Lipidated DNA Nanostructures Target and Rupture Bacterial Membranes.

Chemistry has the power to endow supramolecular nanostructures with new biomedically relevant functions. Here it is reported that DNA nanostructures modified with cholesterol tags disrupt bacterial membranes to cause microbial cell death. The lipidated DNA nanostructures bind more readily to cholesterol-free bacterial membranes than to cholesterol-rich, eukaryotic membranes. These highly negatively charged, lipidated DNA nanostructures cause bacterial cell death by rupturing membranes. Strikingly, killing is mediated by clusters of barrel-shaped nanostructures that adhere to the membrane without the involvement of expected bilayer-puncturing barrels. These DNA nanomaterials may inspire the development of polymeric or small-molecule antibacterial agents that mimic the principles of selective binding and rupturing to help combat antimicrobial resistance.

Phase separation in the outer membrane of Escherichia coli.

Gram-negative bacteria are surrounded by a protective outer membrane (OM) with phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet. The OM is also populated with many ß-barrel outer-membrane proteins (OMPs), some of which have been shown to cluster into supramolecular assemblies. However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane. Here, we reveal how the OM is organized from molecular to cellular length scales, using atomic force microscopy to visualize the OM of live bacteria, including engineered Escherichia coli strains and complemented by specific labeling of abundant OMPs. We find that a predominant OMP in the E. coli OM, the porin OmpF, forms a near-static network across the surface, which is interspersed with barren patches of LPS that grow and merge with other patches during cell elongation. Embedded within the porin network is OmpA, which forms noncovalent interactions to the underlying cell wall. When the OM is destabilized by mislocalization of phospholipids to the outer leaflet, a new phase appears, correlating with bacterial sensitivity to harsh environments. We conclude that the OM is a mosaic of phase-separated LPS-rich and OMP-rich regions, the maintenance of which is essential to the integrity of the membrane and hence to the lifestyle of a gram-negative bacterium.

Measuring Thousands of Single-Vesicle Leakage Events Reveals the Mode of Action of Antimicrobial Peptides.

Host defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a detailed understanding of their structure-activity relationships. However, classical characterization assays often lack the ability to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quantitative long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resolution. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concentrations and related the observed modes to the molecular activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner and for assigning specific modes of action to the corresponding antimicrobial mechanisms.

Investigating Membrane-Mediated Antimicrobial Peptide Interactions with Synchrotron Radiation Far-Infrared Spectroscopy.

Synchrotron radiation-based Fourier transform infrared spectroscopy enables access to vibrational information from mid over far infrared to even terahertz domains. This information may prove critical for the elucidation of fundamental bio-molecular phenomena including folding-mediated innate host defence mechanisms. Antimicrobial peptides (AMPs) represent one of such phenomena. These are major effector molecules of the innate immune system, which favour attack on microbial membranes. AMPs recognise and bind to the membranes whereupon they assemble into pores or channels destabilising the membranes leading to cell death. However, specific molecular interactions responsible for antimicrobial activities have yet to be fully understood. Herein we probe such interactions by assessing molecular specific variations in the near-THz 400-40 cm-1 range for defined helical AMP templates in reconstituted phospholipid membranes. In particular, we show that a temperature-dependent spectroscopic analysis, supported by 2D correlative tools, provides direct evidence for the membrane-induced and folding-mediated activity of AMPs. The far-FTIR study offers a direct and information-rich probe of membrane-related antimicrobial interactions.

Annexin V Drives Stabilization of Damaged Asymmetric Phospholipid Bilayers.

