Characterization of N-Acetylglucosamine Biosynthesis in Pneumocystis species. A New Potential Target for Therapy

Autor: Deanne Hebrink, Jorge H. Ramirez-Prado, Andrew H. Limper, Paige E. Jenson, Theodore J. Kottom
Rok vydání: 2017
Předmět:
0301 basic medicine
Pulmonary and Respiratory Medicine
In silico
Blotting
Western
Genes
Fungal
Clinical Biochemistry
Saccharomyces cerevisiae
Fluorescent Antibody Technique
Biology
Pneumocystis pneumonia
Gas Chromatography-Mass Spectrometry
Acetylglucosamine
Microbiology
Fungal Proteins
Cell wall
Mice
03 medical and health sciences
chemistry.chemical_compound
Cell Wall
Lectins
N-Acetylglucosamine
medicine
Animals
Pneumocystis jirovecii
Molecular Targeted Therapy
RNA
Messenger
Molecular Biology
Gene
Original Research
Pneumocystis
Pneumonia
Pneumocystis
04 agricultural and veterinary sciences
Cell Biology
biology.organism_classification
medicine.disease
Biosynthetic Pathways
Rats
carbohydrates (lipids)
Disease Models
Animal
Aminoglycosides
030104 developmental biology
chemistry
Biochemistry
040103 agronomy & agriculture
0401 agriculture
forestry
and fisheries
Peptidoglycan
Zdroj: American Journal of Respiratory Cell and Molecular Biology. 56:213-222
ISSN: 1535-4989
1044-1549
Popis: N-acetylglucosamine (GlcNAc) serves as an essential structural sugar on the cell surface of organisms. For example, GlcNAc is a major component of bacterial peptidoglycan, it is an important building block of fungal cell walls, including a major constituent of chitin and mannoproteins, and it is also required for extracellular matrix generation by animal cells. Herein, we provide evidence for a uridine diphospho (UDP)–GlcNAc pathway in Pneumocystis species. Using an in silico search of the Pneumocystis jirovecii and P. murina (Pm) genomic databases, we determined the presence of at least four proteins implicated in the Saccharomyces cerevisiae UDP-GlcNAc biosynthetic pathway. These genes, termed GFA1, GNA1, AGM1, and UDP-GlcNAc pyrophosphorylase (UAP1), were either confirmed to be present in the Pneumocystis genomes by PCR, or, in the case of Pm uap1 (Pmuap1), functionally confirmed by direct enzymatic activity assay. Expression analysis using quantitative PCR of Pneumocystis pneumonia in mice demonstrated abundant expression of the Pm uap1 transcript. A GlcNAc-binding recombinant protein and a novel GlcNAc-binding immune detection method both verified the presence of GlcNAc in P. carinii (Pc) lysates. Studies of Pc cell wall fractions using high-performance gas chromatography/mass spectrometry documented the presence of GlcNAc glycosyl residues. Pc was shown to synthesize GlcNAc in vitro. The competitive UDP-GlcNAc substrate synthetic inhibitor, nikkomycin Z, suppressed incorporation of GlcNAc by Pc preparations. Finally, treatment of rats with Pneumocystis pneumonia using nikkomycin Z significantly reduced organism burdens. Taken together, these data support an important role for GlcNAc generation in the cell surface of Pneumocystis organisms.
Databáze: OpenAIRE
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