scholarly journals Genetic and structural validation of Aspergillus fumigatus N-acetylphosphoglucosamine mutase as an antifungal target

2013 ◽  
Vol 33 (5) ◽  
Author(s):  
Wenxia Fang ◽  
Ting Du ◽  
Olawale G. Raimi ◽  
Ramón Hurtado-Guerrero ◽  
Karina Mariño ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
High Throughput Screening ◽  
Uridine Diphosphate ◽  
Chitin Synthesis ◽  
Wall Ultrastructure ◽  
Key Enzyme ◽  
A Cell ◽  
Altered Cell ◽  
Structural Validation

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.

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

2021 ◽  
Author(s):  
Yuanwei Zhang ◽  
Wenxia Fang ◽  
Olawale G. Raimi ◽  
Deborah E. A. Lockhart ◽  
Andrew T. Ferenbach ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
A Cell ◽  
Structural Validation

scholarly journals Targeting a critical step in fungal hexosamine biosynthesis

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.

scholarly journals Characterization of extracellular vesicles produced by Aspergillus fumigatus protoplasts

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.

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

2002 ◽  
Vol 40 (5) ◽  
pp. 455-464 ◽  
Author(s):  
I. Mouyna ◽  
J. Sarfati ◽  
P. Recco ◽  
T. Fontaine ◽  
B. Henrissat ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Molecular Characterization ◽  
A Cell

A highly selective fluorescent probe for real-time imaging of bacterial NAT2 and high-throughput screening of natural inhibitors for tuberculosis therapy

2019 ◽  
Vol 3 (1) ◽  
pp. 145-150 ◽  
Author(s):  
Yinzhu Jin ◽  
Zhenhao Tian ◽  
Xiangge Tian ◽  
Lei Feng ◽  
Jingnan Cui ◽  
...  
Keyword(s):  
Cell Wall ◽  
Real Time ◽  
Fluorescent Probe ◽  
High Throughput ◽  
High Throughput Screening ◽  
Molecular Target ◽  
Cell Wall Synthesis ◽  
Key Enzyme ◽  
Tuberculosis Therapy ◽  
Natural Inhibitors

Fluorescent probeARHBis developed for detecting in various bacteria the activity ofN-acetyltransferase 2 (NAT2), a key enzyme in cell wall synthesis and widely considered to be a molecular target for anti-mycobacterial therapy.

scholarly journals Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies

2015 ◽  
Vol 59 (10) ◽  
pp. 5932-5941 ◽  
Author(s):  
Louise A. Walker ◽  
Keunsook K. Lee ◽  
Carol A. Munro ◽  
Neil A. R. Gow
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Dynamic Imaging ◽  
Chitin Synthase ◽  
Response To Treatment ◽  
Chitin Synthesis ◽  
Calcofluor White ◽  
Content Type ◽  
Chitin Content

ABSTRACTTreatment ofAspergillus fumigatuswith echinocandins such as caspofungin inhibits the synthesis of cell wall β-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca2+-calcineurin signaling pathways.A. fumigatusmutants with thechsgene (encoding chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ΔAfchsGmutant was hypersensitive to caspofungin, and all other ΔAfchsmutants tested remained capable of increasing their chitin content in response to treatment with CaCl2and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ΔAfchsmutants tested, with the exception of the ΔAfchsGmutant, which remained sensitive to caspofungin.In vitroexposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was againAfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. Thesein vitrodata demonstrate thatA. fumigatushas the potential to survive echinocandin treatmentin vivobyAfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections.

scholarly journals N-Myristoyltransferase Is a Cell Wall Target in Aspergillus fumigatus

2015 ◽  
Vol 10 (6) ◽  
pp. 1425-1434 ◽  
Author(s):  
Wenxia Fang ◽  
David A. Robinson ◽  
Olawale G. Raimi ◽  
David E. Blair ◽  
Justin R. Harrison ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
A Cell

scholarly journals CELL WALL COMPOSITION AND VIRULENCE IN ESCHERICHIA COLI

1968 ◽  
Vol 128 (3) ◽  
pp. 399-414 ◽  
Author(s):  
Donald N. Medearis ◽  
Bruce M. Camitta ◽  
Edward C. Heath
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Uridine Diphosphate ◽  
E Coli ◽  
Endotoxin Activity ◽  
A Cell ◽  
The Difference ◽  
Definition Of

Uridine diphosphate galactose 4-epimerase and phosphomannose isomerase-deficient mutants of Escherichia coli O111:B4 were studied to test the hypothesis that in E. coli a specific relationship exists between O antigenicity, virulence, and capacity to resist phagocytosis. The first mutant, designated J-5, produces a cell wall lipopolysaccharide, the side chains of which do not contain galactose, glucose, N-acetylglucosamine, or colitose. The second mutant produces a cell wall lipopolysaccharide which lacks only colitose. The capacity of these various organisms to kill mice was strikingly different. E. coli O111 was 1000 times as virulent as J-5, and 100 times as virulent as L-2. The capacity of the organisms to kill mice was correlated with their ability to resist phagocytosis and to persist in the peritoneal cavity. The parent strain of O111 resisted phagocytosis by macrophages in vivo and polymorphonuclear leukocytes in vitro. The mutants did not, and the organism most deficient in the saccharide component of its LPS was most susceptible to phagocytosis and least virulent. These results were corroborated by growing the mutants in appropriately supplemented media which permitted the synthesis of complete LPS, reversed the susceptibility to phagocytosis, and restored virulence. Finally, serological reactivity was consistent with previous observations which had demonstrated that the O antigenicity of E. coli is determined by the saccharide composition of its cell wall lipopolysaccharide. Despite the difference in the capacity of the various log-phase organisms to kill mice when injected intraperitoneally, purified lipopolysaccharides extracted from them did not differ significantly in their capacity to kill or produce fever. Thus virulence was shown to be independent of endotoxin activity which in turn seemed to be unrelated to the saccharide composition of the cell wall LPS. Collectively, these data provide at least a partial molecular definition of virulence in E. coli by demonstrating that the presence or absence of specific sugars in its cell wall lipopolysaccharide is a determinant of its antiphagocytic capacity and its virulence.

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