Search

Vesna Spasovski,1,2,* Anna Romolo,1,3,* Urška Zagorc,4 Vesna Arrigler,4 Matic Kisovec,5 Apolonija Bedina Zavec,5 Matevž Arko,1 Adrienn Molnár,6,7 Gitta Schlosser,7 Aleš Iglič,3,8 Ksenija Kogej,4 Veronika Kralj-Iglič1 1University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia; 2Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia; 3University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Physics, Ljubljana, Slovenia; 4University of Ljubljana, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia; 5National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia; 6Hevesy György PhD School of Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary; 7MTA-ELTE Lendület Ion Mobility Mass Spectrometry Research Group, Faculty of Science, Institute of Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary; 8University of Ljubljana, Faculty of Medicine, Laboratory of Clinical Biophysics, Ljubljana, Slovenia*These authors contributed equally to this workCorrespondence: Veronika Kralj-Iglič, Email veronika.kralj-iglic@zf.uni-lj.siIntroduction: Lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called nano-sized hybridosomes or nanohybridosomes, NSHs) were considered.Methods: We formed NSHs by mixing appropriate amounts of lecithin, glycerol and supernatant of isolation of extracellular vesicles from spruce needle homogenate. We visualized NSHs by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet–visual spectroscopy, interferometric light microscopy and liquid chromatography–mass spectrometry.Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well on the average hydrodynamic radius of the particles Rh (between 140 and 180 nm), while the concentrations of the particles were in the range between 1013 and 1014/mL indicating that NSHs present a considerable (more than 25%) of the sample which is much more than the yield of natural extracellular vesicles (EVs) from spruce needle homogenate (estimated less than 1%). Spruce specific lipids and proteins were found in hybridosomes.Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large-scale production of NSHs from lipids and spruce needle homogenate is feasible.Plain Language Summary: Cells shed into their exterior nanoparticles (here referred to as extracellular vesicles – EVs) that are free to move, reach distant cells and are taken up by them. As they carry bioactive constituents, EVs may have important impact on the recipient cells. The mechanisms of EV formation and mediation can be employed in designing therapeutic, prophylactic and diagnostic methods for various medical issues. EVs can be harvested from biological samples; however, their yield is small,12 and there are potential side effects. Artificial vesicles – liposomes – have high yield; however, in vivo, they can be degraded before reaching the target and their reproducibility is yet insufficient. In order to combine advantages of both types of nanoparticles, we have composed nanohybridosomes (NSHs) from soya lecithin, water and supernatant of isolation of EVs from spruce needle homogenate, visualized them by cryogenic electron microscopy and characterized them with respect to their size, concentration and protein/nucleic acid content. We have applied a recently developed interferometric light microscopy to determine the hydrodynamic radius and the concentration of EVs. We found that the majority of composed particles are nano-sized and that they enclose more than 25% of the incoming volume of liquid, which is considerably more than about 1% that can be harvested by isolation of EVs from spruce needle homogenate by (ultra)centrifugation.Keywords: hybridosomes, liposomes, nanovesicles, extracellular particles, small cellular particles, drug delivery

Extracellular nanoparticles (EPs) are a subject of increasing interest for their biological role as mediators in cell-cell communication; however, their harvesting and assessment from bodily fluids are challenging, as processing can significantly affect samples. With the aim of minimizing processing artifacts, we assessed the number density ( n ) and hydrodynamic diameter ( D h ) of EPs directly in diluted plasma and blood using the following recently developed technique: interferometric light microscopy (ILM). We analyzed 613 blood and plasma samples from human patients with inflammatory bowel disease (IBD), collected in trisodium citrate and ethylenediaminetetraacetic acid (EDTA) anticoagulants, and 163 blood and plasma samples from canine patients with brachycephalic obstructive airway syndrome (BOAS). We found a highly statistically significant correlation between n in the plasma and n in the blood only in the human (i.e., but not canine) blood samples, between the samples with trisodium citrate and EDTA, and between the respective D h for both species (all p < 10 -3 ). In the human plasma, the average < D h > was 139 ± 31 nm; in the human blood, < D h > was 158 ± 11 nm; in the canine plasma, < D h > was 155 ± 32 nm; and in the canine blood, < D h > was 171 ± 33 nm. The differences within species were statistically significant ( p < 10 -2 ), with sufficient statistical power (P > 0.8). For < n >, we found no statistically significant differences between the human plasma and blood samples or between the samples with trisodium citrate and EDTA. Our results prove that measuring n and D h of EPs in minimally processed fresh blood and in diluted fresh plasma by means of ILM is feasible for large populations of samples.

