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1.
Int J Mol Sci ; 23(9)2022 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-35563308

RESUMEN

We introduce a new family of fungal protease inhibitors with ß-trefoil fold from the mushroom Coprinopsis cinerea, named cocaprins, which inhibit both cysteine and aspartic proteases. Two cocaprin-encoding genes are differentially expressed in fungal tissues. One is highly transcribed in vegetative mycelium and the other in the stipes of mature fruiting bodies. Cocaprins are small proteins (15 kDa) with acidic isoelectric points that form dimers. The three-dimensional structure of cocaprin 1 showed similarity to fungal ß-trefoil lectins. Cocaprins inhibit plant C1 family cysteine proteases with Ki in the micromolar range, but do not inhibit the C13 family protease legumain, which distinguishes them from mycocypins. Cocaprins also inhibit the aspartic protease pepsin with Ki in the low micromolar range. Mutagenesis revealed that the ß2-ß3 loop is involved in the inhibition of cysteine proteases and that the inhibitory reactive sites for aspartic and cysteine proteases are located at different positions on the protein. Their biological function is thought to be the regulation of endogenous proteolytic activities or in defense against fungal antagonists. Cocaprins are the first characterized aspartic protease inhibitors with ß-trefoil fold from fungi, and demonstrate the incredible plasticity of loop functionalization in fungal proteins with ß-trefoil fold.


Asunto(s)
Agaricales , Proteasas de Ácido Aspártico , Proteasas de Cisteína , Lotus , Agaricales/química , Ácido Aspártico Endopeptidasas , Proteasas de Ácido Aspártico/genética , Cisteína , Proteasas de Cisteína/genética , Lotus/metabolismo , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/química
2.
Appl Environ Microbiol ; 84(23)2018 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-30242007

RESUMEN

Resistance of fungi to predation is thought to be mediated by toxic metabolites and proteins. Many of these fungal defense effectors are highly abundant in the fruiting body and not produced in the vegetative mycelium. The defense function of fruiting body-specific proteins, however, including cytoplasmically localized lectins and antinutritional proteins such as biotin-binding proteins, is mainly based on toxicity assays using bacteria as a heterologous expression system, with bacterivorous/omnivorous model organisms as predators. Here, we present an ecologically more relevant experimental setup to assess the toxicity of potential fungal defense proteins towards the fungivorous, stylet-feeding nematodes Aphelenchus avenae and Bursaphelenchus okinawaensis As a heterologous expression host, we exploited the filamentous fungus Ashbya gossypii Using this new system, we assessed the toxicity of six previously characterized, cytoplasmically localized, potential defense proteins from fruiting bodies of different fungal phyla against the two fungivorous nematodes. We found that all of the tested proteins were toxic against both nematodes, albeit to various degrees. The toxicity of these proteins against both fungivorous and bacterivorous nematodes suggests that their targets have been conserved between the different feeding groups of nematodes and that bacterivorous nematodes are valid model organisms to assess the nematotoxicity of potential fungal defense proteins.IMPORTANCE Our results support the hypothesis that cytoplasmic proteins abundant in fungal fruiting bodies are involved in fungal resistance against predation. The toxicity of these proteins toward stylet-feeding nematodes, which are also capable of feeding on plants, and the abundance of these proteins in edible mushrooms, may open possible avenues for biological crop protection against parasitic nematodes, e.g., by expression of these proteins in crops.


Asunto(s)
Proteínas Fúngicas/toxicidad , Hongos/química , Tylenchida/efectos de los fármacos , Animales , Conducta Alimentaria/efectos de los fármacos , Cuerpos Fructíferos de los Hongos/química , Tylenchida/fisiología
3.
Proc Natl Acad Sci U S A ; 111(27): E2787-96, 2014 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-24879441

RESUMEN

Effector proteins of innate immune systems recognize specific non-self epitopes. Tectonins are a family of ß-propeller lectins conserved from bacteria to mammals that have been shown to bind bacterial lipopolysaccharide (LPS). We present experimental evidence that two Tectonins of fungal and animal origin have a specificity for O-methylated glycans. We show that Tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) agglutinates Gram-negative bacteria and exerts toxicity toward the model nematode Caenorhabditis elegans, suggesting a role in fungal defense against bacteria and nematodes. Biochemical and genetic analysis of these interactions revealed that both bacterial agglutination and nematotoxicity of Lb-Tec2 depend on the recognition of methylated glycans, namely O-methylated mannose and fucose residues, as part of bacterial LPS and nematode cell-surface glycans. In addition, a C. elegans gene, termed samt-1, coding for a candidate membrane transport protein for the presumptive donor substrate of glycan methylation, S-adenosyl-methionine, from the cytoplasm to the Golgi was identified. Intriguingly, limulus lectin L6, a structurally related antibacterial protein of the Japanese horseshoe crab Tachypleus tridentatus, showed properties identical to the mushroom lectin. These results suggest that O-methylated glycans constitute a conserved target of the fungal and animal innate immune system. The broad phylogenetic distribution of O-methylated glycans increases the spectrum of potential antagonists recognized by Tectonins, rendering this conserved protein family a universal defense armor.


