Growth and metabolism of Pneumocystis carinii in axenic culture

Abstract

Recent analyses of rRNA have suggested a taxonomic relationship of Pneumocystis carinii with fungi. We investigated various liquid and solid media without feeder cells for the ability to support growth of P. carinii. Organisms were obtained from the lungs of immunosuppressed rats and separated from host cells by treatment with hypotonic solutions and a series of microfiltrations. The growth of P. carinii was quantitated by staining nuclei with Diff-Quik. Optimal replication was observed in media with neopeptone and N-acetylglucosamine at low pH. Increases of at least 8- to 10-fold occurred during the first 24 to 72 h. P. carinii replication was influenced by pH, temperature, inoculum size, and anti-P. carinii drugs. The viability of organisms was confirmed by incorporation of radiolabeled uracil, methionine, and N-acetylglucosamine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trichloroacetic acid-precipitable compounds showed that [35S]methionine was incorporated into specific P. carinii proteins. Thus, P. carinii can metabolize and replicate in the absence of mammalian cells. This system should have applications for in vitro drug screening and other molecular and metabolic analyses.