GH10 xylanase D from Penicillium funiculosum: biochemical studies and xylooligosaccharide production

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus <it>Penicillium funiculosum </it>produces a range of glycoside hydrolases (GH). The <it>XynD </it>gene, encoding the sole <it>P. funiculosum </it>GH10 xylanase described so far, was cloned into the pPICZαA vector and expressed in methylotrophe yeast <it>Pichia pastoris</it>, in order to compare the results obtained with the <it>P. funiculosum </it>GH11 xylanases data.</p> <p>Results</p> <p>High level expression of recombinant XynD was obtained with a secretion of around 60 mg.L^-1. The protein was purified to homogeneity using one purification step. The apparent size on SDS-PAGE was around 64 kDa and was 46 kDa by mass spectrometry thus higher than the expected molecular mass of 41 kDa. The recombinant protein was N- and O-glycosylated, as demonstrated using glycoprotein staining and deglycosylation reactions, which explained the discrepancy in molecular mass. Enzyme-catalysed hydrolysis of low viscosity arabinoxylan (LVAX) was maximal at pH 5.0 with <it>K</it>m_(app)and <it>k_cat</it>/<it>K</it>m_(app)of 3.7 ± 0.2 (mg.mL^-1) and 132 (s^-1mg^-1.mL), respectively. The activity of XynD was optimal at 80°C and the recombinant enzyme has shown an interesting high thermal stability at 70°C for at least 180 min without loss of activity. The enzyme had an endo-mode of action on xylan forming mainly xylobiose and short-chain xylooligosaccharides (XOS). The initial rate data from the hydrolysis of short XOS indicated that the catalytic efficiency increased slightly with increasing their chain length with a small difference of the XynD catalytic efficiency against the different XOS.</p> <p>Conclusion</p> <p>Because of its attractive properties XynD might be considered for biotechnological applications. Moreover, XOS hydrolysis suggested that XynD possess four catalytic subsites with a high energy of interaction with the substrate and a fifth subsite with a small energy of interaction, according to the GH10 xylanase literature data.</p

High-throughput functional metagenomics for the discovery of glycan metabolizing pathways

Glycans are widely distributed in nature. Produced by almost all organisms, they are involved in numerous cellular processes, such as energy supply and storage, cell structuration, protein maturation and signalling, and cell recognition. Glycans are thus key elements mediating the interactions between mammals, plants, bacteria, fungi and even viruses. They also represent a reliable source of carbon for microbes, which have developed complex strategies to face their structural diversity and to harvest them. However between 70 and 99% of these microorganisms are still uncultured, while they represent a goldmine for the discovery of new enzymes. In order to boost their identification and characterization, a functional metagenomic approach was developed, based on the design of various high-throughput, robust and sensitive screening strategies. The functional potential of Gbp of metagenomic DNA from various origins was explored, revealing dozens of novel enzyme families and functions. Integration of biochemical, structural, meta-omic and omic data allowed us to decipher, from the molecular to the ecosystemic scale, novel mechanisms of plant, microbial and mammal glycan metabolization. These new metabolic pathways involve batteries of glycoside-hydrolases, glycoside-phosphorylases and sugar transporters. These fascinating proteins appear as new targets to control host-microbe interactions. They also constitute very efficient biotechnological tools for biorefineries and synthetic biology

Investigating Host Microbiota Relationships Through Functional Metagenomics

The human Intestinal mucus is formed by glycoproteins, the O- and N-linked glycans which constitute a crucial source of carbon for commensal gut bacteria, especially when deprived of dietary glycans of plant origin. In recent years, a dozen carbohydrate-active enzymes from cultivated mucin degraders have been characterized. But yet, considering the fact that uncultured species predominate in the human gut microbiota, these biochemical data are far from exhaustive. In this study, we used functional metagenomics to identify new metabolic pathways in uncultured bacteria involved in harvesting mucin glycans. First, we performed a high-throughput screening of a fosmid metagenomic library constructed from the ileum mucosa microbiota using chromogenic substrates. The screening resulted in the isolation of 124 clones producing activities crucial in the degradation of human O- and N-glycans, namely sialidases, beta-D-N-acetyl-glucosaminidase, beta-D-N-acetyl-galactosaminidase, and/or beta-D-mannosidase. Thirteen of these clones were selected based on their diversified functional profiles and were further analyzed on a secondary screening. This step consisted of lectin binding assays to demonstrate the ability of the clones to degrade human intestinal mucus. In total, the structural modification of several mucin motifs, sialylated mucin ones in particular, was evidenced for nine clones. Sequencing their metagenomic loci highlighted complex catabolic pathways involving the complementary functions of glycan sensing, transport, hydrolysis, deacetylation, and deamination, which were sometimes associated with amino acid metabolism machinery. These loci are assigned to several Bacteroides and Feacalibacterium species highly prevalent and abundant in the gut microbiome and explain the metabolic flexibility of gut bacteria feeding both on dietary and human glycans

