Papel de la N-glicosilación de proteinas en la virulencia de Fusarium oxysporum

Protein glycosylation is a process widely studied in eukaryotes, localized in two different cellular compartments but functionally connected: the endoplasmic reticulum and the Golgi apparatus. Due to the complexity and diversity of its reactions, some enzymatic pathways are still unknown in many organisms, emerging as new research topics in filamentous fungi. The initial glycosylation steps localized in the ER are highly conserved in eukaryotes, by contrast GA steps show wide variations in the sequential addition of monosaccharides that conform the elongation of the previously synthesized glycans, being some of these enzymatic reactions substrate specific. Protein glycosylation in mammalian species presents a high structural diversity in which glycans are decorated by fucose, xilose, glucose, sialic acid and mannose in terminal positions. Fungal and yeast have less diversity and only present galactose, glucose, uronic acids and mannose as terminal residues. N-acetylglucosamine transferases participate in terminal glycosylation, attaching GlcNAc to enlarge glycan molecules, but considering other studies on eukaryotes, this monosaccharide can be localized in terminal position or at bisecting points in the polysaccharide chains. With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and the host during infection and to understand the molecular bases that govern fungal pathogenicity, we have analyzed the genes encoding N-acetylglucosaminyl transferases, presumably involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes in the F. oxysporum f.sp. lycopersici genome. Deletion mutants in the N-acetylglucosaminyl transferase gene gnt2 showed a dramatic reduction in virulence on both plant and animal hosts. The Δgnt2 mutants had alterations in cell wall properties related to terminal α- or β-linked N-acetylglucosamine. In the present study we conclude that is indeed a terminal monosaccharide, based on the cytometry analyses by fluorescence quantification of the lectin GSII-FITC bound to germling cell walls, although we can not discard a bisecting position as well. Mutant conidia and germlings also differed from the wild type in surface structure and physicochemical surface properties. Spore and hyphal aggregation in liquid culture differ between the mutant and the wild type, in a pH independent manner. Transmission electron micrographs of mutant germlings show strong cell-to-cell adherence likely caused by increased formation of an extracellular chemical matrix. Δgnt2 cell walls showed a significant reduction in N-linked oligosaccharides...El proceso de glicosilación de proteínas, ampliamente estudiado en eucariotas, tiene lugar en dos compartimentos celulares separados aunque funcionalmente conectados: el retículo endoplásmico (RE) y el aparato de Golgi (AG). Debido a su complejidad, aún se desconocen algunas de las rutas enzimáticas que lo constituyen en muchos organismos, resultando un tema muy novedoso de estudio en hongos filamentosos. Las etapas de glicosilación iniciales localizadas en el RE están muy conservadas en eucariotas, mientras que las etapas del AG son más variables y corresponden a la adición secuencial de monosacáridos que alargan el glicano sintetizado, siendo algunas específicas de sustrato. En mamíferos la elongación de glicanos es muy diversa, dando lugar a cadenas oligosacarídicas decoradas con fucosa, xilosa, glucosa, ácido siálico o manosa terminales. En hongos y levaduras la diversidad es menor, presentando tan solo residuos galactosa, glucosa, ácidos urónicos o manosa en los extremos. El análisis in silico del genoma de F. oxysporum harevelado la existencia de seis posibles genes de N-acetilglucosamina transferasas (Gnt) ortólogos a GNT1 de Saccharomyces cerevisiae, proteína identificada como enzima de glicosilación anclada a la membrana del AG. Para estudiar el papel de gnt2 en la fisiología y virulencia de F. oxysporum, en este trabajo se ha llevado a cabo la deleción de ambas copias del gen y la posterior complementación de los mutantes resultantes, con el alelo silvestre. El proceso de infección con el mutante deficiente .gnt2, tanto en plantas de tomate como en la larva de la polilla de la cera Galleria mellonella, ha resultado en una reducción de la capacidad de invasión de los tejidos del huésped y en un retraso significativo en el desarrollo de enfermedad. Por el contrario, los individuos infectados con el transformante complementado han mostrado un desarrollo de la enfermedad similar a la estirpe silvestre, con la recuperación total de la virulencia. La anulación de gnt2 produce cambios en la pared celular que conllevan la alteración de la sensibilidad y la afinidad a compuestos que interaccionan con la pared celular fúngica y a enzimas líticas de pared, así como una reducción en la formación de puentes de fusión entre conidias e hifas, dando como resultado un micelio menos cohesionado. Estos análisis han revelado en el mutante un aumento en la sensibilidad a dichos compuestos tóxicos, y mayor resistencia a la acción de glucanasas y quitinasas, así como menor afinidad por el colorante Alcian Blue

