A molecular vision of fungal cell wall organization by functional genomics and solid-state NMR

AbstractVast efforts have been devoted to the development of antifungal drugs targeting the cell wall, but the supramolecular architecture of this carbohydrate-rich composite remains insufficiently understood. Here we compare the cell wall structure of a fungal pathogen Aspergillus fumigatus and four mutants depleted of major structural polysaccharides. High-resolution solid-state NMR spectroscopy of intact cells reveals a rigid core formed by chitin, β-1,3-glucan, and α-1,3-glucan, with galactosaminogalactan and galactomannan present in the mobile phase. Gene deletion reshuffles the composition and spatial organization of polysaccharides, with significant changes in their dynamics and water accessibility. The distribution of α-1,3-glucan in chemically isolated and dynamically distinct domains supports its functional diversity. Identification of valines in the alkali-insoluble carbohydrate core suggests a putative function in stabilizing macromolecular complexes. We propose a revised model of cell wall architecture which will improve our understanding of the structural response of fungal pathogens to stresses.

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Up against the Wall: Is Yeast Cell Wall Integrity Ensured by Mechanosensing in Plasma Membrane Microdomains?

Applied and Environmental Microbiology ◽

10.1128/aem.03273-14 ◽

2014 ◽

Vol 81 (3) ◽

pp. 806-811 ◽

Cited By ~ 42

Author(s):

Christian Kock ◽

Yves F. Dufrêne ◽

Jürgen J. Heinisch

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Yeast Cell ◽

Antifungal Drugs ◽

Cell Wall Integrity ◽

Yeast Cell Wall ◽

Membrane Microdomains ◽

Wall Structure ◽

Fungal Cell ◽

Membrane Spanning

ABSTRACTYeast cell wall integrity (CWI) signaling serves as a model of the regulation of fungal cell wall synthesis and provides the basis for the development of antifungal drugs. A set of five membrane-spanning sensors (Wsc1 to Wsc3, Mid2, and Mtl1) detect cell surface stress and commence the signaling pathway upon perturbations of either the cell wall structure or the plasma membrane. We here summarize the latest advances in the structure/function relationship primarily of the Wsc1 sensor and critically review the evidence that it acts as a mechanosensor. The relevance and physiological significance of the information obtained for the function of the other CWI sensors, as well as expected future developments, are discussed.

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The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity

Microbiology ◽

10.1099/mic.0.028902-0 ◽

2009 ◽

Vol 155 (6) ◽

pp. 2004-2020 ◽

Cited By ~ 42

Author(s):

Emilia Moreno-Ruiz ◽

Giuseppe Ortu ◽

Piet W. J. de Groot ◽

Fabien Cottier ◽

Céline Loussert ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Fungal Pathogens ◽

High Sensitivity ◽

Wall Structure ◽

Minor Role ◽

Cell Wall Proteins ◽

Calcofluor White ◽

Fungal Cell ◽

A Minor

The fungal cell wall is essential in maintaining cellular integrity and plays key roles in the interplay between fungal pathogens and their hosts. The PGA59 and PGA62 genes encode two short and related glycosylphosphatidylinositol-anchored cell wall proteins and their expression has been previously shown to be strongly upregulated when the human pathogen Candida albicans grows as biofilms. Using GFP fusion proteins, we have shown that Pga59 and Pga62 are cell-wall-located, N- and O-glycosylated proteins. The characterization of C. albicans pga59Δ/pga59Δ, pga62Δ/pga62Δ and pga59Δ/pga59Δ pga62Δ/pga62Δ mutants suggested a minor role of these two proteins in hyphal morphogenesis and that they are not critical to biofilm formation. Importantly, the sensitivity to different cell-wall-perturbing agents was altered in these mutants. In particular, simultaneous inactivation of PGA59 and PGA62 resulted in high sensitivity to Calcofluor white, Congo red and nikkomicin Z and in resistance to caspofungin. Furthermore, cell wall composition and observation by transmission electron microscopy indicated an altered cell wall structure in the mutant strains. Collectively, these data suggest that the cell wall proteins Pga59 and Pga62 contribute to cell wall stability and structure.

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Susceptibility Pattern of Caspofungin-Coated Gold Nanoparticles against Clinically Important Candida Species

Advanced Pharmaceutical Bulletin ◽

10.34172/apb.2021.078 ◽

2020 ◽

Author(s):

Zahra Salehi ◽

Azam Fattahi ◽

Ensieh lotfali ◽

Abdolhassan Kazemi ◽

Ali Shakeri-Zadeh ◽

...

