The Experts below are selected from a list of 18993 Experts worldwide ranked by ideXlab platform
Jean-paul Latgé - One of the best experts on this subject based on the ideXlab platform.
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Revisiting Old Questions and New Approaches to Investigate the Fungal Cell Wall Construction.
Current topics in microbiology and immunology, 2020Co-Authors: Michael Blatzer, Anne Beauvais, Bernard Henrissat, Jean-paul LatgéAbstract:The beginning of our understanding of the Cell Wall construction came from the work of talented biochemists in the 70–80’s. Then came the era of sequencing. Paradoxically, the accumulation of Fungal genomes complicated rather than solved the mystery of Cell Wall construction, by revealing the involvement of a much higher number of proteins than originally thought. The situation has become even more complicated since it is now recognized that the Cell Wall is an organelle whose composition continuously evolves with the changes in the environment or with the age of the Fungal Cell. The use of new and sophisticated technologies to observe Cell Wall construction at an almost atomic scale should improve our knowledge of the Cell Wall construction. This essay will present some of the major and still unresolved questions to understand the Fungal Cell Wall biosynthesis and some of these exciting futurist approaches.
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Potential of Chemically Synthesized Oligosaccharides To Define the Carbohydrate Moieties of the Fungal Cell Wall Responsible for the Human Immune Response, Using Aspergillus fumigatus Galactomannan as a Model.
mSphere, 2020Co-Authors: Sarah Sze Wah Wong, Jean-paul Latgé, Thierry Fontaine, Vadim B. Krylov, Dmitry A. Argunov, Alexander A. Karelin, Jean-phillipe Bouchara, Nikolay E. NifantievAbstract:Methodologies to identify epitopes or ligands of the Fungal Cell Wall polysaccharides influencing the immune response of human pathogens have to date been imperfect. Using the galactomannan (GM) of Aspergillus fumigatus as a model, we have shown that synthetic oligosaccharides of distinct structures representing key fragments of Cell Wall polysaccharides are the most precise tools to study the serological and immunomodulatory properties of a Fungal polysaccharide.
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β glucan grafted microcapsule a tool for studying the immunomodulatory effect of microbial Cell Wall polysaccharides
Bioconjugate Chemistry, 2019Co-Authors: Kawthar Bouchemal, Jean-paul Latgé, Sarah Sze Wah Wong, Janet A. Willment, Nicolas Huang, Vishukumar AimaniandaAbstract:β-(1,3)-Glucan is one of the antigenic components of the bacterial as well as Fungal Cell Wall. We designed microcapsules (MCs) ligated with β-(1,3)-glucan, to study its immunomodulatory effect. Th...
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the Fungal Cell Wall structure biosynthesis and function
Microbiology spectrum, 2017Co-Authors: Jean-paul Latgé, Carol A. MunroAbstract:The molecular composition of the Cell Wall is critical for the biology and ecology of each Fungal species. Fungal Walls are composed of matrix components that are embedded and linked to scaffolds of fibrous load-bearing polysaccharides. Most of the major Cell Wall components of Fungal pathogens are not represented in humans, other mammals, or plants, and therefore the immune systems of animals and plants have evolved to recognize many of the conserved elements of Fungal Walls. For similar reasons the enzymes that assemble Fungal Cell Wall components are exCellent targets for antiFungal chemotherapies and fungicides. However, for Fungal pathogens, the Cell Wall is often disguised since key signature molecules for immune recognition are sometimes masked by immunologically inert molecules. Cell Wall damage leads to the activation of sophisticated fail-safe mechanisms that shore up and repair Walls to avoid catastrophic breaching of the integrity of the surface. The frontiers of research on Fungal Cell Walls are moving from a descriptive phase defining the underlying genes and component parts of Fungal Walls to more dynamic analyses of how the various components are assembled, cross-linked, and modified in response to environmental signals. This review therefore discusses recent advances in research investigating the composition, synthesis, and regulation of Cell Walls and how the Cell Wall is targeted by immune recognition systems and the design of antiFungal diagnostics and therapeutics.
