TY - JOUR
T1 - Characterization of N-Acetylglucosamine Biosynthesis in Pneumocystis species A New Potential Target for Therapy
AU - Kottom, Theodore J.
AU - Hebrink, Deanne M.
AU - Jenson, Paige E.
AU - Ramirez-Prado, Jorge H.
AU - Limper, Andrew H.
N1 - Publisher Copyright:
© 2017 by the American Thoracic Society.
PY - 2017/2
Y1 - 2017/2
N2 - 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, andit is also required for extracellularmatrix 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, andUDP-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 ofGlcNAc in P. carinii (Pc) lysates. Studies of Pc cell wall fractions using highperformance 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.
AB - 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, andit is also required for extracellularmatrix 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, andUDP-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 ofGlcNAc in P. carinii (Pc) lysates. Studies of Pc cell wall fractions using highperformance 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.
KW - Cell wall
KW - N-Acetylglucosamine
KW - Nikkomycin Z
KW - Pathogenesis
KW - Pneumocystis
UR - http://www.scopus.com/inward/record.url?scp=85013130937&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85013130937&partnerID=8YFLogxK
U2 - 10.1165/rcmb.2016-0155OC
DO - 10.1165/rcmb.2016-0155OC
M3 - Article
C2 - 27632412
AN - SCOPUS:85013130937
SN - 1044-1549
VL - 56
SP - 213
EP - 222
JO - American journal of respiratory cell and molecular biology
JF - American journal of respiratory cell and molecular biology
IS - 2
ER -