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.

Kinetically Stable Triglyceride-Based Nanodroplets and Their Interactions with Lipid-Specific Proteins.

Understanding of the interactions between proteins and natural and artificially prepared lipid membrane surfaces and embedded nonpolar cores is important in studies of physiological processes and their pathologies and is applicable to nanotechnologies. In particular, rapidly growing interest in cellular droplets defines the need for simplified biomimetic lipid model systems to overcome in vivo complexity and variability. We present a protocol for the preparation of kinetically stable nanoemulsions with nanodroplets composed of sphingomyelin (SM) and cholesterol (Chol), as amphiphilic surfactants, and trioleoylglycerol (TOG), at various molar ratios. To prepare stable SM/Chol-coated monodisperse lipid nanodroplets, we modified a reverse phase evaporation method and combined it with ultrasonication. Lipid composition, ζ-potential, gyration and hydrodynamic radius, shape, and temporal stability of the lipid nanodroplets were characterized and compared to extruded SM/Chol large unilamellar vesicles. Lipid nanodroplets and large unilamellar vesicles with theoretical SM/Chol/TOG molar ratios of 1/1/4.7 and 4/1/11.7 were further investigated for the orientational order of their interfacial water molecules using a second harmonic scattering technique, and for interactions with the SM-binding and Chol-binding pore-forming toxins equinatoxin II and perfringolysin O, respectively. The surface characteristics (ζ-potential, orientational order of interfacial water molecules) and binding of these proteins to the nanodroplet SM/Chol monolayers were similar to those for the SM/Chol bilayers of the large unilamellar vesicles and SM/Chol Langmuir monolayers, in terms of their surface structures. We propose that such SM/Chol/TOG nanoparticles with the required lipid compositions can serve as experimental models for monolayer membrane to provide a system that imitates the natural lipid droplets.

Pore formation by actinoporins, cytolysins from sea anemones.

Actinoporins (APs) from sea anemones are ~20 kDa pore forming toxins with a ß-sandwich structure flanked by two α-helices. The molecular mechanism of APs pore formation is composed of several well-defined steps. APs bind to membrane by interfacial binding site composed of several aromatic amino acid residues that allow binding to phosphatidylcholine and specific recognition of sphingomyelin. Subsequently, the N-terminal α-helix from the ß-sandwich has to be inserted into the lipid/water interphase in order to form a functional pore. Functional studies and single molecule imaging revealed that only several monomers, 3-4, oligomerise to form a functional pore. In this model the α-helices and surrounding lipid molecules build toroidal pore. In agreement, AP pores are transient and electrically heterogeneous. On the contrary, crystallized oligomers of actinoporin fragaceatoxin C were found to be composed of eight monomers with no lipids present between the adjacent α-helices. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Maur Dalla Serra and Franco Gambale.

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.

Pitfalls in Size Characterization of Soft Particles by Dynamic Light Scattering Online Coupled to Asymmetrical Flow Field-Flow Fractionation.

An asymmetrical flow field-flow fractionation (AF4) technique coupled to a multiangle light scattering (MALS) detector with an embedded dynamic light scattering (DLS) module was introduced to study the size characteristics and shape of soft particles of various size and type: polystyrene nanosphere size standards, lipid droplets (LDs), and large unilamellar vesicles (LUVs). A range of flow velocities through the LS detector, at which accurate hydrodynamic size can be extracted from the DLS in flow mode, was studied since the particles subjected to a longitudinal flow exhibit not only the Brownian motion due to diffusion but also the translational movement. In addition, the impact of the longitudinal flow velocity on the shape of the artificial LUV of two different sizes and two different compositions was studied by MALS. For comparison, the conventional batch DLS and static light scattering (SLS) experiments without prior sample separation by size were performed. From a combination of batch and flow light scattering results, we concluded that the passage flow velocities at the detector used in this study, 0.2, 0.5, and 1 mL/min, have no significant impact on the shape of spherical vesicles; however, the flow DLS experiments give accurate hydrodynamic radius (Rh) only at the lowest investigated passage flow rate at the detector (0.2 mL/min). With increasing rate of passage flow at the DLS detector, the error in the accuracy of the Rh determination rapidly increases. The error in Rh depends solely on the detector flow rate and particle size but not on the type of the soft particle.

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.

Fungal MACPF-like proteins and aegerolysins: bi-component pore-forming proteins?