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undressing the Fungal Cell Wall Cell membrane the antiFungal drug targets
Current Pharmaceutical Design, 2013Co-Authors: Rui Tada, Jean-paul Latgé, Vishukumar AimaniandaAbstract:Being external, the Fungal Cell Wall plays a crucial role in the Fungal life. By covering the underneath Cell, it offers mechanical strength and acts as a barrier, thus protecting the fungus from the hostile environment. Chemically, this Cell Wall is composed of different polysaccharides. Because of their specific composition, the Fungal Cell Wall and its underlying plasma membrane are unique targets for the development of drugs against pathogenic Fungal species. The objective of this review is to consolidate the current knowledge on the antiFungal drugs targeting the Cell Wall and plasma membrane, mainly of Aspergillus and Candida species – the most prevalent Fungal pathogens, and also to present challenges and questions conditioning the development of new antiFungal drugs targeting the Cell Wall.
Carol A. Munro - One of the best experts on this subject based on the ideXlab platform.
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the Fungal Cell Wall structure biosynthesis and function
Microbiology spectrum, 2017Co-Authors: Jean-paul Latgé, Carol A. MunroAbstract:The molecular composition of the Cell Wall is critical for the biology and ecology of each Fungal species. Fungal Walls are composed of matrix components that are embedded and linked to scaffolds of fibrous load-bearing polysaccharides. Most of the major Cell Wall components of Fungal pathogens are not represented in humans, other mammals, or plants, and therefore the immune systems of animals and plants have evolved to recognize many of the conserved elements of Fungal Walls. For similar reasons the enzymes that assemble Fungal Cell Wall components are exCellent targets for antiFungal chemotherapies and fungicides. However, for Fungal pathogens, the Cell Wall is often disguised since key signature molecules for immune recognition are sometimes masked by immunologically inert molecules. Cell Wall damage leads to the activation of sophisticated fail-safe mechanisms that shore up and repair Walls to avoid catastrophic breaching of the integrity of the surface. The frontiers of research on Fungal Cell Walls are moving from a descriptive phase defining the underlying genes and component parts of Fungal Walls to more dynamic analyses of how the various components are assembled, cross-linked, and modified in response to environmental signals. This review therefore discusses recent advances in research investigating the composition, synthesis, and regulation of Cell Walls and how the Cell Wall is targeted by immune recognition systems and the design of antiFungal diagnostics and therapeutics.
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rim pathway mediated alterations in the Fungal Cell Wall influence immune recognition and inflammation
Mbio, 2017Co-Authors: Kyla S Ost, Louise A. Walker, Carol A. Munro, Shannon K. Esher, Chrissy Leopold M Wager, Jeanette Wagener, Floyd L Wormley, Andrew J AlspaughAbstract:Compared to other Fungal pathogens, Cryptococcus neoformans is particularly adept at avoiding detection by innate immune Cells. To explore Fungal Cellular features involved in immune avoidance, we characterized Cell surface changes of the C. neoformans rim101Δ mutant, a strain that fails to organize and shield immunogenic epitopes from host detection. These Cell surface changes are associated with an exaggerated, detrimental inflammatory response in mouse models of infection. We determined that the disorganized strain rim101Δ Cell Wall increases macrophage detection in a contact-dependent manner. Using biochemical and microscopy methods, we demonstrated that the rim101Δ strain shows a modest increase in the levels of both Cell Wall chitin and chitosan but that it shows a more dramatic increase in chito-oligomer exposure, as measured by wheat germ agglutinin staining. We also created a series of mutants with various levels of Cell Wall wheat germ agglutinin staining, and we demonstrated that the staining intensity correlates with the degree of macrophage activation in response to each strain. To explore the host receptors responsible for recognizing the rim101Δ mutant, we determined that both the MyD88 and CARD9 innate immune signaling proteins are involved. Finally, we characterized the immune response to the rim101Δ mutant in vivo, documenting a dramatic and sustained increase in Th1 and Th17 cytokine responses. These results suggest that the Rim101 transcription factor actively regulates the C. neoformans Cell Wall to prevent the exposure of immune stimulatory molecules within the host. These studies further explored the ways in which immune Cells detect C. neoformans and other Fungal pathogens by mechanisms that include sensing N-acetylglucosamine-containing structures, such as chitin and chitosan. IMPORTANCE Infectious microorganisms have developed many ways to avoid recognition by the host immune system. For example, pathogenic fungi alter their Cell surfaces to mask immunogenic epitopes. We have created a Fungal strain with a targeted mutation in a pH response pathway that is unable to properly organize its Cell Wall, resulting in a dramatic immune reaction during infection. This mutant Cell Wall is defective in hiding important Cell Wall components, such as the chito-oligomers chitin and chitosan. By creating a series of Cell Wall mutants, we demonstrated that the degree of chito-oligomer exposure correlates with the intensity of innate immune Cell activation. This activation requires a combination of host receptors to recognize and respond to these infecting microorganisms. Therefore, these experiments explored host-pathogen interactions that determine the degree of the subsequent inflammatory response and the likely outcome of infection.