Asunto(s)
Agaricales/inmunología , Inmunidad Innata , Polisacáridos/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/inmunología , Cangrejos Herradura/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Metilación , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido
4.
Elife ; 132024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38743056

RESUMEN

Mutations in the gene for ß-catenin cause liver cancer cells to release fewer exosomes, which reduces the number of immune cells infiltrating the tumor.


Asunto(s)
Escape del Tumor , Humanos , beta Catenina/metabolismo , beta Catenina/genética , Exosomas/inmunología , Exosomas/metabolismo , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/genética , Mutación , Sistema Inmunológico/inmunología , Neoplasias/inmunología , Neoplasias/genética
5.
J Cell Biol ; 223(7)2024 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-38578285

RESUMEN

IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge ß-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.


Asunto(s)
Toxina del Cólera , Endorribonucleasas , Proteínas Serina-Treonina Quinasas , Respuesta de Proteína Desplegada , Toxina del Cólera/genética , Toxina del Cólera/metabolismo , Estrés del Retículo Endoplásmico , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Humanos , Animales , Ratones , Línea Celular
6.
STAR Protoc ; 4(2): 102335, 2023 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-37243601

RESUMEN

Transcytosis is the primary mechanism by which macro-molecules transverse epithelial cell barriers. Here, we present an assay for measuring transcytosis and recycling of IgG in intestinal epithelial Caco-2 cells and primary human intestinal organoids. We describe steps for establishing human enteroids or Caco-2 cells and plating monolayers. We then provide procedures for a transcytosis and recycling assay and a luciferase assay. The protocol facilitates quantification of membrane trafficking and can be used to probe endosomal compartments unique to polarized epithelia. For complete details on the use and execution of this protocol, please refer to Maeda K et al. (2022).1.

7.
Appl Environ Microbiol ; 78(23): 8485-7, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23001676

RESUMEN

Tamavidins are fungal biotin-binding proteins (BBPs) displaying antifungal activity against phytopathogens. Here we show high toxicity of tamavidins toward nematodes, insects, and amoebae. As these organisms represent important phyla of fungal predators and parasites, we propose that BBPs are part of the chemical defense system of fungi.


Asunto(s)
Agaricales/metabolismo , Antibiosis , Proteínas Portadoras/metabolismo , Proteínas Fúngicas/metabolismo , Parásitos/efectos de los fármacos , Amoeba/efectos de los fármacos , Animales , Insectos/efectos de los fármacos , Nematodos/efectos de los fármacos
8.
Cell Rep ; 40(2): 111063, 2022 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-35830800

RESUMEN

The complex sphingolipids exhibit a diversity of ceramide acyl chain structures that influence their trafficking and intracellular distributions, but it remains unclear how the cell discerns among the different ceramides to affect such sorting. To address the mechanism, we synthesize a library of GM1 glycosphingolipids with naturally varied acyl chains and quantitatively assess their sorting among different endocytic pathways. We find that a stretch of at least 14 saturated carbons extending from C1 at the water-bilayer interface dictate lysosomal sorting by exclusion from endosome sorting tubules. Sorting to the lysosome by the C14∗ motif is cholesterol dependent. Perturbations of the C14∗ motif by unsaturation enable GM1 entry into endosomal sorting tubules of the recycling and retrograde pathways independent of cholesterol. Unsaturation occurring beyond the C14∗ motif in very long acyl chains rescues lysosomal sorting. These results define a structural motif underlying the membrane organization of sphingolipids and implicate cholesterol-sphingolipid nanodomain formation in sorting mechanisms.