Proteins with membrane-attack complex/perforin (MACPF) domains are found in almost all kingdoms of life, and they have a variety of biological roles, including defence and attack, organism development, and cell adhesion and signalling. The distribution of these proteins in fungi appears to be restricted to some Pezizomycotina and Basidiomycota species only, in correlation with another group of proteins with unknown biological function, known as aegerolysins. These two protein groups coincide in only a few species, and they might operate in concert as cytolytic bi-component pore-forming agents. Representative proteins here include pleurotolysin B, which has a MACPF domain, and the aegerolysin-like protein pleurotolysin A, and the very similar ostreolysin A, which have been purified from oyster mushroom (Pleurotus ostreatus). These have been shown to act in concert to perforate natural and artificial lipid membranes with high cholesterol and sphingomyelin content. The aegerolysin-like proteins provide the membrane cholesterol/sphingomyelin selectivity and recruit oligomerised pleurotolysin B molecules, to create a membrane-inserted pore complex. The resulting protein structure has been imaged with electron microscopy, and it has a 13-meric rosette-like structure, with a central lumen that is ~4-5 nm in diameter. The opened transmembrane pore is non-selectively permeable for ions and smaller neutral solutes, and is a cause of cytolysis of a colloid-osmotic type. The biological significance of these proteins for the fungal life-style is discussed.

Membrane damage by an α-helical pore-forming protein, Equinatoxin II, proceeds through a succession of ordered steps.

Actinoporin equinatoxin II (EqtII) is an archetypal example of α-helical pore-forming toxins that porate cellular membranes by the use of α-helices. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal α-helix into the lipid bilayer. We studied these separate steps with an EqtII triple cysteine mutant. The mutant was engineered to monitor the insertion of the N terminus into the lipid bilayer by labeling Cys-18 with a fluorescence probe and at the same time to control the flexibility of the N-terminal region by the disulfide bond formed between cysteines introduced at positions 8 and 69. The insertion of the N terminus into the membrane proceeded shortly after the toxin binding and was followed by oligomerization. The oxidized, non-lytic, form of the mutant was still able to bind to membranes and oligomerize at the same level as the wild-type or the reduced form. However, the kinetics of the N-terminal helix insertion, the release of calcein from erythrocyte ghosts, and hemolysis of erythrocytes was much slower when membrane-bound oxidized mutant was reduced by the addition of the reductant. Results show that the N-terminal region needs to be inserted in the lipid membrane before the oligomerization into the final pore and imply that there is no need for a stable prepore formation. This is different from ß-pore-forming toxins that often form ß-barrel pores via a stable prepore complex.

Ostreopexin: a hemopexin fold protein from the oyster mushroom, Pleurotus ostreatus.

Proteins with hemopexin repeats are widespread in viruses, prokaryotes and eukaryotes. We report here for the first time the existence of a protein in fungi with the four-bladed ß-propeller fold that is typical for hemopexin-like proteins. This protein was isolated from the edible basidiomycetous fungus Pleurotus ostreatus and is named ostreopexin. It binds to Ni(2+)-NTA-agarose, and is structurally and functionally very similar to PA2 albumins isolated from legume seeds and the hemopexin fold protein from rice. Like these plant proteins, ostreopexin shows reversible binding to hemin with moderate affinity, but does not bind to polyamines. We suggest that ostreopexin participates in intracellular management of metal (II or III)-chelates.

Targeted lipid analysis of haemolytic mycelial extracts of Aspergillus niger.

Ethanolic extracts of mycelia from Aspergillus niger (strain N402) grown in liquid media were observed to have haemolytic activity on bovine erythrocytes. This haemolytic activity decreased significantly during the time of growth (1-3 days). Moreover, when A. niger was grown on carbon-deprived medium, the efficiency of this haemolytic activity in the ethanolic extracts was much lower than when grown in carbon-enriched medium, and became almost undetectable after 3 days of growth in carbon-deprived medium. The lipid composition of these ethanolic extracts was analysed by liquid chromatography-electrospray ionisation tandem mass spectrometry. This haemolytic activity can be mainly linked to the relative levels of the molar ratios of the unsaturated fatty acids and lysophosphatidylcholines.

A toxin-based probe reveals cytoplasmic exposure of Golgi sphingomyelin.