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Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance
Mbio, 2015Co-Authors: Louise A. Walker, Marion Schiavone, Hélène Martin-yken, Etienne Dague, Carol A. Munro, Alistair J. P. BrownAbstract:The Fungal Cell Wall confers Cell morphology and protection against environmental insults. For Fungal pathogens, the Cell Wall is a key immunological modulator and an ideal therapeutic target. Yeast Cell Walls possess an inner matrix of interlinked -glucan and chitin that is thought to provide tensile strength and rigidity. Yeast Cells remodel their Walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and Cell integrity (Mkc1, Cek1) signal- ing pathways. These mitogen-activated protein kinase (MAPK) pathways modulate Cell Wall gene expression, leading to the con- struction of a new, modified Cell Wall. We show that the Cell Wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans Cells are more resistant to hyperosmotic shock than glucose-grown Cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most Cell death occurs within 10 min of osmotic shock. Sudden decreases in Cell volume drive rapid increases in Cell Wall thickness. The elevated stress resistance of lactate-grown Cells correlates with reduced Cell Wall elasticity, reflected in slower changes in Cell vol- ume following hyperosmotic shock. The Cell Wall elasticity of lactate-grown Cells is increased by a triple mutation that inactivates the Crh family of Cell Wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown Cells reduces Cell Wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the Cell Wall and with the ability of Cells to withstand osmotic shock. IMPORTANCE The C. albicans Cell Wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antiFungal therapy. Its tensile strength is conferred by a network of Cell Wall polysaccharides, which are remodeled in response to growth conditions and environmental stress. However, little is known about how Cell Wall elasticity is regulated and how it affects adaptation to stresses such as sudden changes in osmolarity. We show that elasticity is critical for survival under conditions of osmotic shock, before stress signaling pathways have time to induce gene expression and drive glycerol accumulation. Critical Cell Wall remodeling enzymes control Cell Wallflexibility, and its regulation is strongly de- pendent on host nutritional inputs. We also demonstrate an entirely new level of Cell Wall dynamism, where significant architec- tural changes and structural realignment occur within seconds of an osmotic shock.
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determination of chitin content in Fungal Cell Wall an alternative flow cytometric method
Cytometry Part A, 2013Co-Authors: Sofia Costadeoliveira, Carol A. Munro, Ana Silva, Isabel M Miranda, Alexandre Salvador, Maria Manuela Azevedo, Acacio G Rodrigues, Cidalia PinavazAbstract:The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of Cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast Cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted GlycosylPhosphatidylInositol (GPI)-anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3Δ/chs3Δ and pga31Δ/Δ especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher Cell Wall chitin content. Although no relationship between chitin content and antiFungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate Cell Wall chitin content of fungi.
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chitin and glucan the yin and yang of the Fungal Cell Wall implications for antiFungal drug discovery and therapy
Advances in Applied Microbiology, 2013Co-Authors: Carol A. MunroAbstract:The structural carbohydrate polymers glucan and chitin compliment and reinforce each other in a dynamic process to maintain the integrity and physical strength of the Fungal Cell Wall. The assembly of chitin and glucan in the Cell Wall of the budding yeast Saccharomyces cerevisiae and the polymorphic human pathogen Candida albicans are essential processes that involve a range of Fungal-specific enzymes and regulatory networks. The Fungal Cell Wall is, therefore, an attractive target for novel therapies as host Cells lack many Cell Wall-related proteins. The most recent class of antiFungal drug approved for clinical use, the echinocandins, targets the synthesis of Cell Wall β(1-3)glucan. The echinocandins are effective at treating invasive and bloodstream Candida infections and are now widely used in the clinic. However, there have been sporadic reports of breakthrough infections in patients undergoing echinocandin therapy. The acquisition of point mutations in the FKS genes that encode the catalytic β(1-3)glucan synthase subunits, the target of the echinocandins, has emerged as a dominant resistance mechanism. Cells with elevated chitin levels are also less susceptible to echinocandins and in addition, treatment with sub-MIC echinocandin activates Cell Wall salvage pathways that increase chitin synthesis to compensate for reduced glucan production. The development of drugs targeting the Cell Wall has already proven to be beneficial in providing an alternative class of drug for use in the clinic. Other Cell Wall targets such as chitin synthesis still hold great potential for drug development but careful consideration should be given to the capacity of fungi to manipulate their Walls in a dynamic response to Cell Wall perturbations.