Keyword(s):

Electron Microscopy ◽

Gold Nanoparticles ◽

Cell Wall ◽

Candida Species ◽

Spherical Shape ◽

Sem Analysis ◽

Antifungal Drugs ◽

Wall Structure ◽

Candida Spp ◽

Average Size

Purpose: The present study was performed to examine whether caspofungin-coated gold nanoparticles (CAS-AuNPs) may offer the right platform for sensitivity induction in resistant isolates. Methods: For the purpose of the study, a total of 58 archived Candida species were enrolled in the research. The identification of Candida spp. was performed using polymerase chain reaction-restriction fragment length polymorphism and HWP1 gene amplification approaches. The conjugated CAS-AuNPs were synthesized and then characterized using transmission electron microscopy (TEM) and Zetasizer system to determine their morphology, size, and charge. Furthermore, the efficacy of CAS, CAS-AuNPs conjugate, and AuNPs against Candida spp. was assessed based on the Clinical and Laboratory Standards Institute M60. Finally, the interaction of CAS-AuNPs with Candida element was evaluated via scanning electron microscopy (SEM). Results: According to the TEM results, the synthesized CAS-AuNPs had a spherical shape with an average size of 20 nm. The Zeta potential of CAS-AuNPs was -38.2 mV. Statistical analyses showed that CAS-AuNPs could significantly reduce the minimum inhibitory concentration against C. albicans (P=0.0005) and non-albicans Candida (NAC) species (P<0.0001). All isolates had a MIC value of ≥ 4 µg/ml for CAS, except for C. glabrata. The results of SEM analysis confirmed the effects of AuNPs on the membrane and cell wall structure of C. globrata exposed to CAS-AuNPs, facilitating the formation of pores on the cell wall and finally cell death. Conclusion: The findings revealed that CAS-AuNPs conjugates had significant antifungal effects against Candida spp. through the degradation of the membrane and cell wall integrity. Therefore, it can be concluded that the encapsulation of antifungal drugs in combination with NPs not only diminishes side effects but also enhances the effectiveness of the medications.

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Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis

Open Biology ◽

10.1098/rsob.130022 ◽

2013 ◽

Vol 3 (9) ◽

pp. 130022 ◽

Cited By ~ 16

Author(s):

Alexander Striebeck ◽

David A. Robinson ◽

Alexander W. Schüttelkopf ◽

Daan M. F. van Aalten

Keyword(s):

Cell Wall ◽

Fungal Pathogens ◽

Enzyme Assay ◽

Antibiotic Sensitivity ◽

Site Directed Mutagenesis ◽

Fungal Cell ◽

Pol I ◽

Conserved Amino Acids ◽

Allosteric Activator ◽

Mannan Synthesis

The fungal cell possesses an essential carbohydrate cell wall. The outer layer, mannan, is formed by mannoproteins carrying highly mannosylated O - and N -linked glycans. Yeast mannan biosynthesis is initiated by a Golgi-located complex (M-Pol I) of two GT-62 mannosyltransferases, Mnn9p and Van1p, that are conserved in fungal pathogens. Saccharomyces cerevisiae and Candida albicans mnn9 knockouts show an aberrant cell wall and increased antibiotic sensitivity, suggesting the enzyme is a potential drug target. Here, we present the structure of Sc Mnn9 in complex with GDP and Mn 2+ , defining the fold and catalytic machinery of the GT-62 family. Compared with distantly related GT-78/GT-15 enzymes, Sc Mnn9 carries an unusual extension. Using a novel enzyme assay and site-directed mutagenesis, we identify conserved amino acids essential for Sc Mnn9 ‘priming’ α-1,6-mannosyltransferase activity. Strikingly, both the presence of the Sc Mnn9 protein and its product, but not Sc Mnn9 catalytic activity, are required to activate subsequent Sc Van1 processive α-1,6-mannosyltransferase activity in the M-Pol I complex. These results reveal the molecular basis of mannan synthesis and will aid development of inhibitors targeting this process.

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Targeting a critical step in fungal hexosamine biosynthesis

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012985 ◽

2020 ◽

Vol 295 (26) ◽

pp. 8678-8691 ◽

Cited By ~ 1

Author(s):

Deborah E. A. Lockhart ◽

Mathew Stanley ◽

Olawale G. Raimi ◽

David A. Robinson ◽

Dominika Boldovjakova ◽

...

Keyword(s):

Cell Wall ◽

Antifungal Drugs ◽

Fungal Cell Wall ◽

Fungal Cell ◽

Hexosamine Biosynthetic Pathway ◽

Hexosamine Biosynthesis ◽

Key Enzyme ◽

Opportunistic Fungal Pathogen ◽

Extracellular Environment ◽

Chemical Evidence

Aspergillus fumigatus is a human opportunistic fungal pathogen whose cell wall protects it from the extracellular environment including host defenses. Chitin, an essential component of the fungal cell wall, is synthesized from UDP-GlcNAc produced in the hexosamine biosynthetic pathway. As this pathway is critical for fungal cell wall integrity, the hexosamine biosynthesis enzymes represent potential targets of antifungal drugs. Here, we provide genetic and chemical evidence that glucosamine 6-phosphate N-acetyltransferase (Gna1), a key enzyme in this pathway, is an exploitable antifungal drug target. GNA1 deletion resulted in loss of fungal viability and disruption of the cell wall, phenotypes that could be rescued by exogenous GlcNAc, the product of the Gna1 enzyme. In a murine model of aspergillosis, the Δgna1 mutant strain exhibited attenuated virulence. Using a fragment-based approach, we discovered a small heterocyclic scaffold that binds proximal to the Gna1 active site and can be optimized to a selective submicromolar binder. Taken together, we have provided genetic, structural, and chemical evidence that Gna1 is an antifungal target in A. fumigatus.

