Synergistic Biophysical Techniques Reveal Structural Mechanisms of Engineered Cationic Antimicrobial Peptides in Lipid Model Membranes

In the quest for new antibiotics, two novel engineered cationic antimicrobial peptides (eCAPs) have been rationally designed. WLBU2 and D8 (all 8 valines are the d-enantiomer) efficiently kill both Gram-negative and -positive bacteria, but WLBU2 is toxic and D8 nontoxic to eukaryotic cells. We explore protein secondary structure, location of peptides in six lipid model membranes, changes in membrane structure and pore evidence. We suggest that protein secondary structure is not a critical determinant of bactericidal activity, but that membrane thinning and dual location of WLBU2 and D8 in the membrane headgroup and hydrocarbon region may be important. While neither peptide thins the Gram-negative lipopolysaccharide outer membrane model, both locate deep into its hydrocarbon region where they are primed for self-promoted uptake into the periplasm. The partially α-helical secondary structure of WLBU2 in a red blood cell (RBC) membrane model containing 50 % cholesterol, could play a role in destabilizing this RBC membrane model causing pore formation that is not observed with the D8 random coil, which correlates with RBC hemolysis caused by WLBU2 but not by D8.Fil: Heinrich, Frank. University of Carnegie Mellon; Estados UnidosFil: Salyapongse, Aria. University of Carnegie Mellon; Estados UnidosFil: Kumagai, Akari. University of Carnegie Mellon; Estados UnidosFil: Dupuy, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Shukla, Karpur. University of Carnegie Mellon; Estados UnidosFil: Penk, Anja. Universitat Leipzig; AlemaniaFil: Huster, Daniel. Universitat Leipzig; AlemaniaFil: Ernst, Robert K.. University of Maryland; Estados UnidosFil: Pavlova, Anna. Georgia Institute Of Techology. School Of Chemical & Biomolecular Engineering; Estados UnidosFil: Gumbart, James C.. Georgia Institute Of Techology. School Of Chemical & Biomolecular Engineering; Estados UnidosFil: Deslouches, Berthony. University of Pittsburgh; Estados UnidosFil: Di, Y. Peter. University of Pittsburgh; Estados UnidosFil: Tristram-Nagle, Stephanie. University of Carnegie Mellon; Estados Unido

Building membrane nanopores

Membrane nanopores—hollow nanoscale barrels that puncture biological or synthetic membranes—have become powerful tools in chemical- and biosensing, and have achieved notable success in portable DNA sequencing. The pores can be self-assembled from a variety of materials, including proteins, peptides, synthetic organic compounds and, more recently, DNA. But which building material is best for which application, and what is the relationship between pore structure and function? In this Review, I critically compare the characteristics of the different building materials, and explore the influence of the building material on pore structure, dynamics and function. I also discuss the future challenges of developing nanopore technology, and consider what the next-generation of nanopore structures could be and where further practical applications might emerge

Review on bibliography related to antimicrobials

In this report, a bibliographic research has been done in the field of antimicrobials.In this report, a bibliographic research has been done in the field of antimicrobials. Not all antimicrobials have been included, but those that are being subject of matter in the group GBMI in Terrassa, and others of interest. It includes chitosan and other biopolymers. The effect of nanoparticles is of great interest, and in this sense, the effect of Ag nanoparticles and antibiotic nanoparticles (nanobiotics) has been revised. The report focuses on new publications and the antimicrobial effect of peptides has been considered. In particular, the influence of antimicrobials on membranes has deserved much attention and its study using the Langmuir technique, which is of great utility on biomimetic studies. The building up of antimicrobials systems with new techniques (bottom-up approach), as the Layer-by-Layer technique, can also be found in between the bibliography. It has also been considered the antibiofilm effect, and the new ideas on quorem sensing and quorum quenching.Preprin

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications

Bacterial mechanosensitive channels: models for studying mechanosensory transduction

Significance: Sensations of touch and hearing are manifestations of mechanical contact and air pressure acting on touch receptors and hair cells of the inner ear, respectively. In bacteria, osmotic pressure exerts a significant mechanical force on their cellular membrane. Bacteria have evolved mechanosensitive (MS) channels to cope with excessive turgor pressure resulting from a hypo-osmotic shock. MS channel opening allows the expulsion of osmolytes and water, thereby restoring normal cellular turgor and preventing cell lysis. Recent Advances: As biological force-sensing systems, MS channels have been identified as the best examples of membrane proteins coupling molecular dynamics to cellular mechanics. The bacterial MS channel of large conductance (MscL) and MS channel of small conductance (MscS) have been subjected to extensive biophysical, biochemical, genetic, and structural analyses. These studies have established MscL and MscS as model systems for mechanosensory transduction. Critical Issues: In recent years, MS ion channels in mammalian cells have moved into focus of mechanotransduction research, accompanied by an increased awareness of the role they may play in the pathophysiology of diseases, including cardiac hypertrophy, muscular dystrophy, or Xerocytosis. Future Directions: A recent exciting development includes the molecular identification of Piezo proteins, which function as nonselective cation channels in mechanosensory transduction associated with senses of touch and pain. Since research on Piezo channels is very young, applying lessons learned from studies of bacterial MS channels to establishing the mechanism by which the Piezo channels are mechanically activated remains one of the future challenges toward a better understanding of the role that MS channels play in mechanobiology