Akira Matsuda - One of the best experts on this subject based on the ideXlab platform.
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Novel nikkomycin analogues: inhibitors of the Fungal Cell Wall biosynthesis enzyme chitin synthase
Bioorganic & Medicinal Chemistry Letters, 2000Co-Authors: Kazuhiko Iwase, Osamu Sugimoto, Hiroyuki Ebisu, Akira MatsudaAbstract:A series of novel nikkomycin analogue inhibitors of the chitin synthase of Fungal Cell Wall was synthesized and evaluated for their inhibitory activities. Among them, the compound having a phenanthrene group at the terminal amino acid was found to possess strong anti-chitin synthase activity.
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synthesis and structure activity relationships of novel nikkomycin analogs inhibitors of the Fungal Cell Wall biosynthesis enzyme chitin synthase
Nucleic acids symposium series, 1999Co-Authors: Junichiro Uda, Kazuhiko Iwase, Osamu Sugimoto, Hiroyuki Ebisu, Kikoh Obi, Akira MatsudaAbstract:A series of novel nikkomycin analogs, which inhibited chitin synthase, the Fungal Cell Wall biosynthesis enzyme, has been synthesized and evaluated their inhibitory activities.
Louise A. Walker - One of the best experts on this subject based on the ideXlab platform.
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the viscoelastic properties of the Fungal Cell Wall allow traffic of ambisome as intact liposome vesicles
Mbio, 2018Co-Authors: Louise A. Walker, Prashant Sood, Megan D Lenardon, Gillian Milne, Jon Olson, Gerard Jensen, Julie M Wolf, Arturo Casadevall, Jill AdlermooreAbstract:ABSTRACT The Fungal Cell Wall is a critically important structure that represents a permeability barrier and protective shield. We probed Candida albicans and Cryptococcus neoformans with liposomes containing amphotericin B (AmBisome), with or without 15-nm colloidal gold particles. The liposomes have a diameter of 60 to 80 nm, and yet their mode of action requires them to penetrate the Fungal Cell Wall to deliver amphotericin B to the Cell membrane, where it binds to ergosterol. Surprisingly, using cryofixation techniques with electron microscopy, we observed that the liposomes remained intact during transit through the Cell Wall of both yeast species, even though the predicted porosity of the Cell Wall (pore size, ~5.8 nm) is theoretically too small to allow these liposomes to pass through intact. C. albicans mutants with altered Cell Wall thickness and composition were similar in both their in vitro AmBisome susceptibility and the ability of liposomes to penetrate the Cell Wall. AmBisome exposed to ergosterol-deficient C. albicans failed to penetrate beyond the mannoprotein-rich outer Cell Wall layer. Melanization of C. neoformans and the absence of amphotericin B in the liposomes were also associated with a significant reduction in liposome penetration. Therefore, AmBisome can reach Cell membranes intact, implying that Fungal Cell Wall viscoelastic properties are permissive to vesicular structures. The fact that AmBisome can transit through chemically diverse Cell Wall matrices when these liposomes are larger than the theoretical Cell Wall porosity suggests that the Wall is capable of rapid remodeling, which may also be the mechanism for release of extraCellular vesicles. IMPORTANCE AmBisome is a broad-spectrum fungicidal antiFungal agent in which the hydrophobic polyene antibiotic amphotericin B is packaged within a 60- to 80-nm liposome. The mode of action involves perturbation of the Fungal Cell membrane by selectively binding to ergosterol, thereby disrupting membrane function. We report that the AmBisome liposome transits through the Cell Walls of both Candida albicans and Cryptococcus neoformans intact, despite the fact that the liposome is larger than the theoretical Cell Wall porosity. This implies that the Cell Wall has deformable, viscoelastic properties that are permissive to transWall vesicular traffic. These observations help explain the low toxicity of AmBisome, which can deliver its payload directly to the Cell membrane without unloading the polyene in the Cell Wall. In addition, these findings suggest that extraCellular vesicles may also be able to pass through the Cell Wall to deliver soluble and membrane-bound effectors and other molecules to the extraCellular space.
