Indicators of Pneumocystis carinii viability in short-term cell culture.

Growth of P. carinii in culture has been difficult to document in the absence of reliable methods for distinguishing live from dead organisms. We studied three markers of cell function in P. carinii during the course of short-term cell culture, and correlated these with the number of P. carinii present in culture supernatants. The markers were glucan synthase activity, esterase activity and cell membrane integrity. The last two were assessed by double staining with fluorescein diacetate and propidium iodide followed by analysis of fluorescence using flow cytometry. The rise in P. carinii number after 5 to 7 days in culture was associated with increased glucan synthase activity. Flow cytometry analysis of day-6 P. carinii cultures confirmed that over 80% of the organisms catalyzed the conversion of fluorescein diacetate to fluorescein and excluded propidium iodide. The demonstration of three indices of metabolic activity in an expanding P. carinii population has confirmed the efficacy of a culture system as a means of sustaining the continued activity, albeit short-lived, of viable P. carinii.     

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.     

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

ABSTRACT. 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 I 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° 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.     

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 immunosuppressed host. It is a major complication of HIV-1 infection in man (AIDS). Using surface radioiodination of rat-derived 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 to numerous column matrices. A combination of gel chromatography and hydroxyapatite chromatography in sodium dodecylsulfate was utilized to purify the glycoprotein. Some preliminary characterization of the glycoprotein is presented.     

Pneumocystis carinii pneumonia in the rat model.

Groups of barrier-raised but not certified virus-free Sprague-Dawley rats, obtained from the same source over the course of several years, were placed on an identical immunosuppressive regimen. This caused reactivation of latent Pneumocystis carinii infection, manifest as P. carinii pneumonia (PCP) of varying severity. Rats were euthanized after 9-12 wk of immunosuppression. An assessment of the severity of the induced PCP was made, based on the total number of organisms extracted from the lungs and their ability to proliferate in short-term cell culture. Serum samples obtained at sacrifice were tested by indirect immunofluorescence for antibodies to coronavirus, parvovirus, Sendai virus, pneumonia virus of mice (PVM) and Mycoplasma pulmonis. A total of 60 rats were examined. Thirty-four of these (57%) developed moderate or severe PCP. No antibodies were detected to either coronavirus or Mycoplasma pulmonis in any of the rats. Although antibodies were detected to parvovirus in 13/60 (22%), to PVM in 29/60 (48%), and to Sendai virus in 47/60 (78%), there was no apparent correlation between the presence or absence of antibodies to these agents and the severity of PCP. Sequential observations during the course of immunosuppression are needed to clarify the role of concomitant infections in the development of PCP.     

Comparison of pulsed field gel electrophoresis karyotypes of Pneumocystis carinii derived from rat lung, cell culture, and ferret lung

Pulsed field gel electrophoretic karyotypes of Pneumocystis carinii derived from three sources were compared: immunosuppressed virus-free rats transtracheally inoculated with Pneumocystis-infected rat lung; WI-38 cell/Cytodex bead cell cultures inoculated with the same material; and immunosuppressed ferrets which reactivated latent Pneumocystis pneumonia. Karyotypes of DNA from Pneumocystis trophozoites or cysts from rat lung, and trophozoites from cell culture were identical. In contrast, ferret Pneumocystis DNA karyotypes were distinctly different. Rat Pneumocystis gene probes reacted with Southern- transferred rat Pneumocystis DNA but not with ferret Pneumocystis DNA. We concluded that neither the source nor life stage of rat Pneumocystis carinii influenced genomic karyotype, and that rat and ferret Pneumocystis are genetically diverse.     

A fifteen-year perspective on the in vitro culture of Pneumocystis carinii

The in vitro cultivation of Pneumocystis carinii in chick lung cell culture made it possible to observe the organism proceeding through its life cycle. It provided the foundation for extensive seroepidemiologic studies, for in vitro drug screening, and for essential biological, metabolic, and morphologic research. In vitro culture made possible the discovery of P. carinii antigenemia, its biochemical nature, and its relevance to subclinical and clinical infection. Its utility in the presumptive diagnosis of P. carinii pneumonia and in monitoring responses to drug therapy illustrate the value and clinical application of basic research.     

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.     

Isolation and characterization of rat lung Pneumocystis carinii gp120

The principal glycoprotein (gp120) of Pneumocystis carinii obtained from infected rat lung was isolated by differential extraction and size-exclusion chromatography. The purified glycoprotein was cleaved with CNBr to two peptides of approximately 27 and 33 kDa. Amino acid sequences were obtained from both peptides. Proteolytic digestion with V8 protease yielded several peptides and sequences were obtained from peptides of 10 and 19 kDa. The cyanogen bromide cleavage results led to the conclusion that gp120 exists as a homodimer.     

Serial propagation of Pneumocystis carinii in cell line cultures.

Pneumocystis carinii was propagated on three cell lines routinely cultured in many laboratories; the method is practical and convenient. Organisms produced were found to be reactive to Pneumocystis antisera. Studies of antigenic relationships, life cycles, and diagnostic methods will be made easier by these cultures.     

