ABSTRACT
We investigated the permeation of molecules across lipid membranes on an open microfluidic platform. An array of droplet pairs was created by spotting aqueous droplets, dispersed in a lipid oil solution, onto a plate with cavities surrounded by a hydrophobic substrate. Droplets in two adjacent cavities come in contact and form an artificial lipid bilayer, called a droplet interface bilayer (DIB). The method allows for monitoring permeation of fluorescently tagged compounds from a donor droplet to an acceptor droplet. A mathematical model was applied to describe the kinetics and determine the permeation coefficient. We also demonstrate that permeation kinetics can be followed over a series of droplets, all connected via DIBs. Moreover, by changing the lipid oil composition after spotting donor droplets, we were able to create asymmetric membranes that we used to mimic the asymmetry of the cellular plasma membrane. Finally, we developed a protocol to separate and extract the droplets for label-free analysis of permeating compounds by liquid chromatography-mass spectrometry. Our versatile platform has the potential to become a new tool for the screening of drug membrane permeability in the future.
Subject(s)
Lipid Bilayers , Water , Cell Membrane , Hydrophobic and Hydrophilic Interactions , MembranesABSTRACT
In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and mimic reactions and processes at or across the membrane. In this context, we employ here a versatile microfluidic platform that enables precise positioning of nanoliter droplets with user-specified lipid compositions and in a defined pattern. Adjacent droplets make contact and form a droplet interface bilayer to simulate cellular membranes. Translocation of molecules across membranes are tailored by the addition of alpha-hemolysin to selected droplets. Moreover, we developed a protocol to analyze the translocation of non-fluorescent molecules between droplets with mass spectrometry. Our method is capable of automated formation of one- and two-dimensional droplet networks, which we demonstrated by connecting droplets containing different compound and enzyme solutions to perform translocation experiments and a multistep enzymatic cascade reaction across the droplet network. Our platform opens doors for creating complex artificial systems for bottom-up synthetic biology.
Subject(s)
Microfluidic Analytical Techniques , Microfluidics , Catalysis , Enzymes/chemistry , Enzymes/metabolism , Microfluidics/instrumentation , Microfluidics/methods , NanotechnologyABSTRACT
Naturally occurring membranolytic antimicrobial peptides (AMPs) are rarely cell-type selective and highly potent at the same time. Template-based peptide design can be used to generate AMPs with improved properties de novo. Following this approach, 18 linear peptides were obtained by computationally morphing the natural AMP Aurein 2.2d2 GLFDIVKKVVGALG into the synthetic model AMP KLLKLLKKLLKLLK. Eleven of the 18 chimeric designs inhibited the growth of Staphylococcus aureus, and six peptides were tested and found to be active against one resistant pathogenic strain or more. One of the peptides was broadly active against bacterial and fungal pathogens without exhibiting toxicity to certain human cell lines. Solution nuclear magnetic resonance and molecular dynamics simulation suggested an oblique-oriented membrane insertion mechanism of this helical de novo peptide. Temperature-resolved circular dichroism spectroscopy pointed to conformational flexibility as an essential feature of cell-type selective AMPs.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Staphylococcus aureus/drug effects , Amino Acid Sequence , Drug Design , HEK293 Cells , Humans , Molecular Dynamics Simulation , Protein Conformation, alpha-Helical , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Staphylococcus aureus/growth & developmentABSTRACT
Protein-membrane interactions that modify the shape of membranes are important for generating curvature, membrane deformation by protein-protein crowding or trafficking of vesicles. Giant vesicles represent a simplified but versatile model for biological membranes and are commonly employed for the study of lipid domains and permeation across compartments. In this study, we investigated the interaction of pneumolysin (PLY), a pore-forming toxin secreted by Streptococcus pneumoniae, with multilamellar and unilamellar membranes. It reveals an enlargement of membrane area due to the insertion of pores into the bilayer and protein-membrane aggregations that induce membrane deformation and wrinkling. Moreover, we demonstrate that PLY peel-off layers from multilamellar giant vesicles in a hitherto unknown layer-by-layer peeling mechanism, which reveals the structure and number of membrane lamellae. We employed microfluidic methods to capture giant vesicles and confocal laser scanning microscopy, transmission microscopy, dynamic light scattering and cryo-electron microscopy to disclose the structure of multilamellar vesicles. Based on our findings we suggest how back-to-back pore arrangements stabilize large PLY-membrane entities and that pore-displaced lipids possibly remain in the membrane.
