Toxic effects of amyloid fibrils on cell membranes: the importance of ganglioside GM1

The interaction of amyloid aggregates with the cell plasma membrane is currently considered among the basic mechanisms of neuronal dysfunction in amyloid neurodegeneration. We used amyloid oligomers and fibrils grown from the yeast prion Sup35p, responsible for the specific prion trait [PSI(+)], to investigate how membrane lipids modulate fibril interaction with the membranes of cultured H-END cells and cytotoxicity. Sup35p shares no homology with endogenous mammalian polypeptide chains. Thus, the generic toxicity of amyloids and the molecular events underlying cell degeneration can be investigated without interference with analogous polypeptides encoded by the cell genome. Sup35 fibrils bound to the cell membrane without increasing its permeability to Ca(2+). Fibril binding resulted in structural reorganization and aggregation of membrane rafts, with GM1 clustering and alteration of its mobility. Sup35 fibril binding was affected by GM1 or its sialic acid moiety, but not by cholesterol membrane content, with complete inhibition after treatment with fumonisin B1 or neuraminidase. Finally, cell impairment resulted from caspase-8 activation after Fas receptor translocation on fibril binding to the plasma membrane. Our observations suggest that amyloid fibrils induce abnormal accumulation and overstabilization of raft domains in the cell membrane and provide a reasonable, although not unique, mechanistic and molecular explanation for fibril toxicity.     

Bacteriocin-producing oral streptococci and inhibition of respiratory pathogens

The use of bacteria as probiotics is in continuous development, thanks to their capacity to maintain or restore a host's natural microbiome by interference with and/or inhibition of other microorganisms mediated by antimicrobial peptide production such as bacteriocins. In the oral cavity, Streptococcus salivarius, a non-pathogenic and predominant oral species, is one of the major bacteriocin producers that is able to coexist in this environment and reduce the frequency of colonization of the main pathogens involved in upper respiratory tract infections. The aim of this study was to screen oral bacteria colonizing healthy children for their use as potential oral probiotics. Eighty-one α-hemolytic streptococci isolated from nasal and/or pharyngeal swabs of 31 healthy children aged between two and twelve years were isolated. Among them, 13 α-hemolytic streptococci were selected for their bacteriocin-like inhibitory activity against potential pathogens. These strains were tested for bacteriocin production and assayed for their capacity to adhere to HEp-2 cell lines. Our data showed that 13 bacteriocin producer strains were able to inhibit different gram-positive pathogens. Among them one strain, S. salivarius 24SMB, deposited as DSM 23307, was selected as a potential oral probiotic, thanks to its safety assessment, ability to inhibit Streptococcus pneumoniae and the absence of virulence and antibiotic resistance genes.     

TAB-1 modulates intracellular localization of p38 MAP kinase and downstream signaling

Stress-activated mitogen-activated protein (MAP) kinase p38 mediates stress signaling in mammalian cells via threonine and tyrosine phosphorylation in its conserved TGY motif by upstream MAP kinase kinases (MKKs). In addition, p38 MAP kinase can also be activated by an MKK-independent mechanism involving TAB-1 (TAK-1-binding protein)-mediated autophosphorylation. Although TAB-1-mediated p38 activation has been implicated in ischemic heart, the biological consequences and downstream signaling of TAB-1-mediated p38 activation in cardiomyocytes is largely unknown. We show here that TAB-1 expression leads to a significant induction of p38 autophosphorylation and consequent kinase activation in cultured neonatal cardiomyocytes. In contrast to MKK3-induced p38 kinase downstream effects, TAB-1-induced p38 kinase activation does not induce expression of pro-inflammatory genes, cardiac marker gene expression, or changes in cellular morphology. Rather, TAB-1 binds to p38 and prevents p38 nuclear localization. Furthermore, TAB-1 disrupts p38 interaction with MKK3 and redirects p38 localization in the cytosol. Consequently, TAB-1 expression antagonizes the downstream activity of p38 kinase induced by MKK3 and attenuates interleukin-1beta-induced inflammatory gene induction in cardiomyocytes. These data suggest that TAB-1 can mediate MKK-independent p38 kinase activation while negatively modulating MKK-dependent p38 function. Our study not only redefines the functional role of TAB-1 in p38 kinase-mediated signaling pathways but also provides the first evidence that intracellular localization of p38 kinase and complex interaction dictates its downstream effects. These results suggest a previously unknown mechanism for stress-MAP kinase regulation in mammalian cells.     

Natively folded HypF-N and its early amyloid aggregates interact with phospholipid monolayers and destabilize supported phospholipid bilayers

Recent data depict membranes as the main sites where proteins/peptides are recruited and concentrated, misfold, and nucleate amyloids; at the same time, membranes are considered key triggers of amyloid toxicity. The N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N) in 30% trifluoroethanol undergoes a complex path of fibrillation starting with initial 2-3-nm oligomers and culminating with the appearance of mature fibrils. Oligomers are highly cytotoxic and permeabilize lipid membranes, both biological and synthetic. In this article, we report an in-depth study aimed at providing information on the surface activity of HypF-N and its interaction with synthetic membranes of different lipid composition, either in the native conformation or as amyloid oligomers or fibrils. Like other amyloidogenic peptides, the natively folded HypF-N forms stable films at the air/water interface and inserts into synthetic phospholipid bilayers with efficiencies depending on the type of phospholipid. In addition, HypF-N prefibrillar aggregates interact with, insert into, and disassemble supported phospholipid bilayers similarly to other amyloidogenic peptides. These results support the idea that, at least in most cases, early amyloid aggregates of different peptides and proteins produce similar effects on the integrity of membrane assembly and hence on cell viability.     

