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

Theodore J. Kottom, Deanne M. Hebrink, Paige E. Jenson, Jorge H. Ramirez-Prado, Andrew Harold Limper

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)213-222
Number of pages10
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume56
Issue number2
DOIs
StatePublished - Feb 1 2017

Fingerprint

Pneumocystis
Murinae
Acetylglucosamine
Uridine
Biosynthesis
Pneumocystis Pneumonia
Cells
Cell Wall
Genes
Pneumocystis carinii
Polymerase Chain Reaction
Chitin
Peptidoglycan
Biosynthetic Pathways
Enzyme Assays
Recombinant Proteins
Sugars
Gas chromatography
Computer Simulation
Yeast

Keywords

  • Cell wall
  • N-Acetylglucosamine
  • Nikkomycin Z
  • Pathogenesis
  • Pneumocystis

ASJC Scopus subject areas

  • Medicine(all)
  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

Cite this

Characterization of N-Acetylglucosamine Biosynthesis in Pneumocystis species A New Potential Target for Therapy. / Kottom, Theodore J.; Hebrink, Deanne M.; Jenson, Paige E.; Ramirez-Prado, Jorge H.; Limper, Andrew Harold.

In: American Journal of Respiratory Cell and Molecular Biology, Vol. 56, No. 2, 01.02.2017, p. 213-222.

Research output: Contribution to journalArticle

Kottom, Theodore J. ; Hebrink, Deanne M. ; Jenson, Paige E. ; Ramirez-Prado, Jorge H. ; Limper, Andrew Harold. / Characterization of N-Acetylglucosamine Biosynthesis in Pneumocystis species A New Potential Target for Therapy. In: American Journal of Respiratory Cell and Molecular Biology. 2017 ; Vol. 56, No. 2. pp. 213-222.
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