The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to the complex makeup of biological membranes. Cholesterol, the major modulator of bilayer structure in mammalian cell membranes, is recognized by various proteins, including the well-studied cholesterol-dependent cytolysins. Here, we use in vitro evolution to investigate the molecular adaptations that preserve the cholesterol specificity of perfringolysin O, the prototypical cholesterol-dependent cytolysin from Clostridium perfringens. We identify variants with altered membrane-binding interfaces whose cholesterol-specific activity exceeds that of the wild-type perfringolysin O. These novel variants represent alternative evolutionary outcomes and have mutations at conserved positions that can only accumulate when epistatic constraints are alleviated. Our results improve the current understanding of the biochemical malleability of the surface of a lipid-binding protein., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Small cellular particles (SCPs) are being considered for their role in cell-to-cell communication. We harvested and characterized SCPs from spruce needle homogenate. SCPs were isolated by differential ultracentrifugation. They were imaged by scanning electron microscope (SEM) and cryogenic transmission electron microscope (cryo TEM), assessed for their number density and hydrodynamic diameter by interferometric light microscopy (ILM) and flow cytometry (FCM), total phenolic content (TPC) by UV-vis spectroscopy, and terpene content by gas chromatography-mass spectrometry (GC-MS). The supernatant after ultracentrifugation at 50,000× g contained bilayer-enclosed vesicles whereas in the isolate we observed small particles of other types and only a few vesicles. The number density of cell-sized particles (CSPs) (larger than 2 μm) and meso-sized particles (MSPs) (cca 400 nm-2 µm) was about four orders of magnitude lower than the number density of SCPs (sized below 500 nm). The average hydrodynamic diameter of SCPs measured in 10,029 SCPs was 161 ± 133 nm. TCP decreased considerably due to 5-day aging. Volatile terpenoid content was found in the pellet after 300× g . The above results indicate that spruce needle homogenate is a source of vesicles to be explored for potential delivery use.

The preparation of autologous platelet and extracellular vesicle-rich plasma (PVRP) has been explored in many medical fields with the aim to benefit from its healing potential. In parallel, efforts are being invested to understand the function and dynamics of PVRP that is complex in its composition and interactions. Some clinical evidence reveals beneficial effects of PVRP, while some report that there were no effects. To optimize the preparation methods, functions and mechanisms of PVRP, its constituents should be better understood. With the intention to promote further studies of autologous therapeutic PVRP, we performed a review on some topics regarding PVRP composition, harvesting, assessment and preservation, and also on clinical experience following PVRP application in humans and animals. Besides the acknowledged actions of platelets, leukocytes and different molecules, we focus on extracellular vesicles that were found abundant in PVRP.

