Cell wall assembly by Pneumocystis carinii. Evidence for a unique gsc-1 subunit mediating beta -1,3-glucan deposition.

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 beta-1,3-glucan synthesis rapidly suppress infection in animal models of P. carinii pneumonia. We, therefore, sought to define the molecular mechanisms of beta-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 beta-glucan synthetases. Using degenerative polymerase chain reaction and library screening, the P. carinii Gsc-1 catalytic subunit of beta-1,3-glucan synthetase was cloned and characterized. P. carinii gsc1 exhibited homology to phylogenetically related fungal beta-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 beta-1,3-glucan synthetase utilized in cyst wall formation. Because synthesis of beta-1,3-glucan is absent in mammalian cells, inhibition of the P. carinii Gsc-1 represents an attractive molecular target for therapeutic exploitation.

• Authors: T J Kottom, A H Limper
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 275 Issue 51 Pg. 40628-34 (Dec 22 2000) ISSN: 0021-9258 [Print] United States
• PMID: 11013231 (Publication Type: Journal Article)

Chemical References

• Anti-Bacterial Agents
• DNA Primers
• Glucans
• L 733560
• Peptides
• Peptides, Cyclic
• beta-Glucans
• Uridine Diphosphate
• beta-1,3-glucan
• Glucosyltransferases
• glucan synthase

Topics

• Anti-Bacterial Agents (pharmacology)
• Base Sequence
• Cell Wall (drug effects, enzymology, metabolism)
• DNA Primers
• Glucans (chemistry, metabolism)
• Glucosyltransferases (chemistry, metabolism)
• Molecular Sequence Data
• Peptides
• Peptides, Cyclic (pharmacology)
• Pneumocystis (enzymology, metabolism)
• Protein Conformation
• Uridine Diphosphate (metabolism)
• beta-Glucans