Protein complexes from edible mushrooms as a sustainable potato protection against coleopteran pests.

Protein complexes from edible oyster mushrooms (Pleurotus sp.) composed of pleurotolysin A2 (PlyA2) and pleurotolysin B (PlyB) exert toxicity in feeding tests against Colorado potato beetle (CPB) larvae, acting through the interaction with insect-specific membrane sphingolipid. Here we present a new strategy for crop protection, based on in planta production of PlyA2/PlyB protein complexes, and we exemplify this strategy in construction of transgenic potato plants of cv Désirée. The transgenics in which PlyA2 was directed to the vacuole and PlyB to the endoplasmic reticulum are effectively protected from infestation by CPB larvae without impacting plant performance. These transgenic plants showed a pronounced effect on larval feeding rate, the larvae feeding on transgenic plants being on average five to six folds lighter than larvae feeding on controls. Further, only a fraction (11%-37%) of the larvae that fed on transgenic potato plants completed their life cycle and developed into adult beetles. Moreover, gene expression analysis of CPB larvae exposed to PlyA2/PlyB complexes revealed the response indicative of a general stress status of larvae and no evidence of possibility of developing resistance due to the functional inactivation of PlyA2/PlyB sphingolipid receptors.

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.

A method for targeting a specified segment of DNA to a bacterial microorganelle.

Encapsulation of a selected DNA molecule in a cell has important implications for bionanotechnology. Non-viral proteins that can be used as nucleic acid containers include proteinaceous subcellular bacterial microcompartments (MCPs) that self-assemble into a selectively permeable protein shell containing an enzymatic core. Here, we adapted a propanediol utilization (Pdu) MCP into a synthetic protein cage to package a specified DNA segment in vivo, thereby enabling subsequent affinity purification. To this end, we engineered the LacI transcription repressor to be routed, together with target DNA, into the lumen of a Strep-tagged Pdu shell. Sequencing of extracted DNA from the affinity-isolated MCPs shows that our strategy results in packaging of a DNA segment carrying multiple LacI binding sites, but not the flanking regions. Furthermore, we used LacI to drive the encapsulation of a DNA segment containing operators for LacI and for a second transcription factor.

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.

Development of potent cholinesterase inhibitors based on a marine pharmacophore.

The management of neurological disorders such as dementia associated with Alzheimer's or Parkinson's disease includes the use of cholinesterase inhibitors. These compounds can slow down the progression of these diseases and can also be used in the treatment of glaucoma and myasthenia gravis. The majority of the cholinesterase inhibitors used in the clinic are derived from natural products and our current paper describes the use of a small marine pharmacophore to develop potent and selective cholinesterase inhibitors. Fourteen small inhibitors were designed based on recent discoveries about the inhibitory potential of a range of related marine secondary metabolites. The compounds were evaluated, in kinetic enzymatic assays, for their ability to inhibit three different cholinesterase enzymes and it was shown that compounds with a high inhibitory activity towards electric eel and human recombinant acetylcholinesterase (IC50 between 20-70 µM) could be prepared. It was also shown that this compound class was particularly active against horse serum butyrylcholinesterase, with IC50 values between 0.8-16 µM, which is an order of magnitude more potent than the clinically used positive control neostigmine. The compounds were further tested for off-target toxicity against both human umbilical vein endothelial cells and bovine and human erythrocytes and were shown to display a low mammalian cellular toxicity. Overall, the study illustrates how the brominated dipeptide marine pharmacophore can be used as a versatile natural scaffold for the design of potent, and selective cholinesterase inhibitors.

Characterization and cytotoxic activity of ribotoxin-like proteins from the edible mushroom Pleurotus eryngii.

