Biology subjects

Fragasso, A.

Publications and source records attributed to Fragasso, A..

11 recordsLinked to original sources

bioRxiv2025

Cross-Molecular Active Learning for the Discovery of Antimicrobial Polyacrylamides

Antimicrobial resistance poses an urgent and increasing threat to global health. The development of new antimicrobials is crucial. Synthetic copolymers are attractive as a potential solution, because they can be produced at scale and designed to mimic antimicrobial peptides and act as broad-spectrum antimicrobials capable of evading resistance mechanisms. This work leverages a cross-molecular machine learning pipeline, trained on antimicrobial peptides, to develop potent antimicrobial polymers to combat Escherichia coli, which were then synthesized and validated experimentally. One candidate copolymer was further characterized and shown to permeabilize the bacterial membrane, which is associated with decreased resistance. Furthermore, this copolymer demonstrated remarkable synergy in eradicating biofilm-associated E. coli when combined with a first-line clinical drug regimen, reducing the amount needed to eradicate bacteria in biofilms by three orders of magnitude. These results demonstrate promise for potentiating antibacterial activity of currently available antibiotics, treating serious and complicated infections, and combatting antimicrobial resistance.

microbiology↗

bioRxiv2025

Spatial control of karyopherin binding avidity within NPC mimics revealed by designer FG-Nucleoporins

Nucleocytoplasmic transport occurs via nuclear pore complexes (NPCs), [~]40-60 nm wide pores lined with intrinsically disordered proteins that are rich in Phe-Gly motifs (FG-Nups) that form a selective barrier. Molecules larger than [~]50 kDa are increasingly blocked for transport unless they are bound to a nuclear transport receptor (NTR). How the amino acid sequence of FG-Nups contribute to this is not fully understood. Here, we present de novo designed artificial FG-Nups with a systematically varied FG-repeat spacing and charge-to-hydrophobicity ratio (C/H). Starting from a reference sequence termed NupY (with the average properties of natural yeast GLFG- Nups), we designed, synthesized, and experimentally tested a library of NupY variants using QCM-D experiments and phase separation assays. We find that the spacing between FG-motifs governs Kap95 absorption into the FG- Nup phase, while increasing C/H results in higher avidity for Kap95 due to an increased accessibility of FG-motifs. Molecular dynamics simulations of transport through NupY-coated pores show a reduced barrier function for noncohesive high-C/H-ratio variants and the highest transport selectivity for designs close to native GLFG-Nups. We postulate that a balance between entropic repulsion and enthalpic gain from multivalent Kap-FG-Nup interactions drives the spatial and temporal partitioning of Kaps in the NPC.

biophysics↗

bioRxiv2025

Time-resolved phenotyping at subcellular resolution reveals shared principles and key trade-offs across antimicrobial peptide activities

Cationic antimicrobial peptides are a broad family of host defense molecules that neutralize bacteria by permeabilizing one or more membranes and/or inhibiting intracellular targets. Here, we present a time-resolved single-cell pipeline for quantifying these effects in Escherichia coli. Applying this pipeline to 18 diverse natural peptides and synthetic peptidomimetics reveals shared core activities, but with different kinetics, defining two classes with opposite trade-offs. Class I peptides cause abrupt growth arrest, predominantly coupled with inner membrane permeabilization and ribosome/DNA reorganization, conferring fast, multipronged action. However, rapid intracellular absorption by the first permeabilized cells depletes the extracellular pool, rendering them ineffective against dense populations, including biofilms. Class II peptides act more gradually, with delayed or absent inner membrane permeabilization, limiting their speed of action. However, this results in slower intracellular absorption and greater efficacy at high cell densities and against biofilms. These opposing functional trade-offs point to important immunological and therapeutic implications.

microbiology↗

bioRxiv2024

Glycogen phase separation drives macromolecular rearrangement and asymmetric division in E. coli

