Propagation and Purification of Rat Pneumocystis carinii in Short-Term Cell

A short-term cell culture is used to propagate and purify rat-derived Pneumocystis carinii (Pc). An aliquot of pelleted material washed out of the lungs of rats with moderate to severe Pc pneumonia is cultured for 7 to 10 days on the adherent mink lung cell line Mv 1 Lu, and the rest of the material is frozen down in medium with 10% glycerol. Although it has not been established that substantial multiplication of Pc occurs in culture, the Pc organisms harvested from the supernatant at the end of the culture period arc relatively free of both host and feeder cells. This is in marked contrast with the lung wash inoculum in which the Pc organisms arc heavily contaminated with rat cells and enmeshed in a highly sticky material. Lung wash preparations frozen down in glycerol and stored at −70.°C for as long as 6 months or more can be successfully cultured upon thawing with no apparent loss of viability of the Pc organisms.     

Transcription Factor Genes from Rat Pneumocystis carinii

Genes encoding the TFIID TATA-box binding protein (TBP) from two probable species of rat Pneumocystis carinii (prototype and variant) were sequenced. The two P. carinii TBP gene sequences were 91% identical to each other, and 65-77% identical to TBP genes from other species. A cDNA from one of the two P. carinii TBP genes was sequenced, which showed that four small introns resided in identical positions within the TBP genes from the prototype and variant rat P. carinii. Conservation of the 180 amino acids that constitute the conserved core of TBP was 97% between the P. carinii TBP, which were 95% and 97% identical to conserved core sequences of TBP from Saccharomyces cerevisiae and Schizosaccharomyces pombe respectively.     

The Challenge of Pneumocystis carinii Culture1

ABSTRACT. Published and unpublished data on the cultivation of P. carinii were reviewed by a panel of investigators convened by the National Institutes of Health. Although several cell culture systems allow propagation of P. carinii for a limited time with modest rates of replication, these have not proved adequate for isolation of P. carinii in sufficient quantity to explore important basic biological investigation. Attempts at cell-free culture have yielded only transient proliferation. Because much of the unsuccessful work on cultivation of the organism has been unpublished, the panel agreed that these data may be useful to other investigators in designing experimental strategies for cultivation. Therefore, the purpose of this report is to make available this information to researchers, lest others unknowingly repeat unsuccessful methods. It is hoped that by documenting the history and the complexities of Pneumocystis culture, renewed interest and efforts will be directed toward this fundamental scientific challenge.     

Propagation and purification of rat Pneumocystis carinii in short-term cell culture

A short-term cell culture is used to propagate and purify rat-derived Pneumocystis carinii (Pc). An aliquot of pelleted material washed out of the lungs of rats with moderate to severe Pc pneumonia is cultured for 7 to 10 days on the adherent mink lung cell line Mv 1 Lu, and the rest of the material is frozen down in medium with 10% glycerol. Although it has not been established that substantial multiplication of Pc occurs in culture, the Pc organisms harvested from the supernatant at the end of the culture period are relatively free of both host and feeder cells. This is in marked contrast with the lung wash inoculum in which the Pc organisms are heavily contaminated with rat cells and enmeshed in a highly sticky material. Lung wash preparations frozen down in glycerol and stored at -70 degrees C for as long as 6 months or more can be successfully cultured upon thawing with no apparent loss of viability of the Pc organisms.     

Purification and characterization of recombinant Pneumocystis carinii thymidylate synthase.

Catalytically active Pneumocystis carinii thymidylate synthase is expressed to the extent of about 4% of the soluble protein in Escherichia coli chi 2913 harboring plasmid pUETS-1.8 (U. Edman, J. C. Edman, B. Lundgren, and D. V. Santi, Proc. Natl. Acad. Sci. USA 86, 6503-6507, 1989). Ion-exchange, affinity, hydrophobic, and reactive dye agarose chromatography steps were explored to devise a large-scale purification protocol for P. carinii thymidylate synthase. Sequential DE52, Q-Sepharose, phenyl-Sepharose, and Cibacron Blue F3GA chromatography yielded enzyme that was homogeneous by SDS-PAGE in a yield of over 50%. The sequence of the first 10 amino acid residues of the purified protein was in accord with that predicted from the DNA sequence. Isoelectric focusing gave a pI of 6.2. Kinetic analysis of the purified enzyme revealed that the Km values were 4.7 +/- 1.3 microM for dUMP and 15.7 +/- 4.3 microM for 5,10-methylenetetrahydrofolate, similar to those of many other thymidylate synthases; the kcat of the most active preparation was 0.8 s-1. The enzyme is stable for at least 2 months when stored at -80 degrees C in the presence of 40% glycerol, Tris-HCl, and thiol.     