Investigating host-microbiome interactions by droplet based microfluidics

Funder: Royal Society Newton fellowshipFunder: CAPES Scholarship - BrazilAbstract: Background: Despite the importance of the mucosal interface between microbiota and the host in gut homeostasis, little is known about the mechanisms of bacterial gut colonization, involving foraging for glycans produced by epithelial cells. The slow pace of progress toward understanding the underlying molecular mechanisms is largely due to the lack of efficient discovery tools, especially those targeting the uncultured fraction of the microbiota. Results: Here, we introduce an ultra-high-throughput metagenomic approach based on droplet microfluidics, to screen fosmid libraries. Thousands of bacterial genomes can be covered in 1 h of work, with less than ten micrograms of substrate. Applied to the screening of the mucosal microbiota for β-N-acetylgalactosaminidase activity, this approach allowed the identification of pathways involved in the degradation of human gangliosides and milk oligosaccharides, the structural homologs of intestinal mucin glycans. These pathways, whose prevalence is associated with inflammatory bowel diseases, could be the result of horizontal gene transfers with Bacteroides species. Such pathways represent novel targets to study the microbiota-host interactions in the context of inflammatory bowel diseases, in which the integrity of the mucosal barrier is impaired. Conclusion: By compartmentalizing experiments inside microfluidic droplets, this method speeds up and miniaturizes by several orders of magnitude the screening process compared to conventional approaches, to capture entire metabolic pathways from metagenomic libraries. The method is compatible with all types of (meta)genomic libraries, and employs a commercially available flow cytometer instead of a custom-made sorting system to detect intracellular or extracellular enzyme activities. This versatile and generic workflow will accelerate experimental exploration campaigns in functional metagenomics and holobiomics studies, to further decipher host-microbiota relationships. BEyZgKg3YsWtKJ_ei8gXkiVideo Abstrac

Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We visualized sequential spread of virus from the nasal cavity to the lungs followed by systemic spread to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days of infection. In addition to direct neutralization, in vivo efficacy required Fc effector functions of NAbs, with contributions from monocytes, neutrophils and natural killer cells, to dampen inflammatory responses and limit immunopathology. Thus, our study highlights the requirement of both Fab and Fc effector functions for an optimal in vivo efficacy afforded by NAbs against SARS-CoV-2

Modulation de la susceptibilité des cellules à l'infection par le VIH-1 : effets des IFN-alpha et de la protéine virale Rev