Small particles in natural sources are a subject of interest for their potential role in intercellular, inter-organism, and inter-species interactions, but their harvesting and assessment present a challenge due to their small size and transient identity. We applied a recently developed interferometric light microscopy (ILM) to assess the number density and hydrodynamic radius (R h ) of isolated small cellular particles (SCPs) from blood preparations (plasma and washed erythrocytes) (B), spruce needle homogenate (S), suspension of flagellae of microalgae Tetraselmis chuii (T), conditioned culture media of microalgae Phaeodactylum tricornutum (P), and liposomes (L). The aliquots were also assessed by flow cytometry (FCM), dynamic light scattering (DLS), ultraviolet-visible spectrometry (UV-vis), and imaging by cryogenic transmission electron microscopy (cryo-TEM). In R h , ILM showed agreement with DLS within the measurement error in 10 out of 13 samples and was the only method used here that yielded particle density. Cryo-TEM revealed that representative SCPs from Tetraselmis chuii flagella (T) did not have a globular shape, so the interpretation by R h of the batch methods was biased. Cryo-TEM showed the presence of thin filaments in isolates from Phaeodactylum tricornutum conditioned culture media (P), which provides an explanation for the considerably larger R h obtained by batch methods than the sizes of particles observed by cryo-TEM images. ILM proved convenient for assessment of number density and R h of SCPs in blood preparations (e.g., plasma); therefore, its use in population and clinical studies is indicated.

We studied the efficiency of three culture series of the microalgae Phaeodactylum tricornutum ( P. tricornutum ) and bacteria Thalassospira sp. (axenic microalgae, bacterial culture and co-culture of the two) in removing bisphenols (BPs) from their growth medium. Bacteria were identified by 16S ribosomal RNA polymerase chain reaction (16S rRNA PCR). The microorganism growth rate was determined by flow cytometry. Cultures and isolates of their small cellular particles (SCPs) were imaged by scanning electron microscopy (SEM) and cryogenic transmission electron microscopy (Cryo-TEM). BPs were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). Our results indicate that some organisms may have the ability to remove a specific pollutant with high efficiency. P. tricornutum in axenic culture and in mixed culture removed almost all (more than 99%) of BPC2. Notable differences in the removal of 8 out of 18 BPs between the axenic, mixed and bacterial cultures were found. The overall removals of BPs in axenic P. tricornutum , mixed and bacterial cultures were 11%, 18% and 10%, respectively. Finding the respective organisms and creating microbe societies seems to be key for the improvement of wastewater treatment. As a possible mediating factor, numerous small cellular particles from all three cultures were detected by electron microscopy. Further research on the mechanisms of interspecies communication is needed to advance the understanding of microbial communities at the nano-level.

Tumor growth and metastasis strongly rely on cell-cell communication. One of the mechanisms by which tumor cells communicate involves the release and uptake of lipid membrane encapsulated particles full of bioactive molecules, called extracellular vesicles (EVs). EV exchange between cancer cells may induce phenotype changes in the recipient cells. Our work investigated the effect of EVs released by teratocarcinoma cells on glioblastoma (GBM) cells. EVs were isolated by differential centrifugation and analyzed through Western blot, nanoparticle tracking analysis, and electron microscopy. The effect of large EVs on GBM cells was tested through cell migration, proliferation, and drug-sensitivity assays, and resulted in a specific impairment in cell migration with no effects on proliferation and drug-sensitivity. Noticeably, we found the presence of the EGF-CFC founder member CRIPTO on both small and large EVs, in the latter case implicated in the EV-mediated negative regulation of GBM cell migration. Our data let us propose a novel route and function for CRIPTO during tumorigenesis, highlighting a complex scenario regulating its effect, and paving the way to novel strategies to control cell migration, to ultimately improve the prognosis and quality of life of GBM patients.

Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4-10), osmolarity (50-1000 mOsm/L), temperature (15-60 °C), and surfactant Triton X-100 (10-500 μM)). Their stability was evaluated by LS by considering the hydrodynamic radius ( R h ), intensity of scattered light ( I ), and the shape parameter ( ρ ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS-citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50-1000 mOsm/L, pH 4-10) had no significant effect on the R h (=100-130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.

