Self-assembly of proteins into a three-dimensional multilayer system: investigation of the surface of the human fungal pathogen Aspergillus fumigatus.

Hydrophobins are small surface active proteins that fulfil a wide spectrum of functions in fungal growth and development. The human fungal pathogen Aspergillus fumigatus expresses RodA hydrophobins that self-assemble on the outer conidial surface into tightly organized nanorods known as rodlets. AFM investigation of the conidial surface allows us to evidence that RodA hydrophobins self-assemble into rodlets through bilayers. Within bilayers, hydrophilic domains of hydrophobins point inward, thus making a hydrophilic core, while hydrophobic domains point outward. AFM measurements reveal that several rodlet bilayers are present on the conidial surface thus showing that proteins self-assemble into a complex three-dimensional multilayer system. The self-assembly of RodA hydrophobins into rodlets results from attractive interactions between stacked β-sheets, which conduct to a final linear cross-β spine structure. A Monte Carlo simulation shows that anisotropic interactions are the main driving forces leading the hydrophobins to self-assemble into parallel rodlets, which are further structured in nanodomains. Taken together, these findings allow us to propose a mechanism, which conducts RodA hydrophobins to a highly ordered rodlet structure. The mechanism of hydrophobin assembly into rodlets offers new prospects for the development of more efficient strategies leading to disruption of rodlet formation allowing a rapid detection of the fungus by the immune system

What’s new on physiology and virulence factors of Scedosporium apiospermum ?

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Spontaneous self-assembly of SC3 hydrophobins into nanorods in aqueous solution

Hydrophobins are small surface active proteins secreted by filamentous fungi. Because of their ability to self-assemble at hydrophilic–hydrophobic interfaces, hydrophobins play a key role in fungal growth and development. In the present work, the organization in aqueous solution of SC3 hydrophobins from the fungus Schizophyllum commune was assessed using Dynamic Light Scattering, Atomic Force Microscopy and fluorescence spectroscopy. These complementary approaches have demonstrated that SC3 hydrophobins are able not only to spontaneously self-assemble at the air–water interface but also in pure water. AFM experiments evidenced that hydrophobins self-assemble in solution into nanorods. Fluorescence assays with thioflavin T allowed establishing that the mechanism governing SC3 hydrophobin self-assembly into nanorods involves β-sheet stacking. SC3 assembly was shown to be strongly influenced by ionic strength and solution pH. The presence of a very low ionic strength significantly favoured the protein self-assembly but a further increase of ions in solution disrupted the protein assembly. It was assessed that solution pH had a significant effect on the SC3 hydrophobins organization. In peculiar, the self-assembly process was considerably reduced at acidic pH. Our findings demonstrate that the self-assembly of SC3 hydrophobins into nanorods of well-defined length can be directly controlled in solution. Such control allows opening the way for the development of new smart self-assembled structures for targeted applications

Cell wall modifications during conidial maturation of the human pathogenic fungus Pseudallescheria boydii

This is the final version of the article. Available from the publisher via the DOI in this record.Progress in extending the life expectancy of cystic fibrosis (CF) patients remains jeopardized by the increasing incidence of fungal respiratory infections. Pseudallescheria boydii (P. boydii), an emerging pathogen of humans, is a filamentous fungus frequently isolated from the respiratory secretions of CF patients. It is commonly believed that infection by this fungus occurs through inhalation of airborne conidia, but the mechanisms allowing the adherence of Pseudallescheria to the host epithelial cells and its escape from the host immune defenses remain largely unknown. Given that the cell wall orchestrates all these processes, we were interested in studying its dynamic changes in conidia as function of the age of cultures. We found that the surface hydrophobicity and electronegative charge of conidia increased with the age of culture. Melanin that can influence the cell surface properties, was extracted from conidia and estimated using UV-visible spectrophotometry. Cells were also directly examined and compared using electron paramagnetic resonance (EPR) that determines the production of free radicals. Consistent with the increased amount of melanin, the EPR signal intensity decreased suggesting polymerization of melanin. These results were confirmed by flow cytometry after studying the effect of melanin polymerization on the surface accessibility of mannose-containing glycoconjugates to fluorescent concanavalin A. In the absence of melanin, conidia showed a marked increase in fluorescence intensity as the age of culture increased. Using atomic force microscopy, we were unable to find rodlet-forming hydrophobins, molecules that can also affect conidial surface properties. In conclusion, the changes in surface properties and biochemical composition of the conidial wall with the age of culture highlight the process of conidial maturation. Mannose-containing glycoconjugates that are involved in immune recognition, are progressively masked by polymerization of melanin, an antioxidant that is commonly thought to allow fungal escape from the host immune defenses.The study was funded by “Région Pays de la Loire” in the frame of “Myco-AFM” research program). BED was supported by the Dutch Virgo Consortium (FES0908, NGI 050-060-452) and CAPES/BRASIL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Cell wall changes during maturation of conidia in Pseudallescheria boydii

