membrane interactions, in the presence or absence of MPER-derived peptides, were evaluated by combined atomic force and confocal microscopies. Results:Both mAbs form lipid-segregated aggregates on SLBs and do not induce other significant membrane perturbations. Furthermore, the affinity of MPER toward membranes is differently affected by both mAbs and correlates with the mAbs–epitope core lipid interactions. 2F5 is able to dock the MPER peptide on the membrane, whereas 4E10 extracts the MPER from the lipid bilayer. Conclusion:The results reveal the molecular details underneath 2F5/4E10 membrane-epitope binding and a model is proposed to explain the differential mAbs neutralization efficacies, which relates to the exposure of the epitopes in the lipid bilayers and the role of the lipids in mAb–epitope binding.