Ribotoxin-like proteins (RL-Ps) represent a novel specific ribonuclease family found in edible mushrooms and are able to inhibit protein synthesis. Here, we report the characterization and cytotoxic effects of four novel RL-Ps, named eryngitins, isolated from fruiting bodies of the king oyster mushroom (Pleurotus eryngii). These proteins induced formation of α-fragment from rabbit ribosomes, characteristic of their enzymatic action. The two 15 kDa eryngitins (3 and 4) are considerably more thermostable than the 21 kDa ones (1 and 2), however their overall structural features, as determined by far-UV CD spectrometry, are similar. Complete in vitro digestibility by pepsin-trypsin, and lack of cytotoxicity towards human HUVEC cells suggest low toxicity of eryngitins, if ingested. However, eryngitins exhibit cytotoxic action against insect Sf9 cells, suggesting their possible use in biotechnological applications as bioinsecticides. This cytotoxicity was not enhanced in the presence of cytolytic protein complexes based on aegerolysin proteins from Pleurotus mushrooms.

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.

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.

GPMVs in variable physiological conditions: could they be used for therapy delivery?

BACKGROUND: Cell based carriers are increasingly recognized as a good system for cargo delivery to cells. One of the reasons is their biocompatibility and low toxicity compared to artificial systems. Giant plasma membrane vesicles (GPMV) derive from the cell plasma membrane. Thus they offer the closest approximation to it, which makes them good candidates for potential drug delivery systems. To evaluate the applicability of GPMVs as carriers, we analyzed their basic biophysical properties to test their robustness in the face of changeable physiological conditions, as well as their ability to translocate across the membrane into cells. RESULTS: GPMVs formed from human umbilical vein endothelial cells (HUVEC) sustain a drastic osmotic challenge (50-500 mOsmoL/kg) unlike giant unilamelar vesicles (GUVs). In hyper-osmotic solutions the average volume decreases and membrane invaginations form, while in the hypo-osmolar buffer the volume of GPMVs increases and these changes were not reversible. The membranes of flaccid GPMVs started to wrinkle unevenly giving rise to buds after exposure to lipopolysaccharide (LPS). The shape changes in GUVs are reversible in contrast to GPMVs after LPS removal. GPMVs exposed to fluorescent LPS exhibited a signal that remained visible in some GPMVs even after LPS removal, which was never the case with GUVs. Calcein penetrated both into GUVs and GPMVs, however after the removal from the bulk solution some of the GPMVs still exhibited very bright signal, while in GUVs only a weak fluorescent signal was detected. We could also see that practically all GPMVs incorporated dextran initially, but after the dextran solution was changed with the initial non-fluorescent solution it remained only in 20% of them. The majority of HUVEC cells displayed a fluorescent signal after the incubation with GPMVs that contained fluorescently labeled dextran. CONCLUSION: Our findings indicate that GPMVs behave quite differently from artificially made giant phospholipid vesicles and the changes induced by the different treatments we subjected them to are not reversible. We also demonstrate that different substances can be both loaded into them and delivered into cells, so GPMVs may be of potential use as cargo/therapy delivery systems.

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.

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.

Cytoskeleton modification and cholesterol depletion affect membrane properties and caveolae positioning of CHO cells.

The formation of protrusions is necessary for numerous biological processes. It involves extension of the plasma membrane, and the force needed for this is provided by the actin cytoskeleton. Tether pulling with optical tweezers can mimic the formation of a protrusion, so we used this method to investigate the effects of modifying not only actin (with latrunculin A) but also microtubules (with nocodazole) and the plasma membrane itself (with methyl-ß-cyclodextrin) on the Chinese hamster ovary cell membrane. After these modifications, the membrane reservoir was supposed to redistribute. Caveolae constitute a small part of the reservoir, so the redistribution of caveolar proteins such as caveolin-1 and cavin-1 that represents caveolae per se was assessed. The main findings concerning protrusion force and membrane reservoir availability were as follows: (1) they correlated inversely, (2) their values underwent the greatest change after microtubule disruption, and (3) membrane composition had a major influence on the parameters studied. F-actin disruption and cholesterol depletion decreased, and microtubule disruption increased the amount of the caveolar proteins (caveolae). Caveolae presented just an example of the membrane reservoir, and from our findings, we suppose that the perturbations caused were too large to be related to caveolae redistribution alone. The integrity of the cytoskeleton and plasma membrane composition are important factors in the formation of protrusions and in determining the availability and distribution of the membrane reservoir.