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Bacterial nanocellulose (BNC) has gained a lot of attention in recent years due to its nano-size-derived properties. Although it is essentially chemically similar to plant-derived cellulose, it has smaller size and is enriched in free hydroxyl groups, which greatly improve mechanical properties of reinforced paper. However, although BNC has some unique features, it comes at a high price. In this paper, we introduce a new solution for BNC production. We have isolated bacterial nanocellulose directly from agro-industrial waste—mother of vinegar—and used it in the production of paper sheets. We show here that paper sheets made with the addition of only 10% bacte-rial nanocellulose from mother of vinegar substantially improved basic mechanical as well as printing properties of paper.

Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (Pleurotus ostreatus). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it forms 13-meric transmembrane pore complexes. Further, OlyA6 binds 1000 times more strongly to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with PlyB, OlyA6 has potent and selective insecticidal activity against the western corn rootworm. We analysed the histological alterations of the midgut wall columnar epithelium of western corn rootworm larvae fed with OlyA6/PlyB, which showed vacuolisation of the cell cytoplasm, swelling of the apical cell surface into the gut lumen, and delamination of the basal lamina underlying the epithelium. Additionally, cryo-electron microscopy was used to explore the membrane interactions of the OlyA6/PlyB complex using lipid vesicles composed of artificial lipids containing CPE, and western corn rootworm brush border membrane vesicles. Multimeric transmembrane pores were formed in both vesicle preparations, similar to those described for sphingomyelin/cholesterol membranes. These results strongly suggest that the molecular mechanism of insecticidal action of OlyA6/PlyB arises from specific interactions of OlyA6 with CPE, and the consequent formation of transmembrane pores in the insect midgut.

Bacterial nanocellulose (BNC) is a unique natural nanomaterial that shares very few similarities with other natural or industrially produced nanomaterials. BNC can be produced by a variety of bacteria, as a survival aid in different ecological niches. BNC is traditionally produced by static or shaking culture methods, and the ‘mother vinegar’, or biofilm, is a typical example of this product after static vinegar fermentation. BNC has great potential in biomedicine, and recent studies have also demonstrated its use in the papermaking industry. It has nanoscale fiber size and large numbers of free hydroxyl groups, which ensure high inter-fiber hydrogen bonding. Thus, BNC has great potential as a reinforcing material, and is especially applicable for recycled paper and for paper made of nonwoody cellulose fiber. As well as enhancing the strength and durability of paper, modified BNC shows great potential for production of fire resistant and specialized papers. However, the biotechnological aspects of BNC need to be improved to minimize the cost of its production, and to thus make this process economically feasible.

Aegerolysins are small lipid-binding proteins particularly abundant in fungi. Aegerolysins from oyster mushrooms interact with an insect-specific membrane lipid and, together with MACPF proteins produced by the same organism, form pesticidal pore-forming complexes. The specific interaction with the same membrane lipid was recently demonstrated for nigerolysin A2 (NigA2), an aegerolysin from Aspergillus niger. In Aspergillus species, the aegerolysins were frequently found as secreted proteins, indicating their function in fungal defense. Using immunocytochemistry and live-cell imaging we investigated the subcellular localization of the nigerolysins A in A. niger, while their secretion was addressed by secretion prediction and Western blotting. We show that both nigerolysins A are leaderless proteins that reach the cell exterior by an unconventional protein secretion. NigA proteins are evenly distributed in the cytoplasm of fungal hyphae. A detailed bioinformatics analysis of Aspergillus aegerolysins suggests that the same function occurs only in a limited number of aegerolysins. From alignment, analysis of chromosomal loci, orthology, synteny, and phylogeny it follows that the same or a similar function described for pairs of pesticidal proteins of Pleurotus sp. can be expected in species of the subgenus Circumdati, section Nigri, series Nigri, and some other species with adjacent pairs of putative pesticidal proteins.

