Genetic and structural validation of phosphomannomutase as a cell wall target in Aspergillus fumigatus

Aspergillus fumigatus is the causative agent of IA (invasive aspergillosis) in immunocompromised patients. It possesses a cell wall composed of chitin, glucan and galactomannan, polymeric carbohydrates synthesized by processive glycosyltransferases from intracellular sugar nucleotide donors. Here we demonstrate that A. fumigatus possesses an active AfAGM1 (A. fumigatus N-acetylphosphoglucosamine mutase), a key enzyme in the biosynthesis of UDP (uridine diphosphate)–GlcNAc (N-acetylglucosamine), the nucleotide sugar donor for chitin synthesis. A conditional agm1 mutant revealed the gene to be essential. Reduced expression of agm1 resulted in retarded cell growth and altered cell wall ultrastructure and composition. The crystal structure of AfAGM1 revealed an amino acid change in the active site compared with the human enzyme, which could be exploitable in the design of selective inhibitors. AfAGM1 inhibitors were discovered by high-throughput screening, inhibiting the enzyme with IC50s in the low μM range. Together, these data provide a platform for the future development of AfAGM1 inhibitors with antifungal activity.

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Characterization of extracellular vesicles produced by Aspergillus fumigatus protoplasts

10.1101/2020.05.21.109926 ◽

2020 ◽

Author(s):

Juliana Rizzo ◽

Thibault Chaze ◽

Kildare Miranda ◽

Robert W. Roberson ◽

Olivier Gorgette ◽

...

Keyword(s):

Electron Microscopy ◽

Cell Wall ◽

Aspergillus Fumigatus ◽

Extracellular Vesicles ◽

Fungal Pathogen ◽

Fungal Pathogens ◽

Sugar Metabolism ◽

Biologically Active ◽

Thick Cell Wall ◽

A Cell

AbstractExtracellular vesicles (EVs) are outer membranous compartments produced by yeast and mycelial forms of several fungal species. One of the difficulties to perceive the role of EVs during the fungal life is the fact that an active secretion of these EVs has not been clearly demonstrated in situ due to the presence of a thick cell wall. One alternative to have a better access to these vesicles is to use protoplasts. This approach has been investigated here with Aspergillus fumigatus, one of the most common opportunistic fungal pathogens worldwide. Analysis of regenerating protoplasts by scanning electron microscopy and fluorescence microscopy indicated the occurrence of outer membrane projections in association with surface components and the release of particles with properties resembling those of fungal EVs. EVs in culture supernatants were characterized by transmission electron microscopy and nanoparticle tracking analysis. Proteomic and glycome analysis of EVs revealed the presence of a complex array of enzymes related to lipid / sugar metabolism, pathogenic processes, and cell wall biosynthesis. Our data indicate that i) EV production is a common feature of different morphological stages of this major fungal pathogen, and ii) protoplastic EVs are a promising tool to undertake studies of vesicle functions in fungal cells.IMPORTANCEFungal cells use extracellular vesicles (EVs) to export biologically active molecules to the outer space. Since fungal cells are encaged in a thick cell wall, it is reasonable to expect that this structure might impact the vesicle-mediated molecular export. In this study, we used protoplasts of Aspergillus fumigatus, a major fungal pathogen, as a model to evaluate EV production in the absence of a cell wall. Our results demonstrated that wall-less A. fumigatus exports plasma membrane-derived EVs containing a complex combination of proteins and glycans. Our study is the first to characterize fungal EVs in the absence of a cell wall. Our results suggest that protoplasts are a promising model for functional studies of fungal vesicles.

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Faculty Opinions recommendation of Molecular characterization of a cell wall-associated beta(1-3)endoglucanase of Aspergillus fumigatus.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1010881.160407 ◽

2002 ◽

Author(s):

Sybren de Hoog

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

Molecular Characterization ◽

A Cell

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Molecular characterization of a cell wall-associated ß(1-3)endoglucanase of Aspergillus fumigatus

Medical Mycology ◽

10.1080/714031140 ◽

2002 ◽

Vol 40 (5) ◽

pp. 455-464 ◽

Cited By ~ 1

Author(s):

I. Mouyna ◽

J. Sarfati ◽

P. Recco ◽

T. Fontaine ◽

B. Henrissat ◽

...

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

Molecular Characterization ◽

A Cell

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Targeting a critical step in fungal hexosamine biosynthesis

10.1101/2020.01.07.896944 ◽

2020 ◽

Author(s):

Deborah E.A. Lockhart ◽

Mathew Stanley ◽

Olawale G. Raimi ◽

David A. Robinson ◽

Dominika Boldovjakova ◽

...

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

Drug Target ◽

Hexosamine Biosynthetic Pathway ◽

Hexosamine Biosynthesis ◽

Key Enzyme ◽

A Cell ◽

Opportunistic Fungal Pathogen ◽

Extracellular Environment ◽

Chemical Evidence

SummaryAspergillus fumigatus is a human opportunistic fungal pathogen with a cell wall that protects it from the extracellular environment. Chitin, an essential cell wall component, is synthesised from UDP-GlcNAc that is produced by the hexosamine biosynthetic pathway. 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. Deletion of GNA1 results in loss of viability and disruption of the cell wall, phenotypes that can be rescued by the product of the enzyme. In a murine model of aspergillosis, the Δgna1 mutant strain attenuates virulence. Using a fragment-based approach, we discovered a small heterocyclic scaffold that binds proximal to the active site and can be optimised to a selective sub-micromolar binder. Taken together, we have provided genetic, structural and chemical evidence for Gna1 as an antifungal target in Aspergillus fumigatus.

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N-Myristoyltransferase Is a Cell Wall Target in Aspergillus fumigatus

ACS Chemical Biology ◽

10.1021/cb5008647 ◽

2015 ◽

Vol 10 (6) ◽

pp. 1425-1434 ◽

Cited By ~ 22

Author(s):

Wenxia Fang ◽

David A. Robinson ◽

Olawale G. Raimi ◽

David E. Blair ◽

Justin R. Harrison ◽

...

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

A Cell

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α1,3 Glucans Are Dispensable in Aspergillus fumigatus

Eukaryotic Cell ◽

10.1128/ec.05270-11 ◽

2011 ◽

Vol 11 (1) ◽

pp. 26-29 ◽

Cited By ~ 58

Author(s):

Christine Henry ◽

Jean-Paul Latgé ◽

Anne Beauvais

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

Vegetative Growth ◽

Conidial Germination ◽

Glucan Synthase ◽

Content Type ◽

Chitin Content ◽

A Cell

ABSTRACTA triple α1,3 glucan synthase mutant ofAspergillus fumigatusobtained by successive deletions of the three α1,3 glucan synthase genes (AGS1,AGS2, andAGS3) has a cell wall devoid of α1,3 glucans. The lack of α1,3 glucans affects neither conidial germination nor mycelial vegetative growth and is compensated by an increase in β1,3 glucan and/or chitin content.

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