Disruption of cell membranes is a fundamental host defence response found in virtually all forms of life. The molecular mechanisms vary but generally lead to energetically favored circular nanopores. Here we report an elaborate fractal rupture pattern induced by a single side-chain mutation in ultrashort (8-11-mers) helical peptides, which otherwise form transmembrane pores. In contrast to known mechanisms, this mode of membrane disruption is restricted to the upper leaflet of the bilayer where it exhibits propagating fronts of peptide-lipid interfaces that are strikingly similar to viscous instabilities in fluid flow. The two distinct disruption modes, pores and fractal patterns, are both strongly antimicrobial but only the fractal rupture is non-hemolytic. The results offer wide implications for elucidating differential membrane targeting phenomena defined at the nanoscale.
Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, polypeptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions—their membranes. However, suitable approaches to quantify the efficacy of polypeptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure–activity relationships of two experimental series of polypeptide antibiotics. We reveal strong correlations between each peptide’s physicochemical activity at the membrane level and biological activity at the cellular level. We achieve this knowledge by assaying the membranolytic activities of the compounds on hundreds of individual giant lipid vesicles, and by quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.
Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and byproducts, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We have developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a vesicles suspension is partitioned into three fractions: purified GUVs, residual components, and a washing solution. Using our purification approach, we show that giant vesicles can be separated from various residueswhich range in size and chemical compositionwith a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.
The 18-kDa cationic protein CAP18 is an antimicrobial protein isolated from rabbit granulocytes that binds lipopolysaccharide (LPS) and inhibits many of its biological activities. We covalently coupled a synthetic peptide representing amino acids 106 to 138 of CAP18 to human immunoglobulin G (IgG) by using the heterobifunctional linker N-succinimidyl-3-(2-pyridyldithio)propionate. The ability of CAP18 106-138-IgG to bind and neutralize LPS in whole blood in the presence and absence of anticoagulants was studied. Both CAP18 106-138 and CAP18 106-138-IgG significantly suppressed LPS-induced tumor necrosis factor (TNF) production in whole blood in the absence of anticoagulants. EDTA potentiated the ability of CAP18 106-138 and CAP18 106-138-IgG to decrease LPS-induced TNF production in a dose-dependent manner. In contrast, heparin inhibited the ability of CAP18 106-138 and CAP18 106-138-IgG to suppress LPS-induced TNF production. EDTA also enhanced LPS capture in a fluid-phase binding assay that utilizes magnetic anti-IgG beads to capture CAP18 106-138-IgG (and bound [ 3 H]LPS) in whole blood. In contrast, heparin inhibited the binding dose dependently. We conclude that CAP18 106-138-IgG binds to and neutralizes LPS in whole blood in the absence of anticoagulants. Further studies of its protective efficacy in animal models are warranted. Caution should be used in interpreting assays that measure the binding and neutralization of LPS in whole blood in the presence of calcium-binding anticoagulants or heparin.
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