Asunto(s)
Gangliósido G(M1) , Glicoesfingolípidos , Ceramidas/metabolismo , Colesterol/metabolismo , Gangliósido G(M1)/metabolismo , Esfingolípidos/metabolismo
9.
Commun Biol ; 5(1): 180, 2022 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-35233064

RESUMEN

The direct delivery of molecules and the sampling of endogenous compounds into and from living cells provide powerful means to modulate and study cellular functions. Intracellular injection and extraction remain challenging for fungal cells that possess a cell wall. The most common methods for intracellular delivery into fungi rely on the initial degradation of the cell wall to generate protoplasts, a step that represents a major bottleneck in terms of time, efficiency, standardization, and cell viability. Here, we show that fluidic force microscopy enables the injection of solutions and cytoplasmic fluid extraction into and out of individual fungal cells, including unicellular model yeasts and multicellular filamentous fungi. The approach is strain- and cargo-independent and opens new opportunities for manipulating and analyzing fungi. We also perturb individual hyphal compartments within intact mycelial networks to study the cellular response at the single cell level.


Asunto(s)
Hongos , Hifa , Pared Celular/metabolismo , Hongos/fisiología , Micelio , Levaduras
10.
Cell Host Microbe ; 30(2): 216-231.e5, 2022 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-35143768

RESUMEN

Polarized epithelial cells form an essential barrier against infection at mucosal surfaces. Many pathogens breach this barrier to cause disease, often by co-opting cellular endocytosis mechanisms to enter the cell through the lumenal (apical) cell surface. We recently discovered that the loss of the cell polarity gene PARD6B selectively diminishes apical endosome function. Here, we find that in response to the entry of certain viruses and bacterial toxins into the epithelial cells via the apical membrane, PARD6B and aPKC, two components of the PARD6B-aPKC-Cdc42 apical polarity complex, undergo rapid proteasome-dependent degradation. The perturbation of apical membrane glycosphingolipids by toxin- or virus-binding initiates degradation of PARD6B. The loss of PARD6B causes the depletion of apical endosome function and renders the cell resistant to further infection from the lumenal cell surface, thus enabling a form of cell-autonomous host defense.


Asunto(s)
Toxinas Bacterianas , Virus , Toxinas Bacterianas/metabolismo , Polaridad Celular/fisiología , Endosomas/metabolismo , Células Epiteliales , Proteína Quinasa C/metabolismo , Virus/metabolismo
11.
Toxins (Basel) ; 13(8)2021 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-34437414

RESUMEN

Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1.


Asunto(s)
Toxina del Cólera/metabolismo , Receptores de Superficie Celular/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitosis
12.
Commun Biol ; 4(1): 262, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33637874

RESUMEN

Routinely, fungal-fungal interactions (FFI) are studied on agar surfaces. However, this format restricts high-resolution dynamic imaging. To gain experimental access to FFI at the hyphal level in real-time, we developed a microfluidic platform, a FFI device. This device utilises microchannel geometry to enhance the visibility of hyphal growth and provides control channels to allow comparisons between localised and systemic effects. We demonstrate its function by investigating the FFI between the biological control agent (BCA) Clonostachys rosea and the plant pathogen Fusarium graminearum. Microscope image analyses confirm the inhibitory effect of the necrotrophic BCA and we show that a loss of fluorescence in parasitised hyphae of GFP-tagged F. graminearum coincides with the detection of GFP in mycelium of C. rosea. The versatility of our device to operate under both water-saturated and nutrient-rich as well as dry and nutrient-deficient conditions, coupled with its spatio-temporal output, opens new opportunities to study relationships between fungi.


Asunto(s)
Fusarium/fisiología , Hifa/fisiología , Hypocreales/fisiología , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Microscopía Fluorescente , Control Biológico de Vectores , Fusarium/genética , Fusarium/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hypocreales/genética , Hypocreales/metabolismo , Viabilidad Microbiana , Factores de Tiempo
13.
Nat Commun ; 12(1): 3675, 2021 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-34135326

RESUMEN

Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes.


Asunto(s)
Membrana Celular/química , Ceramidas/química , Actinas/química , Antígenos CD59/química , Membrana Celular/efectos de los fármacos , Toxina del Cólera/química , Toxina del Cólera/farmacología , Colesterol/química , Gangliósido G(M1)/química , Glicoesfingolípidos/química , Glicosilfosfatidilinositoles/química , Modelos Biológicos , Simulación de Dinámica Molecular , Fosfatidilserinas/química
14.
Curr Biol ; 29(2): 217-228.e4, 2019 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-30612903