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rim pathway mediated alterations in the Fungal Cell Wall influence immune recognition and inflammation
Mbio, 2017Co-Authors: Kyla S Ost, Louise A. Walker, Carol A. Munro, Shannon K. Esher, Chrissy Leopold M Wager, Jeanette Wagener, Floyd L Wormley, Andrew J AlspaughAbstract:Compared to other Fungal pathogens, Cryptococcus neoformans is particularly adept at avoiding detection by innate immune Cells. To explore Fungal Cellular features involved in immune avoidance, we characterized Cell surface changes of the C. neoformans rim101Δ mutant, a strain that fails to organize and shield immunogenic epitopes from host detection. These Cell surface changes are associated with an exaggerated, detrimental inflammatory response in mouse models of infection. We determined that the disorganized strain rim101Δ Cell Wall increases macrophage detection in a contact-dependent manner. Using biochemical and microscopy methods, we demonstrated that the rim101Δ strain shows a modest increase in the levels of both Cell Wall chitin and chitosan but that it shows a more dramatic increase in chito-oligomer exposure, as measured by wheat germ agglutinin staining. We also created a series of mutants with various levels of Cell Wall wheat germ agglutinin staining, and we demonstrated that the staining intensity correlates with the degree of macrophage activation in response to each strain. To explore the host receptors responsible for recognizing the rim101Δ mutant, we determined that both the MyD88 and CARD9 innate immune signaling proteins are involved. Finally, we characterized the immune response to the rim101Δ mutant in vivo, documenting a dramatic and sustained increase in Th1 and Th17 cytokine responses. These results suggest that the Rim101 transcription factor actively regulates the C. neoformans Cell Wall to prevent the exposure of immune stimulatory molecules within the host. These studies further explored the ways in which immune Cells detect C. neoformans and other Fungal pathogens by mechanisms that include sensing N-acetylglucosamine-containing structures, such as chitin and chitosan. IMPORTANCE Infectious microorganisms have developed many ways to avoid recognition by the host immune system. For example, pathogenic fungi alter their Cell surfaces to mask immunogenic epitopes. We have created a Fungal strain with a targeted mutation in a pH response pathway that is unable to properly organize its Cell Wall, resulting in a dramatic immune reaction during infection. This mutant Cell Wall is defective in hiding important Cell Wall components, such as the chito-oligomers chitin and chitosan. By creating a series of Cell Wall mutants, we demonstrated that the degree of chito-oligomer exposure correlates with the intensity of innate immune Cell activation. This activation requires a combination of host receptors to recognize and respond to these infecting microorganisms. Therefore, these experiments explored host-pathogen interactions that determine the degree of the subsequent inflammatory response and the likely outcome of infection.
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Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance
Mbio, 2015Co-Authors: Louise A. Walker, Marion Schiavone, Hélène Martin-yken, Etienne Dague, Carol A. Munro, Alistair J. P. BrownAbstract:The Fungal Cell Wall confers Cell morphology and protection against environmental insults. For Fungal pathogens, the Cell Wall is a key immunological modulator and an ideal therapeutic target. Yeast Cell Walls possess an inner matrix of interlinked -glucan and chitin that is thought to provide tensile strength and rigidity. Yeast Cells remodel their Walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and Cell integrity (Mkc1, Cek1) signal- ing pathways. These mitogen-activated protein kinase (MAPK) pathways modulate Cell Wall gene expression, leading to the con- struction of a new, modified Cell Wall. We show that the Cell Wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans Cells are more resistant to hyperosmotic shock than glucose-grown Cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most Cell death occurs within 10 min of osmotic shock. Sudden decreases in Cell volume drive rapid increases in Cell Wall thickness. The elevated stress resistance of lactate-grown Cells correlates with reduced Cell Wall elasticity, reflected in slower changes in Cell vol- ume following hyperosmotic shock. The Cell Wall elasticity of lactate-grown Cells is increased by a triple mutation that inactivates the Crh family of Cell Wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown Cells reduces Cell Wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the Cell Wall and with the ability of Cells to withstand osmotic shock. IMPORTANCE The C. albicans Cell Wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antiFungal therapy. Its tensile strength is conferred by a network of Cell Wall polysaccharides, which are remodeled in response to growth conditions and environmental stress. However, little is known about how Cell Wall elasticity is regulated and how it affects adaptation to stresses such as sudden changes in osmolarity. We show that elasticity is critical for survival under conditions of osmotic shock, before stress signaling pathways have time to induce gene expression and drive glycerol accumulation. Critical Cell Wall remodeling enzymes control Cell Wallflexibility, and its regulation is strongly de- pendent on host nutritional inputs. We also demonstrate an entirely new level of Cell Wall dynamism, where significant architec- tural changes and structural realignment occur within seconds of an osmotic shock.
