scholarly journals The Dynamic Influence of Olorofim (F901318) on the Cell Morphology and Organization of Living Cells of Aspergillus fumigatus

2020 ◽  
Vol 6 (2) ◽  
pp. 47
Author(s):  
Saskia du Pré ◽  
Mike Birch ◽  
Derek Law ◽  
Nicola Beckmann ◽  
Graham E. M. Sibley ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
De Novo ◽  
Dynamic Structure ◽  
Building Blocks ◽  
Uridine Diphosphate ◽  
Dna And Rna ◽  
Diphosphate Glucose ◽  
Cycle Regulation ◽  
Mitotracker Red

The first characterized antifungal in the orotomide class is olorofim. It targets the de novo pyrimidine biosynthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH). The pyrimidines uracil, thymine and cytosine are the building blocks of DNA and RNA; thus, inhibition of their synthesis is likely to have multiple effects, including affecting cell cycle regulation and protein synthesis. Additionally, uridine-5′-triphosphate (UTP) is required for the formation of uridine-diphosphate glucose (UDP-glucose), which is an important precursor for several cell wall components. In this study, the dynamic effects of olorofim treatment on the morphology and organization of Aspergillus fumigatus hyphae were analyzed microscopically using confocal live-cell imaging. Treatment with olorofim led to increased chitin content in the cell wall, increased septation, enlargement of vacuoles and inhibition of mitosis. Furthermore, vesicle-like structures, which could not be stained or visualized with a range of membrane- or vacuole-selective dyes, were found in treated hyphae. A colocalization study of DHODH and MitoTracker Red FM confirmed for the first time that A. fumigatus DHODH is localized in the mitochondria. Overall, olorofim treatment was found to significantly influence the dynamic structure and organization of A. fumigatus hyphae.

THE BIOSYNTHESIS OF CELL WALL CARBOHYDRATES: III. FURTHER STUDIES ON FORMATION OF CELLULOSE AND XYLAN FROM LABELED MONOSACCHARIDES IN WHEAT PLANTS

1956 ◽  
Vol 34 (1) ◽  
pp. 405-413 ◽  
Author(s):  
H. A. Altermatt ◽  
A. C. Neish
Keyword(s):  
Cell Wall ◽  
Leuconostoc Mesenteroides ◽  
Carbon Skeleton ◽  
The Other ◽  
Uridine Diphosphate ◽  
Uridine Diphosphate Glucose ◽  
Polysaccharide Synthesis ◽  
Diphosphate Glucose ◽  
Cell Wall Carbohydrates

D-Glucose-1-C14, D-glucose-2-C14, D-xylose-2-C14, D-xylose-5-C14, D-arabinose-1-C14, D-glucuronolactone-1-C14, D-glucitol-1-C14, D-mannitol-1-C14, D-arabitol-1-C14, and D-arabitol-5-C14 were administered to wheat plants. The cellulose and xylan were isolated after a period of metabolism varying from 2 to 23 hr. D-Mannitol and D-arabitol were not converted to either cellulose or xylan while D-arabinose was utilized slightly. The other compounds gave rise to both labelled cellulose and xylan. The glucose and xylose, obtained from the cellulose and xylan respectively, were degraded by fermentation with Leuconostoc mesenteroides. Glucose and glucuronolactone were equally good precursors of xylan and were superior to the other compounds tried. They appeared to give rise to units for xylan formation by loss of carbon-6. Free xylose was converted to xylan units only after an extensive rearrangement of the carbon skeleton, such as occurred in the conversion of xylose to cellulose units. A hypothetical outline of polysaccharide synthesis, involving uridine diphosphate glucose as the central intermediate, is suggested to explain the data.

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 Identification of Leishmania major UDP-Sugar Pyrophosphorylase Inhibitors Using Biosensor-Based Small Molecule Fragment Library Screening

Molecules ◽  
2019 ◽  
Vol 24 (5) ◽  
pp. 996 ◽  
Author(s):  
Ohm Prakash ◽  
Jana Führing ◽  
John Post ◽  
Sharon Shepherd ◽  
Thomas Eadsforth ◽  
...  
Keyword(s):  
Leishmania Major ◽  
De Novo ◽  
Protozoan Parasite ◽  
Library Screening ◽  
Uridine Diphosphate ◽  
Fragment Library ◽  
Antileishmanial Drug ◽  
Small Set ◽  
Diphosphate Glucose ◽  
Nucleotide Sugars

Leishmaniasis is a neglected disease that is caused by different species of the protozoan parasite Leishmania, and it currently affects 12 million people worldwide. The antileishmanial therapeutic arsenal remains very limited in number and efficacy, and there is no vaccine for this parasitic disease. One pathway that has been genetically validated as an antileishmanial drug target is the biosynthesis of uridine diphosphate-glucose (UDP-Glc), and its direct derivative UDP-galactose (UDP-Gal). De novo biosynthesis of these two nucleotide sugars is controlled by the specific UDP-glucose pyrophosphorylase (UGP). Leishmania parasites additionally express a UDP-sugar pyrophosphorylase (USP) responsible for monosaccharides salvage that is able to generate both UDP-Gal and UDP-Glc. The inactivation of the two parasite pyrophosphorylases UGP and USP, results in parasite death. The present study reports on the identification of structurally diverse scaffolds for the development of USP inhibitors by fragment library screening. Based on this screening, we selected a small set of commercially available compounds, and identified molecules that inhibit both Leishmania major USP and UGP, with a half-maximal inhibitory concentration in the 100 µM range. The inhibitors were predicted to bind at allosteric regulation sites, which were validated by mutagenesis studies. This study sets the stage for the development of potent USP inhibitors.