Although sphingomyelin is an important cellular lipid, its subcellular distribution is not precisely known. Here we use a sea anemone cytolysin, equinatoxin II (EqtII), which specifically binds sphingomyelin, as a new marker to detect cellular sphingomyelin. A purified fusion protein composed of EqtII and green fluorescent protein (EqtII-GFP) binds to the SM rich apical membrane of Madin-Darby canine kidney (MDCK) II cells when added exogenously, but not to the SM-free basolateral membrane. When expressed intracellularly within MDCK II cells, EqtII-GFP colocalizes with markers for Golgi apparatus and not with those for nucleus, mitochondria, endoplasmic reticulum or plasma membrane. Colocalization with the Golgi apparatus was confirmed by also using NIH 3T3 fibroblasts. Moreover, EqtII-GFP was enriched in cis-Golgi compartments isolated by gradient ultracentrifugation. The data reveal that EqtII-GFP is a sensitive probe for membrane sphingomyelin, which provides new information on cytosolic exposure, essential to understand its diverse physiological roles.

EPR and FTIR studies reveal the importance of highly ordered sterol-enriched membrane domains for ostreolysin activity.

Ostreolysin is a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), which recognizes specifically and binds to raft-like sterol-enriched membrane domains that exist in the liquid-ordered phase. Its binding can be abolished by micromolar concentrations of lysophospholipids and fatty acids. The membrane activity of ostreolysin, however, does not completely correlate with the ability of a certain sterol to induce the formation of a liquid-ordered phase, suggesting that the protein requires an additional structural organization of the membrane to exert its activity. The aim of this study was to further characterize the lipid membranes that facilitate ostreolysin binding by analyzing their lipid phase domain structure. Fourier-transformed infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR) were used to analyze the ordering and dynamics of membrane lipids and the membrane domain structure of a series of unilamellar liposomes prepared by systematically changing the lipid components and their ratios. Our results corroborate the earlier conclusion that the average membrane fluidity of ostreolysin-susceptible liposomes alone cannot account for the membrane activity of the protein. Combined with previous data computer-aided interpretation of EPR spectra strongly suggests that chemical properties of membrane constituents, their specific distribution, and physical characteristics of membrane nanodomains, resulting from the presence of sterol and sphingomyelin (or a highly ordered phospholipid, dipalmitoylphosphatidylcholine), are essential prerequisites for ostreolysin membrane binding and pore-formation.

Cytolytic proteins from cnidarians - an overview.

Cnidarians, mostly soft-bodied water organisms, produce several classes of toxins deployed in biological warfare or signalling. Cytolytic toxins, that form pores in cell membranes, form a significant part of their "weaponry". Here, we describe the physiological relevance of membrane permeabilization, and present basic data on those proteinaceous cnidarian cytolysins proven or presumed to damage cell membranes by pore formation. We describe cytolysins that have been at least partially characterized, both functionally and structurally.

Crystal structure of RahU, an aegerolysin protein from the human pathogen Pseudomonas aeruginosa, and its interaction with membrane ceramide phosphorylethanolamine.

Aegerolysins are proteins produced by bacteria, fungi, plants and protozoa. The most studied fungal aegerolysins share a common property of interacting with membranes enriched with cholesterol in combination with either sphingomyelin or ceramide phosphorylethanolamine (CPE), major sphingolipids in the cell membranes of vertebrates and invertebrates, respectively. However, genome analyses show a particularly high frequency of aegerolysin genes in bacteria, including the pathogenic genera Pseudomonas and Vibrio; these are human pathogens of high clinical relevance and can thrive in a variety of other species. The knowledge on bacterial aegerolysin-lipid interactions is scarce. We show that Pseudomonas aeruginosa aegerolysin RahU interacts with CPE, but not with sphingomyelin-enriched artificial membranes, and that RahU interacts with the insect cell line producing CPE. We report crystal structures of RahU alone and in complex with tris(hydroxymethyl)aminomethane (Tris), which, like the phosphorylethanolamine head group of CPE, contains a primary amine. The RahU structures reveal that the two loops proximal to the amino terminus form a cavity that accommodates Tris, and that the flexibility of these two loops is important for this interaction. We show that Tris interferes with CPE-enriched membranes for binding to RahU, implying on the importance of the ligand cavity between the loops and its proximity in RahU membrane interaction. We further support this by studying the interaction of single amino acid substitution mutants of RahU with the CPE-enriched membranes. Our results thus represent a starting point for a better understanding of the role of P. aeruginosa RahU, and possibly other bacterial aegerolysins, in bacterial interactions with other organisms.