Many studies evaluated the short-term in vitro toxicity of nanoparticles (NPs), however, long-term effects are still not adequately understood. Here, we investigated the potential toxic effects of biomedical (polyacrylic acid and polyethylenimine coated magnetic NPs) and two industrial (SiO2 and TiO2) NPs following different short-term and long-term exposure protocols on two physiologically different in vitro models that are able to differentiate: L6 rat skeletal muscle cell line and biomimetic normal porcine urothelial (NPU) cells. We show that L6 cells are more sensitive to NP exposure then NPU cells. Transmission electron microscopy revealed an uptake of NPs into L6 cells but not NPU cells. In L6 cells, we obtained a dose-dependent reduction in cell viability and increased reactive oxygen species (ROS) formation after 24 h. Following continuous exposure, more stable TiO2 and polyacrylic acid (PAA) NPs increased levels of nuclear factor Nrf2 mRNA, suggesting an oxidative damage-associated response. Furthermore, internalized magnetic PAA and TiO2 NPs hindered the differentiation of L6 cells. We propose the use of L6 skeletal muscle cells and NPU cells as a novel approach for assessment of the potential long-term toxicity of relevant NPs that are found in the blood and/or can be secreted into the urine.

The aegerolysin proteins ostreolysin A6, pleurotolysin A2 and erylysin A are produced by mushrooms of the genus Pleurotus. These aegerolysins can interact specifically with sphingolipid-enriched membranes. In particular, they strongly bind insect cells and to artificial lipid membranes that contain physiologically relevant concentrations of the main invertebrate-specific sphingolipid, ceramide phosphoethanolamine. Moreover, the aegerolysins permeabilise these membranes when combined with their protein partner pleurotolysin B, which contains a membrane-attack-complex/perforin domain. These aegerolysin/ pleurotolysin B complexes show strong and selective toxicity towards western corn rootworm larvae and adults and Colorado potato beetle larvae. Their insecticidal activities arise through aegerolysin binding to ceramide phosphoethanolamine in the insect midgut. This mode of membrane binding is different from those described for similar aegerolysin-based complexes of bacterial origin (e.g., Cry34Ab1/Cry35Ab1), or other Bacillus thuringiensis proteinaceous crystal toxins, which associate with protein receptors. The ability of Pleurotus aegerolysins to specifically interact with sphingolipid-enriched domains in mammalian cells can be further exploited to visualize lipid rafts in living cells, and to treat certain types of tumours and metabolic disorders. Finally, these proteins can strongly enhance fruiting initiation of P. ostreatus even when applied externally. In this review, we summarise the current knowledge of the potential biotechnological and biomedical applications of the Pleurotus aegerolysins, either alone or when complexed with pleurotolysin B, with special emphasis on their bioinsecticidal effects.

The growth of oyster mushroom (Pleurotus ostreatus) on pulp and paper industry wastes was studied. Specifically, the question was investigated whether solid-state fermentation ofP. ostreatuson paper-mill deinking sludge and primary sludge substrates is appropriate for production of enzymes, relevant to the pulp and paper industry. Following fermentation, extracellular protein was extracted and the specific activities of four enzymes were determined, namely, the cellulase, xylanase, lipase and peroxidase. Furthermore, the effects of the pH of the extraction buffer on these enzyme activities were determined, along with the effects of the incubation time. The data show thatP. ostreatuscan grow on solid wastes from the pulp and paper industry, which could help to minimize the waste volume and to decrease the ecological impact. Furthermore, the solid wastes in focus are good substrates for the production of commercially interesting enzymes.

Aegerolysins ostreolysin A (OlyA) and pleurotolysin A (PlyA), and pleurotolysin B (PlyB) with the membrane-attack-complex/perforin domain are proteins from the mushroom genus Pleurotus. Upon binding to sphingomyelin/cholesterol-enriched membranes, OlyA and PlyA can recruit PlyB to form multimeric bi-component transmembrane pores. Recently, Pleurotus aegerolysins OlyA, PlyA2 and erylysin A (EryA) were demonstrated to preferentially bind to artificial lipid membranes containing 50 mol% ceramide phosphoethanolamine (CPE), the main sphingolipid in invertebrate cell membranes. In this study, we demonstrate that OlyA6, PlyA2 and EryA bind to insect cells and to artificial lipid membranes with physiologically relevant CPE concentrations. Moreover, these aegerolysins permeabilize these membranes when combined with PlyB. These aegerolysin/PlyB complexes show selective toxicity toward western corn rootworm larvae and adults and Colorado potato beetle larvae. These data strongly suggest that these aegerolysin/PlyB complexes recognize CPE as their receptor molecule in the insect midgut. This mode of binding is different from those described for similar aegerolysin-based bacterial complexes, or other Bacillus thuringiensis Cry toxins, which have protein receptors. Targeting of Pleurotus aegerolysins to CPE and formation of transmembrane pores in concert with PlyB suggest the use of aegerolysin/PlyB complexes as novel biopesticides for the control of western corn rootworm and Colorado potato beetle.

