Pneumocystis is a genus that causes a type of pneumonia, known as PcP, in immune deficient mammals. In contrast to most fungi, whose major membrane sterol is ergosterol, cholesterol scavenged from the mammalian host is the predominant sterol in Pneumocystis. Since Pneumocystis lacks ergosterol, and most traditional anti-fungal therapies target ergosterol biosynthesis, an alternate drug target is needed.
Although cholesterol is the major sterol in organisms grown in animals or culture media supplemented with mammalian sera, Pneumocystis produces de novo unique 28 and 29 carbon sterols with alkylations at the C-24 position of the sterol side chain. These 24-alkyl sterols have been shown to be important for Pneumocystis viability and proliferation. The S-adenosyl-L- methionine: sterol C-24 methyltransferase enzyme (SMT), coded by the erg6 gene, catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to the C-24 position of the sterol side chain. This enzyme, critical to Pneumocystis, is not present in the mammalian host, and therefore represents an intriguing chemotherapeutic target.
The Pneumocystis carinii erg6 gene has been sequenced, cloned, and the recombinant SMT expressed in E. coli, and T. thermophila. These expression systems were of limited utility, so Saccharomyces cerevisiae was examined as a potential expression host. An erg6 null strain of S. cerevisiae was commercially available and chosen as an expression host to eliminate the potential for endogenous SMT activity. Initial attempts to express the P. carinii erg6 in Saccharomyces showed that the gene was transcribed, but the recombinant P. carinii SMT was not clearly present.
Codon usage was one of the potential factors affecting the production of the recombinant P. carinii SMT in S. cerevisiae. It was found that although Pneumocystis and Saccharomyces both have A-T rich genomes, codon usage was actually quite different. Therefore a synthetic P. carinii erg6 gene, optimized for expression in S. cerevisiae was constructed, cloned into a S. cerevisiae expression vector, and transformed into an erg6 null strain of Saccharomyces cerevisiae. The native P. carinii erg6 gene, and the S. cerevisiae erg6 gene were also cloned and transformed in the same manner as controls. It was demonstrated that the P. carinii SMT was expressed in S. cerevisiae. This system will be useful for the production of sterols and recombinant SMT for further studies.