Bacteria often experience nutrient limitation. While the exponential and stationary growth phases have been characterized in the model bacterium Escherichia coli, little is known about what transpires inside individual cells during the transition between these two phases. Through quantitative cell imaging, we found that the positions of nucleoids and cell division sites become increasingly asymmetric during transition phase. These asymmetries were coupled with asymmetric reorganization of protein, ribosome, and RNA probes in the cytoplasm. Results from live-cell imaging experiments, complemented with genetic and 13C whole-cell nuclear magnetic resonance spectroscopy studies, show that preferential accumulation of the storage polymer glycogen at the old cell pole leads to the observed rearrangements and asymmetric divisions. Live-cell atomic force microscopy analysis, combined with in vitro biochemical experiments, suggests that these phenotypes are due to the propensity of glycogen to phase separate into soft condensates in the crowded cytoplasm. Glycogen-associated differences in cell sizes between strains and future daughter cells suggest that glycogen phase separation allows cells to store large glucose reserves without counting them as cytoplasmic space.

microbiology↗

bioRxiv2023

Diameter Dependence of Transport through Nuclear Pore Complex Mimics Studied Using Optical Nanopores

The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode waveguides with the FG-nucleoporin Nsp1. This approach enables the measurement of single-molecule translocations through individual pores using optical detection. We probe the selectivity of Nsp1-coated pores by quantitatively comparing the translocation rates of the nuclear transport receptor Kap95 to the inert probe BSA over a wide range of pore sizes from 35 nm to 160 nm. Pores below 55 {+/-} 5 nm show significant selectivity that gradually decreases for larger pores. This finding is corroborated by coarse-grained molecular-dynamics simulations of the Nsp1 mesh within the pore, which suggest that leakage of BSA occurs by diffusion through transient openings within the dynamic mesh. Furthermore, we experimentally observe a modulation of the BSA permeation when varying the concentration of Kap95. The results demonstrate the potential of single-molecule fluorescence measurements on biomimetic NPCs to elucidate the principles of nuclear transport.

biophysics↗

bioRxiv2022

Dynamin A as a one-component division machinery for synthetic cells

Membrane abscission, the final cut of the last connection between emerging daughter cells, is an indispensable event in the last stage of cell division, as well as in other cellular processes such as endocytosis, virus release, or bacterial sporulation. However, its mechanism remains poorly understood, which also impedes its application as a cell-division machinery for synthetic cells. Here, we use fluorescence microscopy and Fluorescence Recovery After Photobleaching (FRAP) to study the in vitro reconstitution of the bacterial protein Dynamin A (DynA) inside liposomes. Upon external reshaping of the liposomes into dumbbells, DynA self-assembles at the membrane neck, resulting in membrane hemi-scission and even full scission. DynA proteins constitute a simple one-component division machinery that is capable of splitting dumbbell-shaped liposomes, marking an important step towards building a synthetic cell.

synthetic biology↗

bioRxiv2021

Transport receptor occupancy in Nuclear Pore Complex mimics

Nuclear Pore Complexes (NPCs) regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells. Intrinsically disordered Phe-Gly nucleoporins (FG Nups) line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor (karyopherin; Kap). Here, we assess Kap-centric NPC models, which postulate that Kaps participate in establishing the selective barrier. We combine biomimetic nanopores, formed by tethering Nsp1 to the inner wall of a solid-state nanopore, with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores: one population that is transported across the pore in milliseconds, and a second population that is stably assembled within the FG mesh of the pore. Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh. Modeling reveals an accumulation of Kap95 near the pore wall, yielding a conductance decrease. We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that, even at high concentrations, Kaps only bind at most 8% of the FG-motifs in the nanopore, indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs. Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps, where a stable population of Kaps binds avidly to the NPC periphery, while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.