Clinical Pharmacist Services on Admission to a Large Correctional Center.

The clinical role of pharmacists in a correctional facility has not been fully described. We report the proportion of patients assessed by a pharmacist within 48 hours of admission to a large correctional facility. Of those assessed, the frequency and type of pharmacist interventions were described. A retrospective chart review was conducted for patients admitted to the Edmonton Remand Center (ERC) from September to November 2017. From 1,500 patients, 518 (34.5%) were assessed by a pharmacist, and 511 (98.6%) of those received one or more pharmacist interventions. Interventions were most commonly health care provider interactions (89.0%) and drug therapy interventions (76.1%). ERC pharmacists assessed a sizable proportion of admitted patients, with most receiving at least one pharmacist intervention.

Kinetics of cholesterol extraction from lipid membranes by methyl-beta-cyclodextrin--a surface plasmon resonance approach.

The kinetics of cholesterol extraction from cellular membranes is complex and not yet completely understood. In this paper we have developed an experimental approach to directly monitor the extraction of cholesterol from lipid membranes by using surface plasmon resonance and model lipid systems. Methyl-beta-cyclodextrin was used to selectively remove cholesterol from large unilamellar vesicles of various compositions. The amount of extracted cholesterol is highly dependent on the composition of lipid membrane, i.e. the presence of sphingomyelin drastically reduced and slowed down cholesterol extraction by methyl-beta-cyclodextrin. This was confirmed also in the erythrocyte ghosts system, where more cholesterol was extracted after erythrocytes were treated with sphingomyelinase. We further show that the kinetics of the extraction is mono-exponential for mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The kinetics is complex for ternary lipid mixtures composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine, bovine brain sphingomyelin and cholesterol. Our results indicate that the complex kinetics observed in experiments with cells may be the consequence of lateral segregation of lipids in cell plasma membrane.

Clinical Pharmacists in Correctional Facilities: A Literature Review and Future Directions.

The role of pharmacists has undergone a significant transformation. Expanding clinical roles in the community and hospital settings have led to opportunity for correctional facility pharmacists to expand their practice. This literature review identifies past and present roles of correctional pharmacists, along with areas for growth. Peer-reviewed and gray literature is described, outlining current and expanding pharmacist roles from 1997 to 2017. The literature reveals that health care provided in correctional facilities is shifting from a basic level of care to a greater role in inmate health and identifies the challenges and barriers that pharmacists meet. There is strong evidence to support the expanding role of pharmacists as primary care providers in the corrections setting through activities such as direct patient care, health care clinics, and medication management.

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.

Lysophospholipids prevent binding of a cytolytic protein ostreolysin to cholesterol-enriched membrane domains.

Ostreolysin, a 15kDa pore-forming protein from the oyster mushroom (Pleurotus ostreatus), binds specifically to cholesterol-enriched membrane domains existing in the liquid-ordered phase, and lyses cells and lipid vesicles made of cholesterol and sphingomyelin. We have monitored binding of sub-lytic concentrations of ostreolysin to membranes of Chinese Hamster Ovary cells and rat somatotrophs, using primary anti-ostreolysin and fluorescence-labeled secondary antibodies detected by confocal microscopy. Depletion of more than 40% membrane cholesterol content by methyl-beta-cyclodextrin dramatically decreased ostreolysin binding. Immunostaining showed that ostreolysin is not co-localized with raft-binding proteins, cholera toxin B-subunit or caveolin, suggesting that natural membranes display heterogeneity of cholesterol-enriched raft-like microdomains. Impaired ostreolysin binding was also observed after treating the cells with lysophosphatidylinositol. Effects of lysophosphatidylinositol on binding of ostreolysin to immobilized large sphingomyelin/cholesterol (1/1, mol/mol) unilamellar vesicles were studied by a surface plasmon resonance technique. Injection of ostreolysin during the lysophosphatidylinositol dissociation phase showed an inverse relationship between ostreolysin binding and the quantity of lysophosphatidylinositol in the membranes of lipid vesicles. It was concluded that lysophospholipids prevent binding of ostreolysin to cell and to artificial lipid membranes resembling lipid rafts, by partitioning into the lipid bilayer and altering the properties of cholesterol-rich microdomains.