Six Monascus purpureus (red mould) strains were cultivated on brown rice and millet as substrates. They underwent strain selection for high lovastatin and pigment production, and low citrinin mycotoxin production, with particular reference to potential for millet as substrate. For most of these strains, substrate dry matter loss was 54–60% on rice and 46–48% on millet, although the ‘MOPU GS1’ strain showed 18% and 17% dry matter loss, respectively. ‘MOPU GS1’ was also the only strain with detectable levels of lovastatin (1.3 and 1.6 mg lovastatin/g substrate dry weight [dw], respectively) and citrinin under the limit of detection. In the other strains, citrinin varied from 0.3 to 18.2 mg/g substrate dw. Among the six strains, ‘EBY3’ provided high pigment production when grown on rice, although it produced 1.1 mg citrinin/g substrate dw. Millet showed good potential as an alternative substrate to rice, due to higher lovastatin and lower citrinin production; however, rice was the better substrate for production of M. purpureus pigments. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-019-03820-8) contains supplementary material, which is available to authorized users.

The lipid raft hypothesis emerged as a need to explain the lateral organization and behavior of lipids in the environment of biological membranes. The idea, that lipids segregate in biological membranes to form liquid-disordered and liquid-ordered states, was faced with a challenge: to show that lipid-ordered domains, enriched in sphingomyelin and cholesterol, actually exist in vivo. A great deal of indirect evidence and the use of lipid-binding probes supported this idea, but there was a lack of tools to demonstrate the existence of such domains in living cells. A whole new toolbox had to be invented to biochemically characterize lipid rafts and to define how they are involved in several cellular functions. A potential solution came from basic biochemical experiments in the late 1970s, showing that some mushroom extracts exert hemolytic activities. These activities were later assigned to aegerolysin-based sphingomyelin/cholesterol-specific cytolytic protein complexes. Recently, six sphingomyelin/cholesterol binding proteins from different mushrooms have been identified and have provided some insight into the nature of sphingomyelin/cholesterol-rich domains in living vertebrate cells. In this review, we dissect the accumulated knowledge and introduce the mushroom lipid raft binding proteins as molecules of choice to study the dynamics and origins of these liquid-ordered domains in mammalian cells.

Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membrane nanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise both microvesicles with diameters between 100nm and 1μm, and larger vesicles of around 10-μm diameter in Madin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusion protein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+ signalling, and does not compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclear origin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid species were identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholesterol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelin membrane nanodomains induces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV shedding from the cells. We hypothesize that these effects are accounted for by changes of local membrane curvature upon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interesting model for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.

Ceramide phosphoethanolamine (CPE) is the major sphingolipid in invertebrates and in some bacterial species. It has been also detected in mammalian cells, although only in trace amounts. Complete understanding of the biophysical and physiological relevance of CPE is still lacking, and its biological role is still an open question. CPE differs in its biosynthetic mechanisms from sphingomyelin, due to the specific CPE synthase in invertebrates. In contrast to well-established sphingomyelin/cholesterol interactions that result in the formation of ordered membrane domains, the formation of ordered CPE/cholesterol domains is not favored. CPE might be crucial for the early development of Drosophila melanogaster, and it might be involved in the developmental stages of Trypanosoma brucei. As a Bacteroidetes-associated sphingolipid, CPE might also be involved in maintenance of these bacteria in their ecological niches. Therefore, efficient detection of CPE in biological systems is needed to better define its distribution and biological role(s).