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A flavoprotein supports cell wall properties in the necrotrophic fungus Alternaria brassicicola

Background Flavin-dependent monooxygenases are involved in key biological processes as they catalyze a wide variety of chemo-, regio- and enantioselective oxygenation reactions. Flavoprotein monooxygenases are frequently encountered in micro-organisms, most of which require further functional and biocatalytic assessment. Here we investigated the function of the AbMak1 gene, which encodes a group A flavin monooxygenase in the plant pathogenic fungus Alternaria brassicicola, by generating a deficient mutant and examining its phenotype. Results Functional analysis indicates that the AbMak1 protein is involved in cell wall biogenesis and influences the melanization process. We documented a significant decrease in melanin content in the Δabmak1 strain compared to the wild-type and complemented strains. We investigated the cell wall morphology and physical properties in the wild-type and transformants using electron and atomic force microscopy. These approaches confirmed the aberrant morphology of the conidial wall structure in the Δabmak1 strain which had an impact on hydrophilic adhesion and conidial surface stiffness. However, there was no significant impairment in growth, conidia formation, pathogenicity or susceptibility to various environmental stresses in the Δabmak1 strain. Conclusion This study sheds new light on the function of a fungal flavin-dependent monooxygenase, which plays an important role in melanization

A nanostructural view of the cell wall disassembly process during fruit ripening and postharvest storage by atomic force microscopy

Background: The mechanical properties of parenchyma cell walls and the strength and extension of adhesion areas between adjacent cells, jointly with cell turgor, are main determinants of firmness of fleshy fruits. These traits are modified during ripening leading to fruit softening. Cell wall modifications involve the depolymerisation of matrix glycans and pectins, the solubilisation of pectins and the loss of neutral sugars from pectin side chains. These changes weaken the cell walls and increase cell separation, which in combination with a reduction in cell turgor, bring about textural changes. Atomic force microscopy (AFM) has been used to characterize the nanostructure of cell wall polysaccharides during the ripening and postharvest storage of several fruits. This technique allows the imaging of individual polymers at high magnification with minimal sample preparation. Scope and approach: This paper reviews the main features of the cell wall disassembly process associated to fruit softening from a nanostructural point of view, as has been provided by AFM studies. Key findings and conclusions: AFM studies show that pectin size, ramification and complexity is reduced during fruit ripening and storage, and in most cases these changes correlate with softening. Postharvest treatments that improve fruit quality have been proven to preserve pectin structure, suggesting a clear link between softening and pectin metabolism. Nanostructural characterization of cellulose and hemicellulose during ripening has been poorly explored by AFM and the scarce results available are not conclusive. Globally, AFM could be a powerful tool to gain insights about the bases of textural fruit quality in fresh and stored fruits

Immunolocalization of cell wall polymers in grapevine (Vitis vinifera) internodes under nitrogen, phosphorus or sulfur deficiency

Abstract The impact on cell wall (CW) of the deficiency in nitrogen (–N), phosphorus (–P) or sulphur (–S), known to impair essential metabolic pathways, was investigated in the economically important fruit species Vitis vinifera L. Using cuttings as an experimental model a reduction in total internode number and altered xylem shape was observed. Under –N an increased internode length was also seen. CW composition, visualised after staining with calcofluor white, Toluidine blue and ruthenium red, showed decreased cellulose in all stresses and increased pectin content in recently formed internodes under –N compared to the control. Using CW-epitope specific monoclonal antibodies (mAbs), lower amounts of extensins incorporated in the wall were also observed under –N and –P conditions. Conversely, increased pectins with a low degree of methyl-esterification and richer in long linear 1,5-arabinan rhamnogalacturonan-I (RG-I) side chains were observed under –N and –P in mature internodes which, in the former condition, were able to form dimeric association through calcium ions. –N was the only condition in which 1,5-arabinan branched RG- content was not altered, as –P and –S older internodes showed, respectively, lower and higher amounts of this polymer. Higher xyloglucan content in older internodes was also observed under –N. The results suggest that impairments of specific CW components led to changes in the deposition of other polymers to promote stiffening of the CW. The unchanged extensin amount observed under –S may contribute to attenuating the effects on the CW integrity caused by this stress. Our work showed that, in organized V. vinifera tissues, modifications in a given CW component can be compensated by synthesis of different polymers and/or alternative linking between polymers. The results also pinpoint different strategies at the CW level to overcome mineral stress depending on how essential they are to cell growth and plant development