biophysics↗

bioRxiv2021

A synthetic membrane shaper for controlled liposome deformation

Shape defines the structure and function of cellular membranes. In cell division, the cell membrane deforms into a dumbbell shape, while organelles such as the autophagosome exhibit stomatocyte shapes. Bottom-up in vitro reconstitution of protein machineries that stabilize or resolve the membrane necks in such deformed liposome structures is of considerable interest to characterize their function. Here we develop a DNA-nanotechnology-based approach that we call Synthetic Membrane Shaper (SMS), where cholesterol-linked DNA structures attach to the liposome membrane to reproducibly generate high yields of stomatocytes and dumbbells. In silico simulations confirm the shape-stabilizing role of the SMS. We show that the SMS is fully compatible with protein reconstitution by assembling bacterial divisome proteins (DynaminA, FtsZ:ZipA) at the catenoidal neck of these membrane structures. The SMS approach provides a general tool for studying protein binding to complex membrane geometries that will greatly benefit synthetic cell research.

synthetic biology↗

bioRxiv2021

Reconstitution of ultrawide DNA origami pores in liposomes for transmembrane transport of macromolecules

Molecular traffic across lipid membranes is a vital process in cell biology that involves specialized biological pores with a great variety of pore diameters, from fractions of a nanometer to >30 nm. Creating artificial membrane pores covering similar size and complexity will aid the understanding of transmembrane molecular transport in cells, while artificial pores are also a necessary ingredient for synthetic cells. Here, we report the construction of DNA origami nanopores that have an inner diameter as large as 30 nm. We developed new methods to successfully insert these ultrawide pores into the lipid membrane of giant unilamellar vesicles (GUVs) by administering the pores concomitantly with vesicle formation in an inverted-emulsion cDICE technique. The reconstituted pores permit the transmembrane diffusion of large macromolecules such as folded proteins, which demonstrates the formation of large membrane-spanning open pores. The pores are size selective as dextran molecules with a diameter up to 22 nm can traverse the pores, whereas larger dextran molecules are blocked. By FRAP measurements and modelling of the GFP influx rate, we find that up to hundreds of pores can be functionally reconstituted into a single GUV. Our technique bears great potential for applications across different fields from biomimetics, synthetic biology, to drug delivery.

biophysics↗

bioRxiv2020

A designer FG-Nup that reconstitutes the selective transport barrier of the Nuclear Pore Complex

Nuclear Pore Complexes (NPCs) regulate bidirectional transport between the nucleus and the cytoplasm. Intrinsically disordered FG-Nups line the NPC lumen and form a selective barrier, where transport of most proteins is inhibited whereas specific transporter proteins freely pass. The mechanism underlying selective transport through the NPC is still debated. Here, we reconstitute the selective behaviour of the NPC bottom-up by introducing a rationally designed artificial FG-Nup that mimics natural Nups. Using QCM-D, we measure a strong affinity of the artificial FG-Nup brushes to the transport receptor Kap95, whereas no binding occurs to cytosolic proteins such as BSA. Solid-state nanopores with the artificial FG-Nups lining their inner walls support fast translocation of Kap95 while blocking BSA, thus demonstrating selectivity. Coarse-grained molecular dynamics simulations highlight the formation of a selective meshwork with densities comparable to native NPCs. Our findings show that simple design rules can recapitulate the selective behaviour of native FG-Nups and demonstrate that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create functional NPCs.

biophysics↗

bioRxiv2019

Comparing current noise in biological and solid-state nanopores

Nanopores bear great potential as single-molecule tools for bioanalytical sensing and sequencing, due to their exceptional sensing capabilities, high-throughput, and low cost. The detection principle relies on detecting small differences in the ionic current as biomolecules traverse the nanopore. A major bottleneck for the further progress of this technology is the noise that is present in the ionic current recordings, because it limits the signal-to-noise ratio and thereby the effective time resolution of the experiment. Here, we review the main types of noise at low and high frequencies and discuss the underlying physics. Moreover, we compare biological and solid-state nanopores in terms of the signal-to-noise ratio (SNR), the important figure of merit, by measuring free translocations of a short ssDNA through a selected set of nanopores under typical experimental conditions. We find that SiNx solid-state nanopores provide the highest SNR, due to the large currents at which they can be operated and the relatively low noise at high frequencies. However, the real game-changer for many applications is a controlled slowdown of the translocation speed, which for MspA was shown to increase the SNR >160-fold. Finally, we discuss practical approaches for lowering the noise for optimal experimental performance and further development of the nanopore technology.

biophysics↗