Cell wall assembly by Pneumocystis carinii: Evidence for a unique Gsc-1 subunit mediating β-1,3-glucan deposition

Theodore J. Kottom, Andrew Harold Limper

Research output: Contribution to journalArticle

76 Citations (Scopus)

Abstract

Pneumocystis carinii remains a persistent cause of severe pneumonia in immune compromised patients. Recent studies indicate that P. carinii is a fungal species possessing a glucan-rich cyst wall. Pneumocandin antagonists of β-1,3-glucan synthesis rapidly suppress infection in animal models of P. carinii pneumonia. We, therefore, sought to define the molecular mechanisms of β-glucan cell wall assembly by P. carinii. Membrane extracts derived from freshly purified P. carinii incorporate uridine 5′-diphosphoglucose into insoluble carbohydrate, in a manner that was completely inhibited by the pneumocandin L733-560, an antagonist of Gsc-1-type β-glucan synthetases. Using degenerative polymerase chain reaction and library screening, the P. carinii Gsc-1 catalytic subunit of β-1,3-glucan synthetase was cloned and characterized. P. carinii gsc1 exhibited homology to phylogenetically related fungal β-1,3-glucan synthetases, encoding a predicted 214-kDa integral membrane protein with 12 transmembrane domain structure. Immunoprecipitation of P. carinii extracts, with a synthetic peptide anti-Gsc-1 antibody, specifically yielded a protein of 219.4 kDa, which was also capable of incorporating 5′-diphosphoglucose into insoluble glucan carbohydrate. As opposed to other fungi, the expression of gsc-1 mRNA is uniquely regulated over P. carinii's life cycle, having minimal expression in trophic forms, but substantial expression in the thick-walled cystic form of the organism. These results indicate that P. carinii contains a unique catalytic subunit of β-1,3-glucan synthetase utilized in cyst wall formation. Because synthesis of β-1,3-glucan is absent in mammalian cells, inhibition of the P. carinii Gsc-1 represents an attractive molecular target for therapeutic exploitation.

Original languageEnglish (US)
Pages (from-to)40628-40634
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number51
DOIs
StatePublished - Dec 22 2000

Fingerprint

Pneumocystis carinii
Glucans
Cell Wall
Cells
Ligases
Cysts
Catalytic Domain
Organism Forms
Carbohydrates
Uridine Diphosphate Glucose
Pneumocystis Pneumonia
Uridine
Polymerase chain reaction
Life Cycle Stages
Fungi
Immunoprecipitation
Life cycle
Pneumonia
Screening
Membrane Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cell wall assembly by Pneumocystis carinii : Evidence for a unique Gsc-1 subunit mediating β-1,3-glucan deposition. / Kottom, Theodore J.; Limper, Andrew Harold.

In: Journal of Biological Chemistry, Vol. 275, No. 51, 22.12.2000, p. 40628-40634.

Research output: Contribution to journalArticle

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