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.
Highlights
Pneumocystis carinii remains a persistent cause of severe pneumonia in immune compromised patients
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-1type -glucan synthetases
P. carinii gsc-1 mRNA Expression Is Largely Restricted to Cystic Forms—Having observed differential abundance of Gsc-1 protein in isolated cyst and trophic forms of the organism, we investigated whether gsc-1 gene expression was regulated over P. carinii’s life cycle
Summary
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 -1,3glucan synthetase utilized in cyst wall formation. The current investigation was undertaken to accomplish the following: 1) to establish whether P. carinii cell wall assembly occurs through action of a Gsc-1 protein mediating -1,3-glucan synthesis; 2) to clone and characterize the respective gsc-1. This paper is available on line at http://www.jbc.org encoding this activity in P. carinii; and 3) to evaluate expression of P. carinii gsc-1 over the life cycle of the organism
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