This work focused on the study of two mechanisms involved in the modulation of cellular susceptibility to HIV-1 infection. First, we studied the effect of the 12 IFN-α subtypes on the inhibition of viral replication. We showed that IFN-α have different inhibition potencies. We initially confirmed that subtype 14 is the most potent. We also examined the effect of each IFN-α on the different steps of the viral replication cycle. We observed that different steps are affected to various extent by IFN-α depending on the subtype. Thus, some subtypes are more potent on the early steps while others seem to be more effective on the late steps. The study of the transcriptome induced by the different subtypes suggests that the inhibition potency of IFN-α14 is partly related to a higher level of induction of several restriction factors. Second, we studied an interference mechanism induced by the virus itself. We had already observed that the viral protein Rev could interfere with a second viral infection. We observed that all functional domains of Rev (nuclear localization, multimerization, RNA binding, nuclear export) are necessary for interference. We then identified the steps of the viral cycle targeted by Rev as nuclear import and/or integration. In addition, we demonstrated that some viral strains are resistant to Rev-mediated restriction. Our results suggest that the viral capsid may be involved in this mechanism and one or several cellular factors could be involved.Ce travail a porté sur l’étude de deux mécanismes impliqués dans la modulation de la susceptibilité cellulaire à l’infection par le VIH-1. Premièrement, nous avons étudié l’effet des 12 sous-types d’IFN-α sur l’inhibition de la réplication virale. Nous avons montré que les IFN-α avaient des puissances d’inhibition différentes. Nous avons initialement confirmé que le sous-type 14 est le plus puissant. Nous avons également décortiqué l’effet de chaque IFN-α sur les différentes étapes du cycle viral. Nous avons observé que différentes étapes du cycle sont affectées par les IFN-α et ce de manières différentes selon le sous-type. Ainsi, certains sous-types sont plus puissants sur les étapes précoces alors que d’autres semblent plus efficaces sur les étapes tardives. L’étude du transcriptome induit par les différents sous-types nous suggère que la plus forte inhibition de l’IFN-α14 serait en partie liée à un plus fort niveau d’induction de plusieurs facteurs de restriction. Deuxièmement, nous avons étudié un mécanisme d’interférence induit par le virus lui-même. Nous avions déjà observé que la protéine virale Rev pouvait interférer avec une seconde infection virale. Nous avons observé que tous les domaines fonctionnels de Rev (localisation nucléaire, multimérisation, liaison à l’ARN, export nucléaire) sont nécessaires pour cette interférence. Nous avons ensuite identifié l’étape du cycle viral ciblée par Rev comme étant l’import nucléaire et/ou l’intégration. De même, nous avons montré que certaines souches virales sont résistantes à la restriction médiée par Rev. Les résultats de notre étude suggèrent que la capside virale jouerait un rôle dans ce mécanisme et un ou plusieurs facteurs cellulaires pourraient également être impliqués

Invertase vacuolaire de Solanum lycopersicum : relations structure-fonction et régulations post-traductionnelles in vitro

Les invertases de plantes (Invs) hydrolysent de manière irréversible le saccharose en fructose et glucose. En fonction de leur pH optimum et de leur localisation subcellulaire, les Invs sont classées en trois groupes : alcaline et neutre (A/N-Inv), vacuolaire (VI) et de paroi (CWI). Le but de notre étude a été de mieux comprendre les mécanismes impliqués dans les régulations post-traductionnelles d'une VI de Solanum lycopersicum (VINV). L'ADNc codant pour VINV a été cloné et exprimé dans le système hétérologue Pichia pastoris. Après purification, la caractérisation biochimique a été réalisée et a montré des résultats comparables à ceux obtenus précédemment pour d'autres Invs. La structure tridimensionnelle de VINV a été résolue par cristallographie aux rayons X à 2,75 Å et il s'agit de la première structure d'une VI décrite jusqu'ici. Des expériences de mutagénèse dirigée ont permis d'identifier certains acides aminés impliqués dans la catalyse : le nucléophile, le catalyseur acide/base, le stabilisateur d'état de transition et un résidu qui module le pKa du catalyseur acide/base. Par ailleurs, la régulation de l'activité de VINV a été étudiée. La N-glycosylation de VINV recombinante semble être importante pour la stabilité de la structure. De plus, l'activité VINV peut aussi être modulée par un inhibiteur protéique spécifique. Une approche de génomique fonctionnelle a été utilisée, et un inhibiteur d'invertase vacuolaire putatif (SolyVIF) de S. lycopersicum a été identifié dans la banque de données des Solanacées. L'ADNc codant pour SolyVIF a été cloné et exprimé dans le système hétérologue Escherichia coli Rosetta gami (DE3).Plant invertases (Invs) hydrolyze irreversibly sucrose into fructose and glucose. Based on their pH optima and subcellular localization, Invs are categorized into three groups: alkaline and neutral invertase (A/N-Inv), vacuolar invertase (VI), and cell wall invertase (CWI). The goal of our study was to better understand mechanisms involved in the molecular regulation of a VI from Solanum lycopersicum (VINV) at post-translational levels. The VINV cDNA was cloned and heterologously expressed in Pichia pastoris. After purification, the biochemical characterization was performed and showed comparable results with those obtained previously for other characterized Invs. The three-dimensional structure of VINV was solved by X-ray crystallography to 2.75 Å resolution and it was the first structure of a plant VI described so far. Mutations experiments allowed to identify important amino acids: the nucleophile, the acid/base catalyst, the transition-state stabilizer and a residue that modulate pKa of the acid/base catalyst. Moreover, the regulation of VINV at different post-translational levels was studied. N-glycosylation of recombinant VINV seems to be important for structure stability. VINV activity can also be modulated by specific proteinaceous inhibitor. A functional genomics approach was used, and a putative vacuolar invertase inhibitor (SolyVIF) of S. lycopersicum was identified in the Solanaceae data bank. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli Rosetta gami (DE3). Recombinant protein was purified and characterized