RESUMEN

Intercellular distribution of nutrients and coordination of responses to internal and external cues via endogenous signaling molecules are hallmarks of multicellular organisms. Vegetative mycelia of multicellular fungi are syncytial networks of interconnected hyphae resulting from hyphal tip growth, branching, and fusion. Such mycelia can reach considerable dimensions and, thus, different parts can be exposed to quite different environmental conditions. Our knowledge about the mechanisms by which fungal mycelia can adjust nutrient gradients or coordinate their defense response to fungivores is scarce, in part due to limitations in technologies currently available for examining different parts of a mycelium over longer time periods at the microscopic level. Here, we combined a tailor-made microfluidic platform with time-lapse fluorescence microscopy to visualize the dynamic response of the vegetative mycelium of a basidiomycete to two different stimuli. The microfluidic platform allows simultaneous monitoring at both the colony and single-hypha level. We followed the dynamics of the distribution of a locally administered nutrient analog and the defense response to spatially confined predation by a fungivorous nematode. Although both responses of the mycelium were constrained locally, we observed long-distance propagation for both the nutrient analog and defense response in a subset of hyphae. This propagation along hyphae occurred in both acropetal and basipetal directions and, intriguingly, the direction was found to alternate every 3 hr in an individual hypha. These results suggest that multicellular fungi have, as of yet, undescribed mechanisms to coordinate the distribution of nutrients and their behavioral response upon attack by fungivores.


Asunto(s)
Agaricales/fisiología , Cadena Alimentaria , Hifa/fisiología , Tylenchida/fisiología , Animales , Antibiosis , Técnicas Analíticas Microfluídicas , Microscopía Fluorescente , Nutrientes/fisiología , Transducción de Señal
15.
mBio ; 9(3)2018 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-29921669

RESUMEN

Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors-a type III secretion system (T3SS) and Shiga toxins (Stxs)-that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats with Cas9) loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were also targeted. Mutations in these loci not only rescued cells from Stx-mediated cell death, but also prevented cytotoxicity associated with the EHEC T3SS. These mutations interfered with early events associated with T3SS and Stx pathogenicity, markedly reducing entry of T3SS effectors into host cells and binding of Stx. The convergence of Stx and T3SS onto overlapping host targets provides guidance for design of new host-directed therapeutic agents to counter EHEC infection.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors-a type III secretion system (T3SS) and Shiga toxins (Stxs)-that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3) and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogen's key virulence factors.


Asunto(s)
Escherichia coli Enterohemorrágica/fisiología , Infecciones por Escherichia coli/metabolismo , Toxina Shiga/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Factores de Virulencia/metabolismo , Vías Biosintéticas/genética , Línea Celular , Supervivencia Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Escherichia coli Enterohemorrágica/genética , Escherichia coli Enterohemorrágica/metabolismo , Escherichia coli Enterohemorrágica/patogenicidad , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Marcación de Gen , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno , Humanos , Mutación , Toxina Shiga/genética , Esfingolípidos/biosíntesis , Trihexosilceramidas/biosíntesis , Sistemas de Secreción Tipo III/genética , Factores de Virulencia/genética
16.
G3 (Bethesda) ; 6(1): 87-98, 2015 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-26585824

RESUMEN

The dung of herbivores, the natural habitat of the model mushroom Coprinopsis cinerea, is a nutrient-rich but also very competitive environment for a saprophytic fungus. We showed previously that C. cinerea expresses constitutive, tissue-specific armories against antagonists such as animal predators and bacterial competitors. In order to dissect the inducible armories against such antagonists, we sequenced the poly(A)-positive transcriptome of C. cinerea vegetative mycelium upon challenge with fungivorous and bacterivorous nematodes, Gram-negative and Gram-positive bacteria and mechanical damage. As a response to the fungivorous nematode Aphelenchus avenae, C. cinerea was found to specifically induce the transcription of several genes encoding previously characterized nematotoxic lectins. In addition, a previously not characterized gene encoding a cytoplasmic protein with several predicted Ricin B-fold domains, was found to be strongly upregulated under this condition. Functional analysis of the recombinant protein revealed a high toxicity toward the bacterivorous nematode Caenorhabditis elegans. Challenge of the mycelium with A. avenae also lead to the induction of several genes encoding putative antibacterial proteins. Some of these genes were also induced upon challenge of the mycelium with the bacteria Escherichia coli and Bacillus subtilis. These results suggest that fungi have the ability to induce specific innate defense responses similar to plants and animals.


Asunto(s)
Agaricales/genética , Antibiosis , Proteínas Fúngicas/genética , Nematodos , Agaricales/metabolismo , Animales , Biología Computacional/métodos , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Estrés Fisiológico/genética , Transcriptoma
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