Subject(s)
Cell Membrane/chemistry , Streptococcus pneumoniae/chemistry , Streptolysins/chemistry , Unilamellar Liposomes/chemistry , Bacterial Proteins/chemistryABSTRACT
High-throughput screening of cell-secreted proteins is essential for various biotechnological applications. In this article, we show a microfluidic approach to perform the analysis of cell-secreted proteins in nanoliter droplet arrays by two complementary methods, fluorescence microscopy and mass spectrometry. We analyzed the secretion of the enzyme phytase, a phosphatase used as an animal feed additive, from a low number of yeast cells. Yeast cells were encapsulated in nanoliter volumes by droplet microfluidics and deposited on spatially defined spots on the surface of a glass slide mounted on the motorized stage of an inverted fluorescence microscope. During the following incubation for several hours to produce phytase, the droplets can be monitored by optical microscopy. After addition of a fluorogenic substrate at a defined time, the relative concentration of phytase was determined in every droplet. Moreover, we demonstrate the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to monitor the multistep conversion of the native substrate phytic acid by phytase secreted in 7 nL droplets containing 50-100 cells. Our method can be adapted to various other protocols. As the droplets are easily accessible, compounds such as assay reagents or matrix molecules can be added to all or to selected droplets only, or part of the droplet volume could be removed. Hence, this platform is a versatile tool for questions related to cell secretome analysis.
Subject(s)
6-Phytase/analysis , Microfluidic Analytical Techniques , Nanoparticles/chemistry , 6-Phytase/metabolism , Particle Size , Surface PropertiesABSTRACT
We investigate the influence of membrane potential on the permeation of cationic peptides. Therefore, we employ a microfluidic chip capable of capturing giant unilamellar vesicles (GUVs) in physical traps and fast exchange of chemical compounds. Control experiments with calcein proved that the vesicle membranes' integrity is not affected by the physical traps and applied shear forces. Combined with fluorescence correlation spectroscopy, permeation of fluorescently labeled peptides across vesicle membranes can be measured down to the nanomolar level. With the addition of a lipophilic ruthenium(II) complex Ru(C17)22+, GUVs consisting of mixed acyl phospholipids are prepared with a negative membrane potential, resembling the membrane asymmetry in cells. The membrane potential serves as a driving force for the permeation of cationic cell-penetrating peptides (CPPs) nonaarginine (Arg9) and the human immunodeficiency virus trans-activator of transcription (TAT) peptide already at nanomolar doses. Hyperpolarization of the membrane by photo-oxidation of Ru(C17)22+ enhances permeation significantly from 55 to 78% for Arg9. This specific enhancement for Arg9 (cf. TAT) is ascribed to the higher affinity of the arginines to the phosphoserine head groups. On the other hand, permeation is decreased by introducing an additional negative charge in close proximity to the N-terminal arginine residue when changing the fluorophore. In short, with the capability to reconstitute membrane potential as well as shear stress, our system is a suitable platform for modeling the membrane permeability of pharmaceutics candidates. The results also highlight the membrane potential as a major cause of discrepancies between vesicular and cellular studies on CPP permeation.