Using \u3cem\u3eCaulobacter crescentus\u3c/em\u3e as a Model to Probe the Environmental Reactivity of Silver Nanoparticles

Silver nanoparticles (AgNPs) are an increasingly common environmental pollutant with antimicrobial and antibacterial properties. The elucidation of their interaction with cellular membranes and subsequent mechanisms of toxicity are critical areas of research that need to be better understood in order to manage potential adverse environmental effects. This work investigates the interaction of AgNPs with large unilamellar vesicles (LUVs) as a model membrane system. Similar studies conducted by others in this field have largely been conducted with uncoated AgNPs, ignoring the effect of environmental conditions on the formation of an eco-corona on AgNPs. Thus, in this study, the spent medium (SM) of a relevant environmental bacterium, Caulobacter crescentus , was used to form a complex eco-corona. We hypothesized that the eco-corona would mediate the in vivo reactivity of AgNPs, specifically through distinct interactions at the cell membrane. The differential reactivity of AgNPs and SM AgNPs is shown through an in vivo toxicity study using C. crescentus. Model membranes were analyzed using dynamic light scattering (DLS), in which AgNP and LUV size and charge are characterized, and fluorescence anisotropy, where changes to LUV membrane fluidity and dynamics are interrogated. Results of the in vivo study are presented in tandem with model membrane studies in order to correlate toxicity effects seen in vivo with potential mechanisms of reactivity at the cell membrane

Binding of bacterial lipopolysaccharide by the cationic amphiphilic peptide WLBU2 at interfaces

Passage of blood through a sorbent device for removal of bacteria and endotoxin by specific binding with immobilized, membrane-active, bactericidal peptides holds promise for treating severe blood infections. Peptide insertion in the target membrane and stable binding is desirable, while membrane disruption and release of degradation products to the circulating blood is not desirable. Here we describe interactions between bacterial endotoxin (lipopolysaccharide, LPS) and the membrane-active, bactericidal peptides WLBU2 and polymyxin B (PmB). Analysis of the interfacial behavior of mixtures of LPS and peptide using air-water interfacial tensiometry and optical waveguide lightmode spectroscopy strongly suggested insertion and stabilization of intact LPS vesicles by WLBU2, while no such peptide-LPS interactions were evident with PmB. Analysis with dynamic light scattering showed in fact that LPS vesicles appear to undergo peptide-induced destabilization in the presence of PmB. Circular dichroism spectra confirmed that WLBU2, which shows disordered structure in aqueous solution and substantially helical structure in membrane-mimetic environments, is stably located within the LPS membrane in peptide-vesicle mixtures. Interactions between LPS and WLBU2 were also evaluated following immobilization of the peptide at uncoated and polyethylene oxide (PEO)-coated hydrophobic surfaces. PEO layers were prepared by radiolytic grafting of selected PEO-polypropylene oxide (PPO)-PEO triblock surfactants to silanized, hydrophobic surfaces. Immobilization of WLBU2 at the PEO layers was achieved by its noncovalent entrapment among the pendant PEO chains and in separate experiments, its covalent coupling to PEO chains that had been end-activated with pyridyl disulfide groups. Analysis of peptide-LPS interactions using a quartz crystal microbalance with dissipation monitoring showed that upon introduction of LPS suspension to a flow cell housing a surface presenting tethered WLBU2, LPS located at the interface in a fashion irreversible to elution. Circular dichroism spectra recorded for suspensions of LPS and (silanized) hydrophobic silica nanoparticles to which WLBU2-triblock constructs had been adsorbed, confirmed that binding of LPS by tethered WLBU2 is mediated through peptide insertion and conformational change within the LPS membrane. LPS capture by tethered WLBU2 was detected in the presence of fibrinogen as well. However, that outcome is best considered tentative, as it was associated with potentially complex interactions between fibrinogen, LPS, and WLBU2, that remain uncharacterized. In summary, the results of this study strongly suggest that presentation of tethered WLBU2 within a sorbent device will enable the capture of endotoxin from suspension without reintroduction of degradation products to the circulating stream. Thus, they provide a rationale for hypotheses to drive further development of perfusion for the treatment of severe blood infections.Keywords: OWLS, Cationic Amphiphilic Peptide, Endotoxin, Peptide antibiotics, WLBU2, Endotoxins, Hemoperfusion, Bactericides, QCM-