Annexins are soluble membrane-binding proteins that associate in a calcium dependent manner with anionic phospholipids. They play roles in membrane organization, signaling and vesicle transport and in several disease states including thrombosis and inflammation. Annexin V is believed to be involved in membrane repair. Mediated through binding to phosphatidylserine exposed at damaged plasma membrane, the protein forms crystalline networks that seal or stabilize small membrane tears. Herein, we model this biochemical mechanism to simulate membrane healing at microcavity array supported, transversally asymmetric, lipid bilayers (MSLBs) comprising 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS). Varying annexin V concentration, lipid composition, and DOPS presence at each leaflet, fluorescence imaging and correlation spectroscopy confirmed that when DOPS was present at the external, annexin V, contacting leaflet, the protein assembled rapidly at the membrane interface to form a layer. From electrochemical impedance studies, the annexin layer decreased membrane capacitance while reducing resistance. With DOPS incorporated only at the lower (proximal) leaflet, no appreciable annexin assembly was observed over the first 21 h. This suggests that membrane asymmetry is preserved over this window and transversal diffusion of DOPS is slow. Intense laser light applied to the membrane, in which DOPS is initially isolated at the lower leaflet, was found to simulate membrane damage, stimulating the rapid assembly of annexin V at the membrane interface confirmed by fluorescence imaging, correlation spectroscopy, and electrochemical impedance measurements. The damage induced by light increased impedance and decreased membrane resistance. The resulting bilayer annexin V patched bilayer showed better temporal stability toward impedance changes when compared with that of the parent membrane. In summary, this simple model of annexin V assembly in a fluidic lipid membrane provides new insights into the assembly of annexins as well as an empirical basis for building patch-repair mechanisms into interfacial bilayer membrane assemblies.

Cholesterol Anchors Enable Efficient Binding and Intracellular Uptake of DNA Nanostructures.

DNA nanostructures constitute a rapidly advancing tool-set for exploring cell-membrane functions and intracellular sensing or advancing delivery of biomolecular cargo  into cells. Chemical conjugation with lipid anchors can mediate binding of DNA nanostructures to synthetic lipid bilayers, yet how such structures interact with biological membranes and internalize cells has not been shown. Here, an archetypal 6-duplex nanobundle is used to investigate how lipid conjugation influences DNA cell binding and internalization kinetics. Cellular interactions of DNA nanobundles modified with one and three cholesterol anchors were assessed using flow cytometry and confocal microscopy. Nuclease digestion was used to distinguish surface-bound DNA, which is nuclease accessible, from internalized DNA. Three cholesterol anchors were found to enhance cellular association by up to 10-fold when compared with unmodified DNA. The bundles were endocytosed efficiently within 24 h. The results can help design controlled DNA binding and trafficking into cells.

Imaging live bacteria at the nanoscale: comparison of immobilisation strategies.

Atomic force microscopy (AFM) provides an effective, label-free technique enabling the imaging of live bacteria under physiological conditions with nanometre precision. However, AFM is a surface scanning technique, and the accuracy of its performance requires the effective and reliable immobilisation of bacterial cells onto substrates. Here, we compare the effectiveness of various chemical approaches to facilitate the immobilisation of Escherichia coli onto glass cover slips in terms of bacterial adsorption, viability and compatibility with correlative imaging by fluorescence microscopy. We assess surface functionalisation using gelatin, poly-l-lysine, Cell-Tak™, and Vectabond®. We describe how bacterial immobilisation, viability and suitability for AFM experiments depend on bacterial strain, buffer conditions and surface functionalisation. We demonstrate the use of such immobilisation by AFM images that resolve the porin lattice on the bacterial surface; local degradation of the bacterial cell envelope by an antimicrobial peptide (Cecropin B); and the formation of membrane attack complexes on the bacterial membrane.

Accelerating molecular discovery through data and physical sciences: Applications to peptide-membrane interactions.

Simulation and data analysis have evolved into powerful methods for discovering and understanding molecular modes of action and designing new compounds to exploit these modes. The combination provides a strong impetus to create and exploit new tools and techniques at the interfaces between physics, biology, and data science as a pathway to new scientific insight and accelerated discovery. In this context, we explore the rational design of novel antimicrobial peptides (short protein sequences exhibiting broad activity against multiple species of bacteria). We show how datasets can be harvested to reveal features which inform new design concepts. We introduce new analysis and visualization tools: a graphical representation of the k-mer spectrum as a fundamental property encoded in antimicrobial peptide databases and a data-driven representation to illustrate membrane binding and permeation of helical peptides.

Modulating charge-dependent and folding-mediated antimicrobial interactions at peptide-lipid interfaces.