Modulation of cell susceptibility to HIV-1 infection : effects of IFN-α and of the viral protein Rev

Ce travail a porté sur l’étude de deux mécanismes impliqués dans la modulation de la susceptibilité cellulaire à l’infection par le VIH-1. Premièrement, nous avons étudié l’effet des 12 sous-types d’IFN-α sur l’inhibition de la réplication virale. Nous avons montré que les IFN-α avaient des puissances d’inhibition différentes. Nous avons initialement confirmé que le sous-type 14 est le plus puissant. Nous avons également décortiqué l’effet de chaque IFN-α sur les différentes étapes du cycle viral. Nous avons observé que différentes étapes du cycle sont affectées par les IFN-α et ce de manières différentes selon le sous-type. Ainsi, certains sous-types sont plus puissants sur les étapes précoces alors que d’autres semblent plus efficaces sur les étapes tardives. L’étude du transcriptome induit par les différents sous-types nous suggère que la plus forte inhibition de l’IFN-α14 serait en partie liée à un plus fort niveau d’induction de plusieurs facteurs de restriction. Deuxièmement, nous avons étudié un mécanisme d’interférence induit par le virus lui-même. Nous avions déjà observé que la protéine virale Rev pouvait interférer avec une seconde infection virale. Nous avons observé que tous les domaines fonctionnels de Rev (localisation nucléaire, multimérisation, liaison à l’ARN, export nucléaire) sont nécessaires pour cette interférence. Nous avons ensuite identifié l’étape du cycle viral ciblée par Rev comme étant l’import nucléaire et/ou l’intégration. De même, nous avons montré que certaines souches virales sont résistantes à la restriction médiée par Rev. Les résultats de notre étude suggèrent que la capside virale jouerait un rôle dans ce mécanisme et un ou plusieurs facteurs cellulaires pourraient également être impliqués.This work focused on the study of two mechanisms involved in the modulation of cellular susceptibility to HIV-1 infection. First, we studied the effect of the 12 IFN-α subtypes on the inhibition of viral replication. We showed that IFN-α have different inhibition potencies. We initially confirmed that subtype 14 is the most potent. We also examined the effect of each IFN-α on the different steps of the viral replication cycle. We observed that different steps are affected to various extent by IFN-α depending on the subtype. Thus, some subtypes are more potent on the early steps while others seem to be more effective on the late steps. The study of the transcriptome induced by the different subtypes suggests that the inhibition potency of IFN-α14 is partly related to a higher level of induction of several restriction factors. Second, we studied an interference mechanism induced by the virus itself. We had already observed that the viral protein Rev could interfere with a second viral infection. We observed that all functional domains of Rev (nuclear localization, multimerization, RNA binding, nuclear export) are necessary for interference. We then identified the steps of the viral cycle targeted by Rev as nuclear import and/or integration. In addition, we demonstrated that some viral strains are resistant to Rev-mediated restriction. Our results suggest that the viral capsid may be involved in this mechanism and one or several cellular factors could be involved

Sucrose and invertases, a part of the plant defense response to the biotic stresses

International audienceSucrose is the main form of assimilated carbon which is produced during photosynthesis and then transported from source to sink tissues via the phloem. This disaccharide is known to have important roles as signaling molecule and it is involved in many metabolic processes in plants. Essential for plant growth and development, sucrose is engaged in plant defense by activating plant immune responses against pathogens. During infection, pathogens reallocate the plant sugars for their own needs forcing the plants to modify their sugar content and triggering their defense responses. Among enzymes that hydrolyze sucrose and alter carbohydrate partitioning, invertases have been reported to be affected during plant-pathogen interactions. Recent highlights on the role of invertases in the establishment of plant defense responses suggest a more complex regulation of sugar signaling in plant-pathogen interaction