Surface Labeling of Pneumocystis carinii from in Vitro Culture

Pneumocystis carinii is an opportunistic pathogen of man, carried as a commensal in healthy subjects. It frequently causes a fatal pneumonia in the immunosupprcssed host. It is a major complication of HIV-1 infection in man (AIDS). Using surface radioiodination of rat-denved P. carinii trophozoites obtained from in vitro culture, a major surface glycoprotein (gp120) has been identified. The glycoprotein exhibits adherent behavior similar to that of the intact organism. Purification of gp120 by conventional methods was unsuccessful as the glycoprotein irreversibly bound lo numerous column matrices. A combination of gel chromatography and hydroxyapatile chromatography in sodium dodecylsulfaie v. as utilized to purify the glycoprotein. Some preliminary characterization of the glycoprotein is presented.     

Cell Wall Antigens of Pneumocystis carinii Trophozoites and Cysts: Purification and Carbohydrate Analysis of these Glycoproteins

We have purified and biochemically analyzed individual cell wall glycoproteins of Pneumocystis carinii. Our results show that corresponding core glycoproteins constitute the cell wall antigens in both trophozoites and cysts, and glycosylation of these glycoproteins does not appear to be significantly altered during development. Cysts and trophozoites in rat-derived organism preparations were separated from each other by counterflow centrifugal elutriation, then treated with Zymolyase to obtain the cell wall fractions. Gel electrophoresis patterns of these fractions from both life-cycle stages were qualitatively similar. Ten major antigenic glycoproteins in these fractions were purified by preparative continuous elution gel electrophoresis. All ten glycoproteins from cysts and trophozoites contained mannose, glucose, galactose. and N-acetylglucosamine, and some contained traces of fucose. The glycoproteins of cysts had more mannose than their trophozoite counterparts. The trophozoite glycoproteins differed from those of the cyst by the presence of xylose. To examine the species-specificity of glycoprotein glycosylation, preparations of human-derived P. carinii (comprised of mixed life-cycle stages) were also examined and found to contain the same sugars as those found in rat-derived organisms. Most of the purified rat-derived glycoproteins bound Concanavalin A, which was abolished by treatment with N-glycanase. This suggested that the majority of the oligosaccharides were N-linked to the proteins, but attempts to identify carbohydrate linkage sites by amino acid sequencing were hampered by apparent modifications of residues. The peptides derived by cyanogen bromide cleavage revealed distinct size patterns for each glycoprotein, suggesting that they were distinct proteins. Most of the glycoproteins reacted with monoclonal antibodies which recognize a highly conserved epitope on rat P. carinii. Four of the individually purified glycoprotein preparations elicited in vitro cellular immune responses, implicating their involvement in the recognition of P. carinii by host T cells. The identification and characterization of P. carinii cell wall proteins will be helpful in analyzing the relationship of the organism to its mammalian host. Supplementary key words. Biochemical analysis, developmental stages, opportunistic pathogen, structure.     

Kinetics of 2 Genetically Distinct Pneumocystis carinii Populations in Rat Colonies

SUMMARY The natural history of prototype and variant Pneumocystis Corinii populations in several colonies of rats were monitored over time periods ranging from 1 to 4 yean. Karyotypic profiles of organism populations from individual rats were evaluated for the proportion of prototype: variant P. carinii and the number of animals harboring co-infections were enumerated. Throughout the observation periods: (1) each P. carinii population retained its distinctive karyotype; (2) the proportions of prototype:variant within the lungs fluctuated; and (3) the number of rats co-infected within a colony varied. These surveys suggest a competition between 2 similarly adapted populations.     

Characterization of Rat CD4 T Cell Clones Specific for the Major Surface Glycoprotein of Pneumocystis carinii

ABSTRACT. Pneumocystis carinii are coated by abundant heterogenous major surface glycoproteins (MSGs), which facilitate interaction with the host. We have produced MSG-specific T-cell clones from the spleens of P. carinii -exposed Lewis rats and analyzed five for antigen specificity to native MSG and a recombinant form of MSG, cell surface markers, and cytokine profiles. All five of the clones were CD4+. All of the clones proliferated specifically to both the native MSG and the recombinant MSG only in the presence of antigen presenting cells demonstrating that the response is antigen/driven rather than mitogen/driven. All five of the clones secreted IL-2 and IFN-γ, although in differing amounts, implicating a Th l response. Only one of the clones produced any detectable IL-4. This is the first report of T cell clones responsive to a specific antigen of P. carinii , MSG. We conclude that the T cell clones will be helpful in mapping protective epitopes present in MSG and in functional studies of MSG.     

