RESUMO
The AIDS-associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S-adenosylmethionine:sterol C24-methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C-24 position of the sterol side chain producing both C28 and C29 24-alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild-type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy ((1)H-NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C28 sterol ergosterol as the major sterol, and also resulted in low levels of C29 sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ(24(28)) -sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C29 sterols as that found in P. carinii.
Assuntos
Técnicas de Inativação de Genes , Metiltransferases/deficiência , Metiltransferases/metabolismo , Pneumocystis carinii/enzimologia , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Esteróis/análise , Cromatografia Líquida , Espectroscopia de Ressonância Magnética , Metiltransferases/genética , Pneumocystis carinii/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Pneumocandins inhibit beta-1,3-glucan synthesis preventing the development of Pneumocystis cysts that are absent from the lungs of treated rats. To determine whether treated trophozoites are capable of DNA replication, cytochemical analyses were performed on 4',6-diamidino-2-phenylindole (DAPI)- and DB181-stained Pneumocystis carinii isolated from pneumocandin L-693-989-treated rats. Fluorescence intensities of trophozoite nuclei from drug-treated rats were greater than those of untreated controls, suggesting that DNA replication was not inhibited but that cytokinesis and perhaps karyokinesis were blocked.