An ELISA method for quantitation of Pneumocystis carinii in culture and lung.

Numbers of Pneumocystis carinii in cultures or tissues traditionally have been determined by counting organisms on Giemsa-stained slides. For cultures, 10 microliters of culture supernatants have been sampled and counted on days 1, 3, 5 and 7. Infectivity scores of P. carinii-infected animal lung have been determined by three examiners scoring lung impression smears stained with Giemsa using a roughly logarithmic scale. Both counting procedures are tedious and time consuming. We have developed an enzyme-linked immunosorbent assay (ELISA) system which uses culture supernatants (in vitro) or homogenized animal lung (in vivo) as antigen, convalescent rat sera as primary antibody, and goat anti-rat alkaline phosphatase-conjugated immunoglobulin G as secondary antibody. The ELISA method shows good correlation with manual counts of Giemsa stains and allows a more rapid, more efficient method for quantitating P. carinii in both culture and infected lung.     

Serial propagation of Pneumocystis carinii in cell line cultures.

Pneumocystis carinii was propagated on three cell lines routinely cultured in many laboratories; the method is practical and convenient. Organisms produced were found to be reactive to Pneumocystis antisera. Studies of antigenic relationships, life cycles, and diagnostic methods will be made easier by these cultures.     

Plasminogen activator production in a rat model of Pneumocystis carinii pneumonia

Several studies have indicated that the serine protease urokinase-plasminogen-activator (uPA) is an important factor in host defense against pulmonary pathogens. To gain a better insight into the role of uPA in Pneumocystis carinii (P. carinii) pneumonia (PCP), we evaluated PA production in alveolar macrophages (AMs) obtained from rats with steroid-induced PCP. Treatment with cortisone acetate favored PCP in 91% of rats. In the bronchoalveolar lavage (BAL) samples of immunosuppressed rats both with and without PCP, we observed a decrease in uPA activity as well as a decrease in cell number. Urokinase-PA production by AMs was reduced in rats treated with cortisone alone. However, an increase in cell-associated uPA was observed in rats with PCP. This increase appears to be produced in response to P carinii infection. In fact, when AMs obtained from untreated healthy or immunosuppressed uninfected rats were challenged with P carinii, a significant increase in PA activity in cell lysates was observed, though a lower response was obtained in cortisone-treated animals. Our results suggest that healthy AMs respond to the presence of P carinii with an increase in uPA production and that this response in immunodepressed rat-AMs is partially impaired.     

In vitro attachment of Pneumocystis carinii from mouse and rat origin

Summary— The attachment of Pneumocystis carinii to lung cells could play a role in the pathophysiology of P carinii pneumonia. The trophozoite attaches to type I alveolar epithelial cells. Physical, chemical, and extracellular matrix factors, involved in the mouseor rat-derived P carinii attachment to fibroblastic cells in culture, were examined using a new model of in vitro adherence. The development of parasite filopodia penetrating deeply the host cell cytoplasm was observed using transmission electronic microscopy. Killed P carinii organisms were unable to attach to cultured cells. Also, parasites were unable to attach to killed target cells. The P carinii in vitro attachment was partially inhibited by cytochalasin B. In contrast, the parasite attachment was not affected when the target cell cytoskeleton was altered. In our work conditions, sialic acids were not involved in the attachment process. Present results showed that fibronectin (Fn) plays a role in the parasite attachment, and suggest that a specific Fn-binding receptor is present at the surface of mouse-derived P carinii organisms.     

Uptake and Metabolism of L-Serine by Pneumocystis carinii carinii

ABSTRACT. Serine is an important amino acid that is utilized in the biosyntheses of proteins and lipids. It is directly incorporated into the head group of phosphatidylserine, which in turn can be converted to other phospholipids. Also, it is required for the formation of long chain bases, precursors of sphingolipids. Uptake and incorporation of radiolabeled serine into both lipids and acid-precipitable material were demonstrated in Pneumocystis carinii carinii organism preparations freshly isolated from infected rat lungs. Radioactivity in proteins was about double that observed in lipids. Liquid scintillation spectrometry of metabolically radiolabeled lipids separated by thin-layer chromatography showed 53% of the total radioactivity were in phosphatidylserine, 12% in phosphatidylethanolamine, 24% in ceramides, and 11% in long chain bases and other compounds. Four long chain bases were detected by thin-layer chromatography in hydrolyzed P. carinii ceramides metabolically labeled with radioactive serine. Phytosphingosine and dihydrosphingosine were tentatively identified by their migrations on thin-layer plates. Radiolabeled ethanolamine was incorporated into P. carinii phosphatidylethanolamine, but relatively low incorporation of radiolabeled choline into phosphatidylcholine occurred. The observations made in this study indicated that P. carinii has the biosynthetic capacity to metabolize phospholipid head groups and to de novo synthesize sphingolipids. L-Cycloserine and β-CI-D-alanine, inhibitors of long chain base synthesis, reduced the incorporation of serine into P. carinii long chain bases and ceramides, which supported the conclusion that the pathogen synthesizes sphingolipids.