The mushroom Pleurotus ostreatus has been reported to produce the hemolytic proteins ostreolysin (OlyA), pleurotolysin A (PlyA) and pleurotolysin B (PlyB). The present study of the native and recombinant proteins dissects out their lipid-binding characteristics and their roles in lipid binding and membrane permeabilization. Using lipid-binding studies, permeabilization of erythrocytes, large unilamellar vesicles of various lipid compositions, and electron microscopy, we show that OlyA, a PlyA homolog, preferentially binds to membranes rich in sterol and sphingomyelin, but it does not permeabilize them. The N-terminally truncated Δ48PlyB corresponds to the mature and active form of native PlyB, and it has a membrane attack complex-perforin (MACPF) domain. Δ48PlyB spontaneously oligomerizes in solution, and binds weakly to various lipid membranes but is not able to perforate them. However, binding of Δ48PlyB to the cholesterol and sphingomyelin membranes, and consequently, their permeabilization is dramatically promoted in the presence of OlyA. On these membranes, Δ48PlyB and OlyA form predominantly 13-meric oligomers. These are rosette-like structures with a thickness of ∼9 nm from the membrane surface, with 19.7 nm and 4.9 nm outer and inner diameters, respectively. When present on opposing vesicle membranes, these oligomers can dimerize and thus promote aggregation of vesicles. Based on the structural and functional characteristics of Δ48PlyB, we suggest that it shares some features with MACPF/cholesterol-dependent cytolysin (CDC) proteins. OlyA is obligatory for the Δ48PlyB permeabilization of membranes rich in cholesterol and sphingomyelin.

Proteins of the aegerolysin family span many kingdoms of life. They are relatively widely distributed in bacteria and fungi, but also appear in plants, protozoa and insects. Despite being produced in abundance in cells at specific developmental stages and present in secretomes, only a few aegerolysins have been studied in detail. In particular, their organism-specific physiological roles are intriguing. Here, we review published findings to date on the distribution, molecular interactions and biological activities of this family of structurally and functionally versatile proteins, the aegerolysins.

Comparative lipidomic studies were performed across the RT4 versus T24 urothelial cancer cell lines, as models for noninvasive urothelial papilloma cells (with a relatively high level of differentiation) and invasive urothelial carcinoma cells (with low level of differentiation), respectively. The aim was to investigate the differences in lipid profile associated with different levels of urothelial cancer cell invasiveness. The cellular lipidomes were characterized using our previously developed joint methodology of liquid chromatography-mass spectrometry and high-resolution nuclear magnetic resonance, which included analysis of the phospholipids and ceramide-based glycosphingolipids. This study shows that the invasive T24 cells have 3-fold lower levels of 1-alkyl (ether)-2-acyl phosphocholine species, which are accompanied by greater length and higher unsaturation of acyl chains of several lipid classes. Moreover, d18:1-based glycosphingolipids show different profiles; in particular, α-hydroxylated glucosylceramides have lower levels in the T24 cells, along with increased lactosyl ceramides. These differences between RT4 and T24 cells suggest significantly different organization of the cellular membranes, which can affect the membrane fluidity and membrane-dependent functions, and contribute to the lower stiffness of plasma membrane and reduced cell-cell adhesion required for movement and invasiveness of these T24 urothelial carcinoma cells with a high metastatic potential.

Mammalian systems can be a subject of cellular engineering in a similar way to bacterial cells. Our team decided to modify the existing mammalian cell signalling network of the innate immune response to bacterial infection. Binding of bacterial components (pathogen associated molecular patterns, or PAMPs) to a family of Toll-like receptors (TLRs) activates the cells of the immune system but an exaggerated response may lead to systemic inflammation and sepsis which is often fatal. We designed a feedback loop, which inhibits the signalling cascade at the weak spot – protein MyD88 – which is the consensus adaptor protein of the surface-expressed TLRs. A mathematical model of cell activation with an engineered feedback loop predicts a decrease of cellular activation after repeated stimulation of TLR. We have prepared 26 BioBricks for mammalian systems and deposited them into the BioBrick Registry at MIT. Mammalian cells transfected with the feedback loop construct performed as designed with a decrease in the cellular response upon repeated stimulation with PAMPs. Cell activation decreased without completely abolishing the responsiveness to the bacterial stimulus, therefore our engineered system represents an artificial type of immunotolerance. The Slovenian team was composed of seven undergraduate students of microbiology and biochemistry and five mentors from the National Institute of Chemistry and University of Ljubljana Faculty of Chemistry and Chemical Technology.