Plant-derived nanovesicles (NVs) have attracted interest due to their anti-inflammatory, anticancer and antioxidative properties and their efficient uptake by human intestinal epithelial cells. Previously we showed that tomato ( Solanum lycopersicum L.) fruit is one of the interesting plant resources from which NVs can be obtained at a high yield. In the course of the isolation of NVs from different batches of tomatoes, using the established differential ultracentrifugation or size-exclusion chromatography methods, we occasionally observed the co-isolation of viral particles. Density gradient ultracentrifugation (gUC), using sucrose or iodixanol gradient materials, turned out to be efficient in the separation of NVs from the viral particles. We applied cryogenic transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM) for the morphological assessment and LC-MS/MS-based proteomics for the protein identification of the gradient fractions. Cryo-TEM showed that a low-density gUC fraction was enriched in membrane-enclosed NVs, while the high-density fractions were rich in rod-shaped objects. Mass spectrometry-based proteomic analysis identified capsid proteins of tomato brown rugose fruit virus, tomato mosaic virus and tomato mottle mosaic virus. In another batch of tomatoes, we isolated tomato spotted wilt virus, potato virus Y and southern tomato virus in the vesicle sample. Our results show the frequent co-isolation of plant viruses with NVs and the utility of the combination of cryo-TEM, SEM and proteomics in the detection of possible viral contamination.

Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes. Therefore, our goal was to compare the cytotoxic effect of LLO Y406A on cancer cells (RT4) and normal urothelial cells (NPU), and to identify which cell membranes are the primary target of LLO Y406A by viability assays, life-cell imaging, fluorescence, and electron microscopy. LLO Y406A decreased viability, altered cell morphology, provoked membrane blebbing, and induced apoptosis in RT4 cells, while it did not affect NPU cells. LLO Y406A did not cause endosomal escape in RT4 cells, while the plasma membrane of RT4 cells was revealed as the primary target of LLO Y406A. It has been concluded that LLO Y406A has the ability to selectively eliminate cancer urothelial cells through pore-forming activity at the plasma membrane, without cytotoxic effects on normal urothelial cells. This promising selective activity merits further testing as an anti-cancer agent.

A major drawback of the IgG capture step is the high cost of the protein A resin. For a better utilization of the resin, a continuous multi-column operation was recently proposed. In this method, accurate detection of leaking IgG is crucial to divert the breakthrough fluid from the waste to the next column and prolong the loading step without product loss. The detection of a breakthrough point as a change in UV absorption is based on a relatively small signal addition of IgGs to the bulk signal of host cell proteins. To achieve specificity, we used a quartz crystal microbalance and immobilized protein A as specific ligand on the sensor surface. We integrated the quartz crystal microbalance sensor in-line after the protein A column for real-time detection of IgGs in the breakthrough fluid. We show that this specific IgG detection in the breakthrough fluid can be more sensitive than with the UV detector. The use of the same product-specific ligand in the affinity column and in the sensor allows simultaneous in-line regeneration of column and sensor in a single step. Such a sensor could support cost-efficient load control during the entire continuous multi-column capture step in downstream processing., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lipid membranes are becoming increasingly popular in synthetic biology due to their biophysical properties and crucial role in communication between different compartments. Several alluring protein-membrane sensors have already been developed, whereas protein logic gates designs on membrane-embedded proteins are very limited. Here we demonstrate the construction of a two-level protein-membrane logic gate with an OR-AND logic. The system consists of an engineered pH-dependent pore-forming protein listeriolysin O and its DARPin-based inhibitor, conjugated to a lipid vesicle membrane. The gate responds to low pH and removal of the inhibitor from the membrane either by switching to a reducing environment, protease cleavage, or any other signal depending on the conjugation chemistry used for inhibitor attachment to the membrane. This unique protein logic gate vesicle system advances generic sensing and actuator platforms used in synthetic biology and could be utilized in drug delivery.