Cell population obtained by bronchoalveolar lavage in Pneumocystis carinii pneumonitis

In the course of bronchoalveolar lavages performed in 115 immunocompromised patients in order to investigate the occurrences of pneumonitis, Pneumocystis carinii pneumonia was diagnosed by demonstration of cysts in bronchoalveolar lavage specimens from 11 patients. The cellular phenomena associated with P. carinii infection at the level of the alveolar space were evaluated. Differential cell counts on bronchoalveolar lavage preparations stained by the May-Grünwald-Giemsa method were performed in immunocompromised patients and in ten nonimmunocompromised patients without any respiratory disease. A decrease in the alveolar macrophage count associated with an increase in the polymorphonuclear neutrophil count and the presence of plasma cells and/or immunoblasts was highly suggestive of P. carinii pneumonia. These cellular changes in bronchoalveolar lavage specimens are discussed in relation to the pathologic features usually described in P. carinii pneumonia.     

S-adenosylmethionine and Pneumocystis carinii

We previously reported that S-adenosylmethionine (AdoMet), a key molecule in methylation reactions and polyamine biosynthesis, enhances axenic culture of the AIDS-associated opportunistic fungal pathogen Pneumocystis carinii. Here we report that AdoMet is absolutely required for continuous growth. Two transporters are present, one high affinity, K(m) = 4.5 microm, and one low affinity, K(m) = 333 microm. The physiologically relevant high affinity transporter has a pH optimum of 7.5 and no related natural compounds compete for uptake. Transport is 98% inhibited at 4 degrees C, 24% inhibited by 20 mm sodium azide, and 95% inhibited by the combination of 20 mm sodium azide and 1 mm salicylhydroxamic acid; thus transport is active and dependent on both a cytochrome chain and an alternative oxidase. In vitro, AdoMet is used at a rate of 1. 40 x 10(7) molecules cell(-1) min(-1). AdoMet synthetase activity was not detected by a sensitive radiolabel incorporation assay capable of detecting 0.1% of the activity in rat liver. In addition, the AdoMet plasma concentration of rats is inversely correlated with the number of P. carinii in the lungs. These findings demonstrate that P. carinii is an AdoMet auxotroph. The uptake and metabolism of this compound are rational chemotherapeutic targets.     

High-Speed Cell Sorting of Infectious Trophic and Cystic Forms of Pneumocystis carinii

ABSTRACT. The separation of Pneumocystis carinii life-cycle stages while preserving infectivity is a hitherto unresolved challenge. We describe an original, reproducible, and efficient method for separating trophic from cystic forms of P. carinii using a high-speed cell sorter. The large amounts of highly purified (99.6±0.3%) infectious trophic and cystic forms can now be used to elucidate the poorly understood P. carinii life cycle.     

Influence of Pneumocystis carinii on Nitrite Production by Rat Alveolar Macrophages1

ABSTRACT Nitrite production by rat alveolar macrophages was studied to determine the role of L-arginine oxidation in the interaction between these cells and Pneumocystis carinii. Alveolar macrophages from rats obtained from two different breeders were used: rats from Janvier breeder had latent P. carinii infection, while those from Charles River breeder were bred in a germ-free environment. Pneumocystis carinii increased in vitro nitrite generation by unstimulated alveolar macrophages from Janvier rats only, and this was blocked by N^G-monomethyl-L-arginine. Incubation of cells from Janvier and Charles River rats with lipopolysaccharide and/or interferon-gamma increased nitrite production to a similar extent. Pneumocystis carinii partially decreased nitrite release by activated alveolar macrophages, and this was still inhibited by N^G-monomethyl-L-arginine. In the presence of P. carinii, superoxide dismutase used as a superoxide anion scavenger had no effect on nitrite production by activated cells. These results show that prior exposure to P. carinii leads to nitric oxide production by rat alveolar macrophages. Although the magnitude of this production seems to be moderate, it is of biological significance since cells of P. curinii-naive rats do not generate nitrite whereas those of latently infected rats do.