THE BIOSYNTHESIS OF CELL WALL CARBOHYDRATES: III. FURTHER STUDIES ON FORMATION OF CELLULOSE AND XYLAN FROM LABELED MONOSACCHARIDES IN WHEAT PLANTS

1956 ◽  
Vol 34 (3) ◽  
pp. 405-413 ◽  
Author(s):  
H. A. Altermatt ◽  
A. C. Neish
Keyword(s):  
Cell Wall ◽  
Leuconostoc Mesenteroides ◽  
Carbon Skeleton ◽  
The Other ◽  
Uridine Diphosphate ◽  
Uridine Diphosphate Glucose ◽  
Polysaccharide Synthesis ◽  
Diphosphate Glucose ◽  
Cell Wall Carbohydrates

D-Glucose-1-C14, D-glucose-2-C14, D-xylose-2-C14, D-xylose-5-C14, D-arabinose-1-C14, D-glucuronolactone-1-C14, D-glucitol-1-C14, D-mannitol-1-C14, D-arabitol-1-C14, and D-arabitol-5-C14 were administered to wheat plants. The cellulose and xylan were isolated after a period of metabolism varying from 2 to 23 hr. D-Mannitol and D-arabitol were not converted to either cellulose or xylan while D-arabinose was utilized slightly. The other compounds gave rise to both labelled cellulose and xylan. The glucose and xylose, obtained from the cellulose and xylan respectively, were degraded by fermentation with Leuconostoc mesenteroides. Glucose and glucuronolactone were equally good precursors of xylan and were superior to the other compounds tried. They appeared to give rise to units for xylan formation by loss of carbon-6. Free xylose was converted to xylan units only after an extensive rearrangement of the carbon skeleton, such as occurred in the conversion of xylose to cellulose units. A hypothetical outline of polysaccharide synthesis, involving uridine diphosphate glucose as the central intermediate, is suggested to explain the data.

scholarly journals Stable isotope informed genome-resolved metagenomics reveals that Saccharibacteria utilize microbially processed plant derived carbon

2017 ◽  
Author(s):  
Evan P. Starr ◽  
Shengjing Shi ◽  
Steven J. Blazewicz ◽  
Alexander J. Probst ◽  
Donald J. Herman ◽  
...  
Keyword(s):  
Rhizosphere Soil ◽  
De Novo ◽  
Plant Root ◽  
Gradient Centrifugation ◽  
Isotopic Labeling ◽  
Building Blocks ◽  
Carbon Utilization ◽  
Nucleotide Synthesis ◽  
Dna And Rna ◽  
Dna And Rna Synthesis

AbstractBackgroundThe transformation of plant photosynthate into soil organic carbon and its recycling to CO2 by soil microorganisms is one of the central components of the terrestrial carbon cycle. There are currently large knowledge gaps related to which soil-associated microorganisms take up plant carbon in the rhizosphere and the fate of that carbon.ResultsWe conducted an experiment in which common wild oats (Avena fatua) were grown in a 13CO2 atmosphere and the rhizosphere and non-rhizosphere soil was sampled for genomic analyses. Density gradient centrifugation of DNA extracted from soil samples enabled distinction of microbes that did and did not incorporate the 13C into their DNA. A 1.45 Mbp genome of a Saccharibacteria (TM7) was identified and, despite the microbial complexity of rhizosphere soil, curated to completion. The genome lacks many biosynthetic pathways, including genes required to synthesize DNA de novo. Rather, it requires externally-derived nucleotides for DNA and RNA synthesis. Given this, we conclude that rhizosphere-associated Saccharibacteria recycle DNA from bacteria that live off plant exudates and/or phage that acquired 13C because they preyed upon these bacteria and/or directly from the labelled plant DNA. Isotopic labeling indicates that the population was replicating during the six-week period of plant growth. Interestingly, the genome is ~30% larger than other complete Saccharibacteria genomes from non-soil environments, largely due to more genes for complex carbon utilization and amino acid metabolism. Given the ability to degrade cellulose, hemicellulose, pectin, starch and 1,3-β-glucan, we predict that this Saccharibacteria generates energy by fermentation of soil necromass and plant root exudates to acetate or lactate. The genome encodes a linear electron transport chain featuring a terminal oxidase, suggesting that this Saccharibacteria may respire aerobically. The genome encodes a hydrolase that could breakdown salicylic acid, a plant defense signaling molecule, and genes to make a variety of isoprenoids, including the plant hormone zeatin.ConclusionsRhizosphere Saccharibacteria likely depend on other bacteria for basic cellular building blocks. We propose that isotopically labeled CO2 is incorporated into plant-derived carbon and then into the DNA of rhizosphere organisms capable of nucleotide synthesis, and the nucleotides are recycled into Saccharibacterial genomes.

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
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