Synthetic Lipid Vesicles Recruit Native-Like Aggregates and Affect the Aggregation Process of the Prion Ure2p: Insights on Vesicle Permeabilization and Charge Selectivity

The yeast prion Ure2p polymerizes into native-like fibrils, retaining the overall structure and binding properties of the soluble protein. Recently we have shown that, similar to amyloid oligomers, the native-like Ure2p fibrils and their precursor oligomers are highly toxic to cultured mammalian cells when added to the culture medium, whereas Ure2p amyloid fibrils generated by heating the native-like fibrils are substantially harmless. We show here that, contrary to the nontoxic amyloid fibrils, the toxic, native-like Ure2p assemblies induce a significant calcein release from negatively charged phosphatidylserine vesicles. A minor and less-specific effect was observed with zwitterionic phosphatidylcholine vesicles, suggesting that the toxic aggregates preferentially bind to negatively charged sites on lipid membranes. We also found that cholesterol-enriched phospholipid membranes are protected against permeabilization by native-like Ure2p assemblies. Moreover, vesicle permeabilization appears charge-selective, allowing calcium, but not chloride, influx to be monitored. Finally, we found that the interaction with phosphatidylserine membranes speeds up Ure2p polymerization into oligomers and fibrils structurally and morphologically similar to the native-like Ure2p assemblies arising in free solution, although less cytotoxic. These data suggest that soluble Ure2p oligomers and native-like fibrils, but not amyloid fibrils, interact intimately with negatively charged lipid membranes, where they allow selective cation influx.     

Impact of Endochitinase-Transformed White Spruce on Soil Fungal Biomass and Ectendomycorrhizal Symbiosis