Peptide-lipid interactions support a variety of biological functions. Of particular interest are those that underpin fundamental mechanisms of innate immunity that are programmed in host defense or antimicrobial peptide sequences found virtually in all multicellular organisms. Here we synthetically modulate antimicrobial peptide-lipid interactions using an archetypal helical antimicrobial peptide and synthetic membranes mimicking bacterial and mammalian membranes in solution. We probe these interactions as a function of membrane-induced folding, membrane stability and peptide-lipid ratios using a correlative approach encompassing light scattering and spectroscopy measurements such as circular dichroism spectroscopy, fluorescence and nuclear magnetic resonance spectroscopy. The peptide behavior is assessed against that of its anionic counterpart having similar propensities for α-helical folding. The results indicate strong correlations between peptide folding and membrane type, supporting folding-responsive binding of antimicrobial peptides to bacterial membranes. The study provides a straightforward approach for modulating structure-activity relationships in the context of membrane-induced antimicrobial action, thus holding promise for the rational design of potent antimicrobial agents.

Linear and orthogonal peptide templating of silicified protein fibres.

Biomineralisation is essential for biology. Specialist proteins use peptide motifs that catalyse mineral deposition into nano-to-microscale inorganic materials. Unlike in native proteins, the motifs incorporated into self-assembled fibres can persistently propagate on the microscopic scale enabling empirically defined silica nanostructures. Herein we show that the two main modes of motif templating - linear and orthogonal - in self-assembling, fibre-forming peptide sequences effectively silicify protein fibres. We show that the mere charge and morphology of protein fibres are not sufficient for silica deposition, but it is the synergy between fibrillogenesis and silica-specific motifs regularly spaced in fibres that ensures silica templating, regardless of the relative orientation of the motifs.

Binary Encoding of Random Peptide Sequences for Selective and Differential Antimicrobial Mechanisms.

Binary encoding of peptide sequences into differential antimicrobial mechanisms is reported. Such sequences are random in composition, but controllable in chain length, are assembled from the same two amino acids, but differ in the stereochemistry of one. Regardless of chirality, the sequences lyse bacteria including the "superbugs" methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). Sequences with the same chirality, so-called homochiral sequences, assemble into antimicrobial pores and form contiguous helices that are biologically promiscuous and hemolytic. By contrast, heterochiral sequences that lack such persistence selectively attack bacterial membranes without oligomerizing into visible pores. These results offer a mechanistic rationale for designing membrane-selective and sequence-independent antimicrobials.

A De Novo Virus-Like Topology for Synthetic Virions.

A de novo topology of virus-like assembly is reported. The design is a trifaceted coiled-coil peptide helix, which self-assembles into ultrasmall, monodisperse, anionic virus-like shells that encapsulate and transfer both RNA and DNA into human cells. Unlike existing artificial systems, these shells share the same physical characteristics of viruses being anionic, nonaggregating, abundant, hollow, and uniform in size, while effectively mediating gene silencing and transgene expression. These are the smallest virus-like structures reported to date, both synthetic and native, with the ability to adapt and transfer small and large nucleic acids. The design thus offers a promising solution for engineering bespoke artificial viruses with desired functions.

What Is the 'Minimum Inhibitory Concentration' (MIC) of Pexiganan Acting on Escherichia coli?-A Cautionary Case Study.

We measured the minimum inhibitory concentration (MIC) of the antimicrobial peptide pexiganan acting on Escherichia coli , and found an intrinsic variability in such measurements. These results led to a detailed study of the effect of pexiganan on the growth curve of E. coli, using a plate reader and manual plating (i.e. time-kill curves). The measured growth curves, together with single-cell observations and peptide depletion assays, suggested that addition of a sub-MIC concentration of pexiganan to a population of this bacterium killed a fraction of the cells, reducing peptide activity during the process, while leaving the remaining cells unaffected. This pharmacodynamic hypothesis suggests a considerable inoculum effect, which we quantified. Our results cast doubt on the use of the MIC as 'a measure of the concentration needed for peptide action' and show how 'coarse-grained' studies at the population level give vital information for the correct planning and interpretation of MIC measurements.