Background: A major bottleneck in drug delivery is the breakdown and degradation of the delivery system through the endosomal/lysosomal network of the host cell, hampering the correct delivery of the drug of interest. In nature, the bacterial pathogen Listeria monocytogenes has developed a strategy to secrete Listeriolysin O (LLO) toxin as a tool to escape the eukaryotic lysosomal system upon infection, allowing it to grow and proliferate unharmed inside the host cell., Results: As a "proof of concept", we present here the use of purified His-LLO H311A mutant protein and its conjugation on the surface of gold nanoparticles to promote the lysosomal escape of 40 nm-sized nanoparticles in mouse embryonic fibroblasts. Surface immobilization of LLO was achieved after specific functionalization of the nanoparticles with nitrile acetic acid, enabling the specific binding of histidine-tagged proteins., Conclusions: Endosomal acidification leads to release of the LLO protein from the nanoparticle surface and its self-assembly into a 300 Å pore that perforates the endosomal/lysosomal membrane, enabling the escape of nanoparticles.

Aerolysin-like pore-forming proteins are an important family of proteins able to efficiently damage membranes of target cells by forming transmembrane pores. They are characterized by a unique domain organization and mechanism of action that involves extensive conformational rearrangements. Although structures of soluble forms of many different members of this family are well understood, the structures of pores and their mechanism of assembly have been described only recently. The pores are characterized by well-defined β-barrels, which are devoid of any vestibular regions commonly found in other protein pores. Many members of this family are bacterial toxins; therefore, structural details of their transmembrane pores, as well as the mechanism of pore formation, are an important base for future drug design. Stability of pores and other properties, such as specificity for some cell surface molecules, make this family of proteins a useful set of molecular tools for molecular recognition and sensing in cell biology.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'., (© 2017 The Author(s).)

Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH., Competing Interests: The authors declare no competing financial interests.

The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6-2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.

Listeria monocytogenes is a food and soil-borne pathogen that secretes a pore-forming toxin listeriolysin O (LLO) as its major virulence factor. We tested the effects of LLO on an intestinal epithelial cell line Caco-2 and compared them to an unrelated pore-forming toxin equinatoxin II (EqtII). Results showed that apical application of both toxins causes a significant drop in transepithelial electrical resistance (TEER), with higher LLO concentrations or prolonged exposure time needed to achieve the same magnitude of response than with EqtII. The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and associated actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was observed. Influx of calcium after pore formation affected the magnitude of the drop in TEER. Both toxins exhibit similar effects on epithelium morphology and physiology. Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concentrations than EqtII. This could favor listerial invasion in hosts resistant to E-cadherin related infection.

Pore formation of cellular membranes is an ancient mechanism of bacterial pathogenesis that allows efficient damaging of target cells. Several mechanisms have been described, however, relatively little is known about the assembly and properties of pores. Listeriolysin O (LLO) is a pH-regulated cholesterol-dependent cytolysin from the intracellular pathogen Listeria monocytogenes, which forms transmembrane β-barrel pores. Here we report that the assembly of LLO pores is rapid and efficient irrespective of pH. While pore diameters at the membrane surface are comparable at either pH 5.5 or 7.4, the distribution of pore conductances is significantly pH-dependent. This is directed by the unique residue H311, which is also important for the conformational stability of the LLO monomer and the rate of pore formation. The functional pores exhibit variations in height profiles and can reconfigure significantly by merging to other full pores or arcs. Our results indicate significant plasticity of large β-barrel pores, controlled by environmental cues like pH.

Membrane Attack Complex/Perforin (MACPF) and Cholesterol-Dependent Cytolysins (CDC) form the MACPF/CDC superfamily of important effector proteins widespread in nature. MACPFs and CDCs were discovered separately with no sequence similarity at that stage being apparent between the two protein families such that they were not, until recently, considered evolutionary related. The breakthrough showing they are came with recent structural work that also shed light on the molecular mechanism of action of various MACPF proteins. Similarity in structural properties and conserved functional features indicate that both protein families have the same evolutionary origin. We will describe the distribution of MACPF/CDC proteins in nature and discuss briefly their similarity and functional role in different biological processes.