New Insights into Interactions between Mushroom Aegerolysins and Membrane Lipids.

Aegerolysins are a family of proteins that recognize and bind to specific membrane lipids or lipid domains; hence they can be used as membrane lipid sensors. Although aegerolysins are distributed throughout the tree of life, the most studied are those produced by the fungal genus Pleurotus. Most of the aegerolysin-producing mushrooms code also for proteins containing the membrane attack complex/perforin (MACPF)-domain. The combinations of lipid-sensing aegerolysins and MACPF protein partners are lytic for cells harboring the aegerolysin membrane lipid receptor and can be used as ecologically friendly bioinsecticides. In this work, we have recombinantly expressed four novel aegerolysin/MACPF protein pairs from the mushrooms Heterobasidion irregulare, Trametes versicolor, Mucidula mucida, and Lepista nuda, and compared these proteins with the already studied aegerolysin/MACPF protein pair ostreolysin A6-pleurotolysin B from P. ostreatus. We show here that most of these new mushroom proteins can form active aegerolysin/MACPF cytolytic complexes upon aegerolysin binding to membrane sphingolipids. We further disclose that these mushroom aegerolysins bind also to selected glycerophospholipids, in particular to phosphatidic acid and cardiolipin; however, these interactions with glycerophospholipids do not lead to pore formation. Our results indicate that selected mushroom aegerolysins show potential as new molecular biosensors for labelling phosphatidic acid.

A single point mutation expands the applicability of ostreolysin A6 in biomedicine.

An aegerolysin protein ostreolysin A6 (OlyA6) binds to cholesterol-complexed sphingomyelin and can be used for specific labelling of lipid rafts. In addition, OlyA6 interacts with even higher affinity with ceramide phosphoethanolamine (CPE), a sphingolipid that dominates in invertebrate cell membranes. In the presence of pleurotolysin B, a protein bearing the membrane-attack complex/perforin domain, OlyA6 forms pores in insect midgut cell membranes and acts as a potent bioinsecticide. It has been shown that a point mutation of glutamate 69 to alanine (E69A) allows OlyA6 to bind to cholesterol-free sphingomyelin. Using artificial lipid membranes and mammalian MDCK cells, we show that this mutation significantly enhances the interaction of OlyA6 with sphingomyelin and CPE, and allows recognition of these sphingolipids even in the absence of cholesterol. Our results suggest that OlyA6 mutant E69A could serve as complementary tool to detect and study cholesterol-associated and free sphingomyelin or CPE in membranes. However, the mutation does not improve the membrane-permeabilizing activity after addition of pleurotolysin B, which was confirmed in toxicity tests on insect and mammalian cell lines, and on Colorado potato beetle larvae.

Towards Understanding the Function of Aegerolysins.

Aegerolysins are remarkable proteins. They are distributed over the tree of life, being relatively widespread in bacteria and fungi, but also present in some insects, plants, protozoa, and viruses. Despite their abundance in cells of certain developmental stages and their presence in secretomes, only a few aegerolysins have been studied in detail. Their function, in particular, is intriguing. Here, we summarize previously published findings on the distribution, molecular interactions, and function of these versatile aegerolysins. They have very diverse protein sequences but a common fold. The machine learning approach of the AlphaFold2 algorithm, which incorporates physical and biological knowledge of protein structures and multisequence alignments, provides us new insights into the aegerolysins and their pore-forming partners, complemented by additional genomic support. We hypothesize that aegerolysins are involved in the mechanisms of competitive exclusion in the niche.

Ceramide Phosphoethanolamine as a Possible Marker of Periodontal Disease.

Periodontal disease is a chronic oral inflammatory disorder initiated by pathobiontic bacteria found in dental plaques-complex biofilms on the tooth surface. The disease begins as an acute local inflammation of the gingival tissue (gingivitis) and can progress to periodontitis, which eventually leads to the formation of periodontal pockets and ultimately results in tooth loss. The main problem in periodontology is that the diagnosis is based on the assessment of the already obvious tissue damage. Therefore, it is necessary to improve the current diagnostics used to assess periodontal disease. Using lipidomic analyses, we show that both crucial periodontal pathogens, Porphyromonas gingivalis and Tannerella forsythia, synthesize ceramide phosphoethanolamine (CPE) species, membrane sphingolipids not typically found in vertebrates. Previously, it was shown that this particular lipid can be specifically detected by an aegerolysin protein, erylysin A (EryA). Here, we show that EryA can specifically bind to CPE species from the total lipid extract from P. gingivalis. Furthermore, using a fluorescently labelled EryA-mCherry, we were able to detect CPE species in clinical samples of dental plaque from periodontal patients. These results demonstrate the potential of specific periodontal pathogen-derived lipids as biomarkers for periodontal disease and other chronic inflammatory diseases.

Dissecting Out the Molecular Mechanism of Insecticidal Activity of Ostreolysin A6/Pleurotolysin B Complexes on Western Corn Rootworm.

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.

What Can Mushroom Proteins Teach Us about Lipid Rafts?

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.

Aegerolysins from the fungal genus Pleurotus - Bioinsecticidal proteins with multiple potential applications.

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.

Unconventional Secretion of Nigerolysins A from Aspergillus Species.

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.

Proposing Urothelial and Muscle In Vitro Cell Models as a Novel Approach for Assessment of Long-Term Toxicity of Nanoparticles.

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.

Comparison of lovastatin, citrinin and pigment production of different Monascus purpureus strains grown on rice and millet.

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.

Ceramide phosphoethanolamine, an enigmatic cellular membrane sphingolipid.

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).

Pore-forming protein complexes from Pleurotus mushrooms kill western corn rootworm and Colorado potato beetle through targeting membrane ceramide phosphoethanolamine.

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.

Aegerolysins: Lipid-binding proteins with versatile functions.

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.

Characterisation of plasmalemmal shedding of vesicles induced by the cholesterol/sphingomyelin binding protein, ostreolysin A-mCherry.

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.

Comparative lipidomic study of urothelial cancer models: association with urothelial cancer cell invasiveness.

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.

Fungal aegerolysin-like proteins: distribution, activities, and applications.

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.

Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein.

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-ß-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.

Membrane cholesterol and sphingomyelin, and ostreolysin A are obligatory for pore-formation by a MACPF/CDC-like pore-forming protein, pleurotolysin B.

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.

Titanium dioxide in our everyday life; is it safe?

BACKGROUND: Titanium dioxide (TiO(2)) is considered as an inert and safe material and has been used in many applications for decades. However, with the development of nanotechnologies TiO(2) nanoparticles, with numerous novel and useful properties, are increasingly manufactured and used. Therefore increased human and environmental exposure can be expected, which has put TiO(2) nanoparticles under toxicological scrutiny. Mechanistic toxicological studies show that TiO(2) 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 TiO(2) nanoparticles, which govern their bioavailability and reactivity. Based on the experimental evidence from animal inhalation studies TiO(2) 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 TiO(2) 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 TiO(2) 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. TiO(2) 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, TiO(2) nanoparticles should be used with great care.