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.

Peptide self-assembly for nanomaterials: the old new kid on the block.

Peptide self-assembly is an increasingly attractive tool for nanomaterials. Perfected in biology peptide self-assembling systems have impacted on nearly any conceivable nanomaterial type. However, with all the information available to us commercialisation of peptide materials remains in its infancy. In an attempt to better understand the reasons behind this shortcoming we categorise peptide self-assembled materials in relation to their non-peptide counterparts. A particular emphasis is placed on the versatility of peptide self-assembly in terms of modularity, responsiveness and functional diversity, which enables direct comparisons with more traditional material chemistries.

Probing label-free intracellular quantification of free peptide by MALDI-ToF mass spectrometry.

Cell-penetrating peptides are promising reagents for gene and drug delivery. They can efficiently traverse the plasma membrane and deliver various cargo materials ranging from genes to nanoparticles. The functional efficiency of cargo often depends on the completeness of intracellular peptide uptake, which can be measured, but its quantification remains largely inconclusive. Existing approaches rely on the use of radioactive and fluorescent labels or tags which allow colorimetric, fluorescent or spectrometric detection, but lack the ability to detect free peptide. Herein we describe a generic label- and tag-free method to measure the concentration of internalised peptide by matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Quantification is preceded by two-dimensional chromatography and is performed at benign temperatures for the lysates of human dermal fibroblasts transfected with cell penetrating peptides in free form. Isotopically labelled peptides of the same structure are used as internal standards to enable accurate determination of concentration of the recovered free peptide. The method offers a minimalistic approach for intracellular quantification, which can be used as a correlative measure for fluorescence-based imaging methods.

Interfacial zippering-up of coiled-coil protein filaments.

Protein self-assembled materials find increasing use in medicine and nanotechnology. A challenge remains in our ability to tailor such materials at a given length scale. Here we report a de novo self-assembly topology which enables the engineering of filamentous protein nanostructures under morphological control. The rationale is exemplified by a ubiquitous self-assembly motif - an α-helical coiled-coil stagger. The stagger incorporates regularly spaced interfacial tryptophan residues, which allows it to zipper up into discrete filaments that bundle together without thickening by maturation. Using a combination of spectroscopy, microscopy, X-ray small-angle scattering and fibre diffraction methods we show that the precise positioning of tryptophan residues at the primary and secondary structure levels defines the extent of coiled-coil packing in resultant filaments. Applicable to other self-assembling systems, the rationale holds promise for the construction of advanced protein-based architectures and materials.

Supramolecular amphipathicity for probing antimicrobial propensity of host defence peptides.

Host defence peptides (HDPs) are effector components of innate immunity that provide defence against pathogens. These are small-to-medium sized proteins which fold into amphipathic conformations toxic to microbial membranes. Here we explore the concept of supramolecular amphipathicity for probing antimicrobial propensity of HDPs using elementary HDP-like amphiphiles. Such amphiphiles are individually inactive, but when ordered into microscopic micellar assemblies, respond to membrane binding according to the orthogonal type of their primary structure. The study demonstrates that inducible supramolecular amphipathicity can discriminate against bacterial growth and colonisation thereby offering a physico-chemical rationale for tuneable targeting of biological membranes.

Anti-antimicrobial peptides: folding-mediated host defense antagonists.

Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance.

Differentially instructive extracellular protein micro-nets.

An ability to construct biological matter from the molecule up holds promise for applications ranging from smart materials to integrated biophysical models for synthetic biology. Biomolecular self-assembly is an efficient strategy for biomaterial construction which can be programmed to support desired function. A challenge remains in replicating the strategy synthetically, that is at will, and differentially, that is for a specific function at a given length scale. Here we introduce a self-assembly topology enabling a net-like architectural mimetic of native extracellular matrices capable of differential responses to cell adhesion--enhanced mammalian cell attachment and proliferation, and enhanced resistance to bacterial colonization--at the native sub-millimeter length scales. The biological performance of such protein micro-nets directly correlates with their morphological and chemical properties, offering thus an application model for differential extracellular matrices.