The Fusarium oxysporum gnt2, Encoding a Putative NAcetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed colocalization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity

The Fusarium oxysporum gnt2, Encoding a Putative NAcetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

21 páginas, 9 figuras, 1 tablaWith the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed colocalization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity.Ministerio de Ciencia y Tecnologia (BIO2010-015505) of Spain, Junta de Andalucía (P08-CVI-3847). L.L.F. was supported by a PhD fellowship from the former Ministerio de Ciencia y Tecnologia.L.L.F. was supported by a PhD fellowship from the former Ministerio de Ciencia y Tecnología. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Validation of the Mini-Z for the detection of burnout in Colombian health personnel

La carga emocional y el estrés psicológico del personal sanitario se incrementa cuando las jornadas de trabajo son extenuantes, hay un desequilibrio entre el trabajo y las horas en casa y una importante disminución en el recurso humano y de gestión para la atención de los pacientes1 como en las actuales circunstancias de pandemia por el Sars-Cov-22. Estos factores afectan al bienestar emocional de los trabajadores y la calidad de los cuidados de los pacientes3, lo que incrementa la aparición de enfermedades psiquiátricas, estrés4 y síndrome de burnout (BO)5

Δ<i>gnt2</i> mutants have reduced colonization ability and induce higher defence response of tomato plants.

<p>(<b>A</b>) Comparative analysis of fungal biomass using quantitative real-time polymerase chain reaction during disease progression caused by the wild type (wt), the Δ<i>gnt2</i> mutant and the complemented cΔ<i>gnt2</i> strains. Data represent nanograms of fungal DNA amplified from 100 ng of DNA extracted from infected stems. Each column represents the mean from three independent inoculation experiments with three replicates each. Standard error bars are indicated. (<b>B</b>) Expression of defence-response genes in tomato plants three days after inoculation with the indicated strains. Transcript abundance was determined by quantitative reverse transcriptase-polymerase chain reaction. Expression levels in each sample were normalized to the expression of the tomato <i>efα</i> gene and were calculated relative to the uninfected control plants by the DDCt method. Error bars indicate the standard error calculated from three independent inoculation experiments with three replicates each.</p

<i>gnt2</i> is part of a gene family expanded in the pathogenic fungus <i>F. oxysporum</i>.

<div><p>Phylogenetic tree constructed using the neighbour-joining method from Clustal W multiple-sequence alignment of 146 aa of GT8 proteins. Numbers at the branch points represent percentage bootstrap values based on 1,000 replicates. <i>F. oxysporum</i> (FOXG_01495; FOXG_12436/FOXG_14101; FOXG_12874; FOXG_12897; FOXG_14149; FOXG_16408)<i>, F. graminearum</i> (FGSG_10936; FGSG_10956*)<i>, F. verticillioides</i> (FVEG_07859; FVEG_11624; FVEG_11649*), <i>Aspergillus fumigatus</i> (AFUB_08390), <i>A. niger</i> (An11g10206; An03g02990), <i>A. oryzae</i> (AO09001000), <i>Chaetomium globosum</i> (CHGG_09837; CHGG_08452), <i>Saccharomyces cerevisiae</i> (GNT1, YOR320C), <i>Nectria haematococca</i> (NHA_39806; NHA_79931; NHA_79932), <i>Verticillium dahliae</i> (VDBG_01023; VDBG_03984), <i>Penicillium chrysogenum</i> (PC_16g00820; PC_22g12650).</p> <p>*These genes were manually cured as follows: FGSG_10956, 120 amino acids were added upstream of the annotated start codon; FVEG_11649, 78 amino acids were added upstream of the annotated start codon.</p></div

Gnt2 co-localizes with Golgi sub-cellular compartment proteins.