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Dissecting the Bacterial Cell Wall with Solid-State NMR

Biophysical Journal ◽

10.1016/j.bpj.2017.11.885 ◽

2018 ◽

Vol 114 (3) ◽

pp. 158a

Author(s):

Joseph A. Romaniuk ◽

Lynette Cegelski

Keyword(s):

Cell Wall ◽

Solid State ◽

Bacterial Cell ◽

Solid State Nmr ◽

Bacterial Cell Wall

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Novel tool to quantify cell wall porosity relates wall structure to cell growth and drug uptake

The Journal of Cell Biology ◽

10.1083/jcb.201810121 ◽

2019 ◽

Vol 218 (4) ◽

pp. 1408-1421 ◽

Cited By ~ 5

Author(s):

Xiaohui Liu ◽

Jiazhou Li ◽

Heyu Zhao ◽

Boyang Liu ◽

Thomas Günther-Pomorski ◽

...

Keyword(s):

Cell Wall ◽

Cell Walls ◽

Dynamic Structure ◽

Cell Types ◽

Antifungal Drugs ◽

Drug Uptake ◽

Wall Structure ◽

Quenching Efficiency ◽

Model Plant Arabidopsis Thaliana ◽

Cell Wall Porosity

Even though cell walls have essential functions for bacteria, fungi, and plants, tools to investigate their dynamic structure in living cells have been missing. Here, it is shown that changes in the intensity of the plasma membrane dye FM4-64 in response to extracellular quenchers depend on the nano-scale porosity of cell walls. The correlation of quenching efficiency and cell wall porosity is supported by tests on various cell types, application of differently sized quenchers, and comparison of results with confocal, electron, and atomic force microscopy images. The quenching assay was used to investigate how changes in cell wall porosity affect the capability for extension growth in the model plant Arabidopsis thaliana. Results suggest that increased porosity is not a precondition but a result of cell extension, thereby providing new insight on the mechanism plant organ growth. Furthermore, it was shown that higher cell wall porosity can facilitate the action of antifungal drugs in Saccharomyces cerevisiae, presumably by facilitating uptake.

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Evaluation of Antifungal Activity and Mode of Action of New Coumarin Derivative, 7-Hydroxy-6-nitro-2H-1-benzopyran-2-one, againstAspergillusspp.

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2015/925096 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 11

Author(s):

Felipe Queiroga Sarmento Guerra ◽

Rodrigo Santos Aquino de Araújo ◽

Janiere Pereira de Sousa ◽

Fillipe de Oliveira Pereira ◽

Francisco J. B. Mendonça-Junior ◽

...

Keyword(s):

Cell Wall ◽

Mode Of Action ◽

Cell Walls ◽

Coumarin Derivative ◽

Antifungal Drugs ◽

Azole Resistance ◽

Subinhibitory Concentration ◽

Fungal Cell ◽

Mycelia Growth

Aspergillusspp. produce a wide variety of diseases. For the treatment of such infections, the azoles and Amphotericin B are used in various formulations. The treatment of fungal diseases is often ineffective, because of increases in azole resistance and their several associated adverse effects. To overcome these problems, natural products and their derivatives are interesting alternatives. The aim of this study was to examine the effects of coumarin derivative, 7-hydroxy-6-nitro-2H-1-benzopyran-2-one (Cou-NO2), both alone and with antifungal drugs. Its mode of action againstAspergillusspp. Cou-NO2was tested to evaluate its effects on mycelia growth and germination of fungal conidia ofAspergillusspp. We also investigated possible Cou-NO2action on cell walls (0.8 M sorbitol) and on Cou-NO2to ergosterol binding in the cell membrane. The study shows that Cou-NO2is capable of inhibiting both the mycelia growth and germination of conidia for the species tested, and that its action affects the structure of the fungal cell wall. At subinhibitory concentration, Cou-NO2enhanced thein vitroeffects of azoles. Moreover, in combination with azoles (voriconazole and itraconazole) Cou-NO2displays an additive effect. Thus, our study supports the use of coumarin derivative 7-hydroxy-6-nitro-2H-1-benzopyran-2-one as an antifungal agent againstAspergillusspecies.

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