Cell wall assembly by Pneumocystis carinii. Evidence for a unique gsc-1 subunit mediating beta -1,3-glucan deposition.
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.
Kottom
T J
TJ
Thoracic Diseases Research Unit, Departments of Medicine and Biochemistry, Mayo Clinic, Rochester, Minnesota 55905, USA.
Limper
A H
AH
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D002473:Cell Wall / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D017931:DNA Primers; D005936:Glucans / Q000737:chemistry / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides; D010456:Peptides, Cyclic / Q000494:pharmacology; D011010:Pneumocystis / Q000201:enzymology / Q000378:metabolism*; D011487:Protein Conformation; D014530:Uridine Diphosphate / Q000378:metabolism; D047071:beta-Glucans
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D005936:Glucans; C089367:L 733560; D010455:Peptides; D010456:Peptides, Cyclic; D047071:beta-Glucans; D014530:Uridine Diphosphate; C033363:beta-1,3-glucan; D005964:Glucosyltransferases; C040232:glucan synthase