The aegerolysin protein family (from aegerolysin of the mushroom Agrocybe aegerita) comprises proteins of ∼15–20 kDa from various eukaryotic and bacterial taxa. Aegerolysins are inconsistently distributed among fungal species, and variable numbers of homologs have been reported for species within the same genus. As such noncore proteins, without a member of a protein family in each of the sequenced fungi, they can give insight into different species-specific processes. Some aegerolysins have been reported to be hemolytically active against mammalian erythrocytes. However, some function as bi-component proteins that have membrane activity in concert with another protein that contains a membrane attack complex/perforin domain. The function of most of aegerolysins is unknown, although some have been suggested to have a role in development of the organism. Potential biotechnological applications of aegerolysins are already evident, despite the limited scientific knowledge available at present. Some mushroom aegerolysins, for example, can be used as markers to detect and label specific membrane lipids. Others can be used as biomarkers of fungal exposure, where their genes can serve as targets for detection of fungi and their progression during infectious diseases. Antibodies against aegerolysins can also be raised as immuno-diagnostic tools. Aegerolysins have been shown to serve as a species determination tool for fungal phytopathogen isolates in terms of some closely related species, where commonly used internal transcribed spacer barcoding has failed. Moreover, strong promoters that regulate aegerolysin genes can promote secretion of heterologous proteins from fungi and have been successfully applied in simultaneous multi-gene expression techniques.

Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-mCherry and the sphingomyelin-specific markers equinatoxin II-Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-mCherry in comparison with these other proteins. Furthermore, we show that OlyA-mCherry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1-positive structures. No binding to membranes could be seen when OlyA-mCherry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-mCherry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

Background. Titanium dioxide (TiO2) is considered as an inert and safe material and has been used in many applications for decades. However, with the development of nanotechnologies TiO2 nanoparticles, with numerous novel and useful properties, are increasingly manufactured and used. Therefore increased human and environmental exposure can be expected, which has put TiO2 nanoparticles under toxicological scrutiny. Mechanistic toxicological studies show that TiO2 nanoparticles predominantly cause adverse effects via induction of oxidative stress resulting in cell damage, genotoxicity, inflammation, immune response etc. The extent and type of damage strongly depends on physical and chemical characteristics of TiO2 nanoparticles, which govern their bioavailability and reactivity. Based on the experimental evidence from animal inhalation studies TiO2 nanoparticles are classified as "possible carcinogenic to humans" by the International Agency for Research on Cancer and as occupational carcinogen by the National Institute for Occupational Safety and Health. The studies on dermal exposure to TiO2 nanoparticles, which is in humans substantial through the use of sunscreens, generally indicate negligible transdermal penetration; however data are needed on long-term exposure and potential adverse effects of photo-oxidation products. Although TiO2 is permitted as an additive (E171) in food and pharmaceutical products we do not have reliable data on its absorption, distribution, excretion and toxicity on oral exposure. TiO2 may also enter environment, and while it exerts low acute toxicity to aquatic organisms, upon long-term exposure it induces a range of sub-lethal effects. Conclusions. Until relevant toxicological and human exposure data that would enable reliable risk assessment are obtained, TiO2 nanoparticles should be used with great care.

Membrane rafts are transient and unstable membrane microdomains that are enriched in sphingolipids, cholesterol, and specific proteins. They are involved in intracellular trafficking, signal transduction, pathogen entry, and attachment of various ligands. Increasing experimental evidence on the crucial biological roles of membrane rafts under normal and pathological conditions require new techniques for their structural and functional characterization. In particular, fluorescence-labeled cytolytic proteins that interact specifically with molecules enriched in rafts are of increasing interest. Cholera toxin subunit B interacts specifically with raft-residing ganglioside G(M1), and it has long been the lipid probe of choice for membrane rafts. Recently, four new pore-forming toxins have been proposed as selective raft markers: (i) equinatoxin II, a cytolysin from the sea anemone Actinia equina, which specifically recognizes free and membrane-embedded sphingomyelin; (ii) a truncated non-toxic mutant of a cytolytic protein, lysenin, from the earthworm Eisenia foetida, which specifically recognizes sphingomyelin-enriched membrane domains; (iii) a non-toxic derivative of the cholesterol-dependent cytolysin perfringolysin O, from the bacterium Clostridium perfringens, which selectively binds to membrane domains enriched in cholesterol; and (iv) ostreolysin, from the mushroom Pleurotus ostreatus, which does not bind to a single raft-enriched lipid component, but requires a specific combination of two of the most important raft-residing lipids: sphingomyelin and cholesterol. Nontoxic, raft-binding derivatives of cytolytic proteins have already been successfully used to explore both the structure and function of membrane rafts, and of raft-associated molecules. Here, we review these four new derivatives of pore-forming toxins as new putative markers of these membrane microdomains.