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. caring 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 8to 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. carinùi 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. * Corresponding author. National Cancer Institute were immunosuppressed with weekly injections of 4 mg of methylprednisolone acetate (Depo-Medrol; The Upjohn Co., Kalamazoo, Mich.) for 6 to 8 weeks (9). P. carinii was obtained by homogenization of minced lungs with a Stomacher Lab Blender (Tekmar, Cincinnati, Ohio) as previously described (10-12). Hypotonic lysis and exhaustive filtrations through 10-,um-poresize Mitex filters (Millipore Corp., Bedford, Mass.) were used to provide a preparation free of intact host cells. Inocula were plated on Mueller-Hinton and Sabouraud dextrose agars for the detection of microbial contaminants. In addition, ail inocula were evaluated for the presence of host and microbial contaminants by the following criteria: (i) examination of 10 oil immersion fields (magnification, x 1,000) of each of three 10-,ul drops (for a total of 30 oil immersion fields) stained with Diff-Quik; (ii) examination of an unstained 1-,ul drop by phase-contrast microscopy; and (iii) examination of 10 oil immersion fields of a 10-pul drop stained with Gram stain. Fungal and bacterial surveillance cultures of all inocula used for culture studies were negative. Organism numbers were assessed by enumeration of P. carinii nuclei per milliliter (9-12; see below). The viability of preparations was always >95% by erythrosin B dye exclusion (12). Culture media and conditions. The following media and additives were evaluated for support of P. carinii growth: Dulbecco minimal essential medium (DMEM), yeast extractpeptone-dextrose (YEPD) broth and agar, yeast extract-malt extract (YM) broth, brain heart infusion broth and agar, wort broth and agar, Sabouraud dextrose broth and agar, modified Sabouraud dextrose broth and agar, and Vogel and Johnson agar. Solid agars were tested at pH 4.0 and 7.0. Additives were purchased from...

Incorporation of Fatty Acids and Amino Acids by Cultured Pneumocystis carinii

“…Studies continue to describe the metabolic capabilities of P. carinii and the effects of drugs on metabolism. preparations of P. carinii from lung lavage, which also contained up to 1,000 bacterialml and 100 host cells/ml, with short term culture [20] and in axenically cultured P. carinii [4]. A mixture of tritiated amino acids was reported to be incorporated into materials precipitated by trichloroacetic acid, which were presumed to be macromolecules [20].…”

Uptake and Metabolism of L‐Serine by Pneumocystis carinii carinii

“…Similarly, Amoeba does not take up radiolabeled glucose unless pinocytotic activity is induced. It appears that Pneumocystis is similar to Paramecium in that they both take up and metabolize exogenous lipids much faster than amino acids [8,12,17,18,211. The rates of uptake and metabolism of exogenous material P21.…”

Typing of Pneumocystis carinii strains that infect humans based on nucleotide sequence variations of internal transcribed spacers of rRNA genes

“…However, restriction fragment length polymorphism analyses are not readily applicable to human clinical specimens because they require enormous numbers of organisms, and there is no culture system in which human P. carinii can be grown. In contrast, P. cannii isolates that infect rats (hereafter referred to as rat P. cafinii) can be grown in tissue culture or passaged in rats (3,5,7,9,20,27,28); therefore, it has been possible to analyze the rat P. cannii genome more extensively. Up to five types of rat P. cannii strains have been described by molecular karyotyping on the basis of the numbers and the sizes of chromosomes separated by pulsed-field gel electrophoresis (6,17,35).…”