The impact of transgenic white spruce [Picea glauca (Moench) Voss] containing the endochitinase gene (ech42) on soil fungal biomass and on the ectendomycorrhizal fungi Wilcoxina spp. was tested using a greenhouse trial. The measured level of endochitinase in roots of transgenic white spruce was up to 10 times higher than that in roots of nontransformed white spruce. The level of endochitinase in root exudates of three of four ech42-transformed lines was significantly greater than that in controls. Analysis soil ergosterol showed that the amount of fungal biomass in soil samples from control white spruce was slightly larger than that in soil samples from ech42-transformed white spruce. Nevertheless, the difference was not statistically significant. The rates of mycorrhizal colonization of transformed lines and controls were similar. Sequencing the internal transcribed spacer rRNA region revealed that the root tips were colonized by the ectendomycorrhizal fungi Wilcoxina spp. and the dark septate endophyte Phialocephala fortinii. Colonization of root tips by Wilcoxina spp. was monitored by real-time PCR to quantify the fungus present during the development of ectendomycorrhizal symbiosis in ech42-transformed and control lines. The numbers of Wilcoxina molecules in the transformed lines and the controls were not significantly different (P > 0.05, as determined by analysis of covariance), indicating that in spite of higher levels of endochitinase expression, mycorrhization was not inhibited. Our results indicate that the higher levels of chitinolytic activity in root exudates and root tissues from ech42-transformed lines did not alter the soil fungal biomass or the development of ectendomycorrhizal symbiosis involving Wilcoxina spp.White spruce [Picea glauca (Moench) Voss] is a tree species with an extensive distribution in boreal and subboreal forests and with significant ecological roles (37, 38). It is also an important commercial species for production of pulpwood and construction-grade lumber. However, in nurseries and plantations, white spruce is sensitive to multiple fungal diseases (23, 29, 42, 62, 76). Climate change scenarios suggest that diseases could result in increased mortality in conifer forests (22, 48). Genetic engineering offers a potential means to mitigate biotic and abiotic stresses.During the last 2 decades, chitinase genes isolated from plants, fungi, or bacteria have been studied and used to transform crops or trees in order to increase their resistance to plant-pathogenic fungi. One potential goal is improving white spruce tolerance to fungal infection through insertion of a chitinase gene. Chitin is a biopolymer of β-(1-4)-linked molecules of N-acetylglucosamine (NAG), a derivative of glucose, and is the primary constituent of the fungal cell wall and arthropod exoskeleton (3, 51). Chitinases are plant defense pathogenesis-related proteins (6, 11) that break down the chitin chain either by cleavage of internal glycoside bonds (endochitinases), by hydrolysis of the nonreducing end of the chitin chain (exochitinases), or by hydrolysis of NAG oligomers and trimers into NAG monomers (chitobiases). Endo- and exochitinase genes have been well characterized using sugar beet (Beta vulgaris) (44) and the filamentous fungal genus Trichoderma (14, 24, 69). Chitinolytic genes have been inserted into the genomes of cultivated plants and trees in an attempt to boost plant chitinase activity. Among the different genes involved in the production of chitinolytic enzymes, the ech42 endochitinase gene from Trichoderma harzianum has been inserted into plant genomes to enhance their resistance against phytopathogenic fungi. In McIntosh apple cultivars transformed with the ech42 gene there was limited attack by the apple scab fungus Venturia inaequalis (5). Transgenic black spruce (Picea mariana) expressing the ech42 gene was more resistant to the root rot pathogen Cylindrocladium floridanum (45).However, field deployment of crops and trees genetically transformed to improve nonspecific resistance against phytopathogenic fungi has raised concerns about the impact on nontarget fungi, including potentially beneficial symbionts. This is particularly worrisome when nonspecific constitutive promoters control expression of the resistance gene and the gene is expressed in all tissues from roots to leaves. As a consequence, the natural colonization of such transformed plants by endophytic or mycorrhizal fungi can be altered.Mycorrhizal fungi play a key role in plant nutrition (55) by mobilizing and transferring nutrients to the host through an intimate and highly organized association with plant roots (52, 63). Furthermore, their involvement in soil nutrient recycling (56) makes mycorrhizal symbiosis a major ecological process that is important for the health of soil and forest ecosystems. Crops, fruits, and forest trees exhibit mycorrhizal colonization by arbuscular mycorrhizae, ectomycorrhizae, and ectendomycorrhizae (EEM). While numerous studies have addressed the impact of transgenic plants on arbuscular mycorrhizae (10, 26, 64, 68, 72, 73) and ectomycorrhizae (32, 43, 50, 60), no previous study focused on EEM.Ectendomycorrhizal fungi can be distinguished from ectomycorrhizae by the presence of a thin or fragmented mantle and intracellular penetration into root cortical cells. All EEM fungi identified so far belong to the Ascomycetes, and these fungi are represented by several genera of Helotiales and Pezizales (77). EEM fungi are prevalent in conifer and deciduous tree nurseries (27, 39, 40, 70) and are also very common on seedling root tips at disturbed sites (15, 16, 19). The prevalence of EEM fungi on seedling roots, from which the genus Wilcoxina is frequently recovered (16, 67), suggests that they can play a significant role in establishment and growth of seedlings (77) and provide protection against root diseases (31, 61). Consequently, the potentially negative effects of chitinase-transformed trees on ectendomycorrhizal fungi could be detrimental to plant health.The present study addressed the potential impact of ech42-transformed white spruce on soil fungal biomass and ectendomycorrhizal symbiosis. It was hypothesized that (i) the soil fungal biomass in a transgenic white spruce rhizosphere is less than the soil fungal biomass in a control tree rhizosphere and (ii) the development of Wilcoxina spp. on root tips of transgenic white spruce is less important than the development of Wilcoxina spp. on root tips of control trees. To test these hypotheses, 5-year-old white spruce trees transformed with the 35S promoter-ech42 construct were analyzed by performing a greenhouse trial. The amount of soil fungal biomass was estimated using measurements of ergosterol in soil. A real-time PCR method was developed to detect changes in the quantity of ectendomycorrhizal hyphae involved in colonization of transgenic white spruce root tips.     

Differing molecular mechanisms appear to underlie early toxicity of prefibrillar HypF-N aggregates to different cell types

Considerable attention has been paid to the high cytotoxic potential of small, prefibrillar aggregates of proteins/peptides, either associated or not associated with amyloid diseases. Recently, we reported that different cell types are variously affected by early aggregates of the N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N), a protein not involved in any disease. In this study, we provide detailed information on a chain of events triggered in Hend murine endothelial cells and IMR90 fibroblasts, which have previously been shown to be highly vulnerable or very resistant, respectively, to HypF-N aggregates. Initially, both cell lines displayed impaired viability upon exposure to HypF-N toxic aggregates; however, at longer exposure times, IMR90 cells recovered completely, whereas Hend cells did not. In particular, significant initial mitochondrial permeability transition (MPT) pore opening was found in IMR90 cells followed by a sudden repair of membrane integrity with rapid and efficient inhibition of cytochrome c and AIF release, and upregulation of Bcl-2. The greater resistance of IMR90 fibroblasts may also be due to a higher cholesterol content in the plasma membrane, which disfavours interaction with the aggregates. In contrast, Hend cells, which have less membrane cholesterol, showed delayed MPT opening with prolonged translocation of cytochrome c into the cytosol. Finally, the caspase 9 active fragment was increased significantly in both Hend and IMR90 cells; however, only Hend cells showed caspase 8 and caspase 3 activation with DNA fragmentation. From our data, the different responses of the two cell types to the same aggregates appear to be associated with two key events: (a) aggregate interaction with the plasma membrane, disfavoured by a high level of membrane cholesterol; and (b) alterations in mitochondrial functionality, leading to the release of pro-apoptotic stimuli, which are counteracted by upregulation of Bcl-2.