Subject(s)
Cell-Penetrating Peptides/chemistry , Fluorescent Dyes/chemistry , Membrane Potentials , Phospholipids/chemistry , Unilamellar Liposomes/chemistry , Fluoresceins/chemistry , Humans , Oligopeptides/chemistryABSTRACT
Nucleated cells eliminate lesions induced by bacterial pore-forming toxins, such as pneumolysin via shedding patches of damaged plasmalemma into the extracellular milieu. Recently, we have shown that the majority of shed pneumolysin is present in the form of inactive pre-pores. This finding is surprising considering that shedding is triggered by Ca2+-influx following membrane perforation and therefore is expected to positively discriminate for active pores versus inactive pre-pores. Here we provide evidence for the existence of plasmalemmal domains that are able to attract pneumolysin at high local concentrations. Within such a domain an immediate plasmalemmal perforation induced by a small number of pneumolysin pores would be capable of triggering the elimination of a large number of not yet active pre-pores/monomers and thus pre-empt more frequent and perilous perforation events. Our findings provide further insights into the functioning of the cellular repair machinery which benefits from an inhomogeneous plasmalemmal distribution of pneumolysin.
Subject(s)
Host-Pathogen Interactions/immunology , Lipid Bilayers/metabolism , Pneumococcal Infections/immunology , Streptococcus pneumoniae/physiology , Bacterial Proteins/metabolism , Bacterial Shedding/immunology , Cell Line, Tumor , Cell Membrane/metabolism , Cell Membrane/microbiology , Cholesterol/metabolism , HEK293 Cells , Humans , Intravital Microscopy , Lipid Bilayers/immunology , Microfluidics , Pneumococcal Infections/microbiology , Streptolysins/metabolismABSTRACT
Because of inhomogeneous matrix-assisted laser desorption/ionization (MALDI) matrix crystallization and laser shot-to-shot variability, quantitation is not generally performed by MALDI mass spectrometry. Here we introduce a high-throughput MALDI method using an innovative high-density microarray for mass spectrometry (MAMS) technology, which allows semiquantitative measurement of cocaine and its metabolites, benzoylecgonine, cocaethylene, and ecgonine methyl ester. A MAMS slide containing lanes of hydrophilic spots and an automated slider to drag a sample droplet over several small spots can accomplish automatic sample aliquoting and lead to homogeneous crystallization of the matrix-analyte mixture and, thus, to a reproducible signal (average RSD 6%). Four hair samples of self-reported drug users were analyzed in parallel by MALDI-MS/MS and by a validated LC-MS/MS method. The consumption profiles as well as the metabolite-parent drug ratios obtained correlated well, confirming the effectiveness of the MALDI-MS/MS method to establish a calendar of consumption in only 1 mg of hair. The analysis time for 10 hair samples is below 40 min, with 12 replicates per sample. Since only 3 µL of a 20 µL extract is analyzed, complementary assays are possible, such as the detection of additional drugs. The semiquantitative MALDI method worked well with only a small amount of hair and gave results in less than 4 min per sample, including replicates. This was made possible by the use of MAMS slides for sample preparation, which thus present significant advantages over traditional methods in cases where results are required urgently or if samples are scarce.
Subject(s)
Cocaine/analysis , Hair/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Adult , Cocaine/analogs & derivatives , Female , Humans , Limit of Detection , Male , Reproducibility of ResultsABSTRACT
Specific interactions of peptides with lipid membranes are essential for cellular communication and constitute a central aspect of the innate host defense against pathogens. A computational method for generating innovative membrane-pore-forming peptides inspired by natural templates is presented. Peptide representation in terms of sequence- and topology-dependent hydrophobic moments is introduced. This design concept proves to be appropriate for the de novo generation of first-in-class membrane-active peptides with the anticipated mode of action. The designed peptides outperform the natural template in terms of their antibacterial activity. They form a kinked helical structure and self-assemble in the membrane by an entropy-driven mechanism to form dynamically growing pores that are dependent on the lipid composition. The results of this study demonstrate the unique potential of natural template-based peptide design for chemical biology and medicinal chemistry.