Deep impact of the inactivation of the SecA2-only protein export pathway on the proteosurfaceome of Listeria monocytogenes.

Listeria monocytogenes presents a dimorphism associated to the SecA2 activity with cells having a normal rod shape or a dysmorphic elongated filamentous form. Besides variation of the cell and colony morphotype, this cell differentiation has profound ecophysiological and physiopathological implications with collateral effects on virulence and pathogenicity, biotope colonisation, bacterial adhesion and biofilm formation. This suggests the SecA2-only protein export could influence the listerial cell surface, which was investigated first by characterising its properties in L. monocytogenes wt and ΔsecA2. The degree of hydrophilicity and Lewis acid-base properties appeared significantly affected upon SecA2 inactivation. As modification of electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteosurfaceome was further investigated by shotgun label-free proteomic analysis with a comparative relative quantitative approach. Following secretomic analysis, the protein secretion routes of the identified proteins were mapped considering the cognate transport and post-translocational maturation systems, as well as protein categories and subcellular localisation. Differential protein abundance profiles coupled to network analysis revealed the SecA2 dependence of 48 proteins, including some related to cell envelope biogenesis, translation and protein export, which could account for modifications of adhesion and surface properties of L. monocytogenes upon SecA2 inactivation. This investigation unravelled the profound influence of SecA2 activity on the cell surface properties and proteosurfaceome of L. monocytogenes, which provides advanced insights about its ecophysiopathology. SIGNIFICANCE: L. monocytogenes is a foodborne zoonotic pathogen and etiological agent of human listeriosis. This species presents a cellular dimorphism associated to the SecA2 activity that has profound physiopathological and ecophysiological implications with collateral effects on bacterial virulence and colonisation. To explore the influence of the SecA2-only protein export on the listerial cell, the surface properties of L. monocytogenes expressing or depleted of SecA2 was characterised by microelectrophoresis, microbial affinity to solvents and contact angles analyses. As modifications of hydrophilicity and Lewis acid-base electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteinaceous subset of the surfaceome, i.e. the proteosurfaceome, was investigated further by shotgun label-free proteomic analysis. This subproteome appeared quite impacted upon SecA2 inactivation with the identification of proteins accounting for modifications in the cell surface properties. The profound influence of SecA2 activity on the cell surface of L. monocytogenes was unravelled, which provides advanced insights about its ecophysiopathology.

Fungal Treatment for the Valorization of Technical Soda Lignin.

Technical lignins produced as a by-product in biorefinery processes represent a potential source of renewable carbon. In consideration of the possibilities of the industrial transformation of this substrate into various valuable bio-based molecules, the biological deconstruction of a technical soda lignin by filamentous fungi was investigated. The ability of three basidiomycetes (Polyporus brumalis, Pycnoporus sanguineus and Leiotrametes menziesii) to modify this material, the resultant structural and chemical changes, and the secreted proteins during growth on this substrate were investigated. The three fungi could grow on the technical lignin alone, and the growth rate increased when the media were supplemented with glucose or maltose. The proteomic analysis of the culture supernatants after three days of growth revealed the secretion of numerous Carbohydrate-Active Enzymes (CAZymes). The secretomic profiles varied widely between the strains and the presence of technical lignin alone triggered the early secretion of many lignin-acting oxidoreductases. The secretomes were notably rich in glycoside hydrolases and H2O2-producing auxiliary activity enzymes with copper radical oxidases being induced on lignin for all strains. The lignin treatment by fungi modified both the soluble and insoluble lignin fractions. A significant decrease in the amount of soluble higher molar mass compounds was observed in the case of P. sanguineus. This strain was also responsible for the modification of the lower molar mass compounds of the lignin insoluble fraction and a 40% decrease in the thioacidolysis yield. The similarity in the activities of P. sanguineus and P. brumalis in modifying the functional groups of the technical lignin were observed, the results suggest that the lignin has undergone structural changes, or at least changes in its composition, and pave the route for the utilization of filamentous fungi to functionalize technical lignins and produce the enzymes of interest for biorefinery applications.

Secretomic analysis of cheap enzymatic cocktails of Aspergillus niger LBM 134 grown on cassava bagasse and sugarcane bagasse.

Currently, agroindustrial wastes are little used for generating value-added products; hence, their use of these waste to produce enzymatic cocktails for the conversion of lignocellulosic biomass to fermentable sugars is a very interesting alternative in the second-generation bioethanol process. The Ascomycota fungus Aspergillus niger LBM 134 produces hydrolytic enzymes in large proportions. In this work, A. niger LBM 134 was grown on sugarcane and cassava bagasses under optimized conditions. To identify the extracellular enzymes involved in the degradation of these agroindustrial wastes, the secretomes of the culture supernatants of the fungus were analyzed and validated by biochemical assays of the enzymatic activities. A. niger LBM 134 secreted higher quantities of xylanases and accessory hemicellulases when it grew on sugarcane bagasse, whereas more cellulases, amylases, and pectinases were secreted when it grew on cassava bagasse. These findings suggest two promising enzyme cocktails for the hydrolysis of lignocellulose carbohydrate polymers to fermentable sugars. These bioinformatic analysis were functional validates through enzymatic biochemical assays that confirm the biotechnological potential of A. niger LBM 134 for the bioconversion of hemicellulosic substrates such as sugarcane and cassava bagasses.

"Pop-slide" patterning: Rapid fabrication of microstructured PDMS gasket slides for biological applications.

We describe a "pop-slide" patterning approach to easily produce thin film microstructures on the surface of glass with varying feature sizes (3 µm - 250 µm) and aspect ratios (0.066 - 3) within 45 minutes. This low cost method does not require specialized equipment while allowing us to produce micro structured gasket layers for sandwich assays and could be readily applied to many biological applications.