<p>(<b>A</b>) Light (left panels) and fluorescence (right panels) micrographs of germlings from the wild type (wt) and the Δ<i>gnt2</i> mutant, both harbouring the GFP::Gnt2 fusion protein, after 5 min treatment with (+) or without (-) Brefeldin A (BFA). Scale bars, 10 µm. (<b>B</b>) Enzymatic activities of sub-cellular fractions (1 to 14) obtained after velocity sucrose gradient ultracentrifugation of cell lysates from the indicated strains. Aliquots of the 10,000 <i>g</i> x 10-min pellet (P10) and the 160,000 <i>g</i> x 90-min pellet (P160) were also included in the analyses. Dashed line, sucrose concentration; black diamonds, NADPH cytochrome c reductase activity (endoplasmic reticulum marker); white diamonds, GDPase activity (Golgi marker). (<b>C</b>) Proteins contained within the indicated fractions were resolved by SDS-PAGE and detected by Western blotting analyses using anti-GFP or anti-Vps10p antibodies, as indicated. (<b>D</b>) Colocalization of GFP::Gnt2 (green) with the Golgi apparatus (red) as stained with BODIPY TR ceramide in the indicated strains. Bar, 10 μm. </p

GS II-FITC lectin binding affinity of the indicated <i>F. oxysporum</i> strains.

<p>Intensity of fluorescence emitted by GS II-FITC bound to cell surface was measured for 20,000 events in microconidia from wild type (wt), Δ<i>gnt2</i> mutant and cΔ<i>gnt2</i> complemented strain, using flow cytometer separation and fluorescence detection. (<b>A</b>) Fluorescence analysis using the auxiliary channel adjusted to allow discrimination of single cell population (H3 in the histograms and white columns in the graph) from aggregated cells population (H4 and stripped columns). The percentages of each cell population are represented for the three strains. (<b>B</b>) Histograms showing relative fluorescence determined by FL1 channel for single cells (H3) or aggregated cells (H4) from the three strains. Columns in the graph represent the relative fluorescence medium values observed for single cells (white) and aggregated cells (stripped). The standard errors from three independent experiments are indicated. (<b>C</b>) Morphological analyses of cell populations from the different strains by cytometer light scattering detection. Forward and side scattered light shape (FSC and SSC, respectively) is represented.</p

<i>Fusarium oxysporum</i> contains six putative <i>N-</i>acetyl glucosaminyltransferase genes.

<p>(<b>A</b>) Alignment of the conserved domains of six predicted Gnts encoded by <i>F. oxysporum</i> f.sp <i>lycopersici</i> genes FOXG_<i>01495</i> (Gnt1), FOXG_<i>12436/FOXG</i>_<i>14101</i> (Gnt2), FOXG_<i>12874</i> (Gnt3), FOXG_<i>12897</i> (Gnt4), FOXG_<i>14149</i> (Gnt6), and <i>FOXG-16408</i> (Gnt7), with the corresponding orthologs from <i>Saccharomyces cerevisiae</i> Gnt1 (YOR320c) and <i>Coprinopsis cinerea</i> Gnt1 (CC1G 14119). Identical amino acids are highlighted on a grey background. Conserved motifs: DXD triads (black rectangle), Asp residues involved in ion binding (filled black circle), and conserved Cys residues (open triangles) are indicated. Amino acid sequences were aligned using Clustal W. (<b>B</b>) Scaled diagrams with conserved features of <i>F. oxysporum</i> f.sp. <i>lycopersici</i> Gnt proteins. Positions of the conserved DXD domains, Cys residues (C), and transmembrane domains (dashed boxes) are indicated. </p

Gnt2 contributes to virulence of <i>F. oxysporum</i> on plant and animal systems.

<p>(<b>A</b>) Groups of ten plants (cultivar Monika) were inoculated by immersing the roots into a suspension of 5 x 10⁶ freshly obtained microconidia mL^-1 of the wild type (wt), the Δ<i>gnt2</i> mutant and cΔ<i>gnt2</i> complemented strains, and planted in minipots. Percentage survival was recorded after different time points. All experiments were performed at least three times with similar results. The data shown are from one representative experiment. (<b>B</b>) Mantel-Cox plots of <i>Galleria mellonella</i> larvae survival after injection of 1.6 x 10⁵ microconidia of the indicated strains into the hemocoel and incubation at 30 °C. Data shown are from one representative experiment. Experiments were performed three times with similar results.</p