Arthur F J Ram - One of the best experts on this subject based on the ideXlab platform.
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a novel screening method for Cell Wall mutants in aspergillus niger identifies udp galactopyranose mutase as an important protein in Fungal Cell Wall biosynthesis
Genetics, 2008Co-Authors: Robbert A Damveld, Frans M Klis, Mark Arentshorst, Cees A M J J Van Den Hondel, Angelique C W Franken, Peter J Punt, Arthur F J RamAbstract:To identify Cell Wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antiFungals, we developed a novel screening method for Cell Wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative α-glucan synthase, is strongly induced in response to Cell Wall stress. By placing the agsA promoter region in front of a selectable marker, the acetamidase (amdS) gene of A. nidulans, we reasoned that Cell Wall mutants with a constitutively active Cell Wall stress response pathway could be identified by selecting mutants for growth on acetamide as the sole nitrogen source. For the genetic screen, a strain was constructed that contained two reporter genes controlled by the same promoter: the metabolic reporter gene PagsA-amdS and PagsA-H2B-GFP, which encodes a GFP-tagged nuclear protein. The primary screen yielded 161 mutants that were subjected to various Cell Wall-related secondary screens. Four calcofluor white-hypersensitive, osmotic-remediable thermosensitive mutants were selected for complementation analysis. Three mutants were complemented by the same gene, which encoded a protein with high sequence identity with eukaryotic UDP-galactopyranose mutases (UgmA). Our results indicate that galactofuranose formation is important for Fungal Cell Wall biosynthesis and represents an attractive target for the development of antiFungals.
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identification of Fungal Cell Wall mutants using susceptibility assays based on calcofluor white and congo red
Nature Protocols, 2006Co-Authors: Arthur F J Ram, Frans M KlisAbstract:The Fungal Cell Wall is an essential organelle and represents a considerable metabolic investment. Its macromolecular composition, molecular organization and thickness can vary greatly depending on environmental conditions. Its construction is also tightly controlled in space and time. Many genes are therefore involved in building the Fungal Cell Wall. Here we present a simple approach for detecting these genes. The method is based on the observation that Cell Wall mutants are generally more sensitive to two related anionic dyes, Calcofluor white (CFW) and Congo red (CR), both of which interfere with the construction and stress response of the Cell Wall. CFW-based and CR-based susceptibility assays identify Cell Wall mutants not only in ascomycetous yeasts (such as Saccharomyces cerevisiae and Candida albicans) but also in mycelial ascomycetes (such as Aspergillus fumigatus and Aspergillus niger), basidiomycetous species (Cryptococcus neoformans) and probably also zygomycetous fungi. The protocol can be completed in 4–6 h (excluding the incubation time required for Fungal growth).
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activity of quinones from teak tectona grandis on Fungal Cell Wall stress
Planta Medica, 2006Co-Authors: Pattarawadee Sumthong, Arthur F J Ram, Robbert A Damveld, Young Hae Choi, Mark Arentshorst, Cees A M J J Van Den Hondel, Robert VerpoorteAbstract:Teak ( Tectona grandis L.f., Verbenaceae) sawdust extract inhibited the growth of Aspergillus niger. Centrifugal partition chromatography was used to isolate the active compounds. By (1)H-NMR the active compounds were identified as deoxylapachol and tectoquinone. Two A. niger transgenic strains which show induction of 1,3 -alpha-D-glucan synthase were used as a Cell Wall damage model. The result showed that deoxylapachol from T. grandis extract induced Fungal Cell Wall stress.
