Pneumocystis carinii f. sp. carinii synthesizes de novo four homologs of ubiquinone

Ubiquinone, coenzyme Q, plays a pivotal role in electron transport and is a target for chemotherapy against a number of eukaryotic infectious agents, including Pneumocystis carinii. Coenzyme Q10 was previously identified as the major ubiquinone homolog in P. carinii isolated and purified from rat lungs; CoQ9 was also present. In contrast, CoQ9 and CoQ8 (but not CoQ10) were detected in the lungs of uninfected rat controls. These observations suggested that the pathogen synthesizes CoQ10, and perhaps CoQ9 as well. In the present study, CoQ biosynthesis in P. carinii was examined in greater detail. Radiolabeled mevalonate, a precursor of the CoQ polyprenyl chain, was incorporated in vitro into P. carinii ubiquinones. Incorporation of radiolabeled mevalonate into P. carinii CoQ was not enhanced by treating cells with lovastatin, suggesting that the cells did not transport the drug, or that a lovastatin-insensitive pathway for de novo synthesis of isoprenoids may also function in this organism. Radiolabeled precursors of the ring moiety, including shikimic acid, p-hydroxybenzoic acid, and tyrosine were also incorporated into P. carinii CoQ. Unexpectedly, it was found that not only CoQ9 and CoQ10, but also CoQ7, and CoQ8, were metabolically radiolabeled by all the precursors tested, indicating that the organism synthesizes CoQ7, CoQ8, CoQ9, and CoQ10. Metabolic radiolabeling of ubiquinones in rat lung controls was not detected in experiments using either radioactive mevalonate or p-hydroxybenzoate. Thus the incorporations measured using purified P. carinii preparations were due to the enzymes of the organism.     

Pneumocystis carinii polyamine catabolism.

DL-alpha-Difluoromethylornithine (DFMO) causes polyamines of the AIDS-associated opportunistic pathogen Pneumocystis carinii to diminish 15 times more rapidly than mammalian host cells. The proposed mechanism was that, unlike mammalian cells, P. carinii is unable to regulate polyamine catabolism when synthesis is blocked. To test this, the responses of the polyamine catabolic enzymes spermidine/spermine acetyltransferase (SSAT) and polyamine oxidase (PAO) were determined using a new high-performance liquid chromatography assay to measure the products of these enzymes. The specific activities in untreated Pneumocystis carinii were 1.78 +/- 0.5 pmol min(-1) mg protein(-1) for SSAT, similar to mammalian cells, and 6.42 +/- 0.8 pmol min(-1) mg protein(-1) for PAO, 19% of that of mammalian cells. DFMO treatment for 12 h caused reductions of only 11 and 4% in SSAT and PAO, respectively, despite polyamine reductions of 94, 96, and 90% for putrescine, spermidine, and spermine, respectively. The P. carinii SSAT K(m) value of 25 microM spermidine is 20% of that of mammalian cells, and the PAO K(m) value of 14 nM N(1)-acetylspermidine is 0.01% of that of mammalian cells. Acetylated polyamines continue to be lost from P. carinii even when exposed to DFMO. Collectively, these results support the hypothesis that P. carinii is unable to regulate polyamine catabolism.     

Differential splicing of Pneumocystis carinii f. sp. carinii inosine 5'-monophosphate dehydrogenase pre-mRNA

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in guanine nucleotide metabolism that has drawn attention as a drug target in several organisms. Pneumocystis carinii f. sp. carinii IMPDH mRNA (GeneBank Accession No: U42442) previously identified from cultured organisms yielded a predicted amino acid sequence about 70 amino acids shorter at the amino terminus than IMPDH from other species. Recent research has shown that the amino terminal region is important for enzyme activity, suggesting that the previous putative P. carinii IMPDH might not represent full length, functional enzyme. To test this hypothesis, RT-PCR was performed with total RNA isolated from P. carinii f. sp. carinii. Three IMPDH splicing variants were found and splicing preference was observed: P. carinii isolated from infected rat lung contained primarily splicing variant one (introns two and four deleted), but organisms from spinner flask culture contained primarily splicing variant three (all four introns deleted). Importantly, splicing variant one (GeneBank Accession No: AF196975) contained an open reading frame for 529 amino acids, a size comparable to that of other eukaryotic IMPDH forms. The other variants contained the same open reading frame (454 amino acids) previously reported. Sequence analysis and complementation studies suggest variant one represents the full length, catalytically active form of P. carinii IMPDH. The differential splicing of the enzyme may reflect a mechanism by which the organism regulates the expression of IMPDH in response to environmental stresses.     

Glucan synthesis in Pneumocystis carinii.

Rat-derived Pneumocystis carinii lysed with sodium deoxycholate catalysed the incorporation of uridine diphosphoglucose into an insoluble polymer. This enzyme activity was present in both the pellet and the supernatant when the P. carinii preparations were centrifuged. The polymer whose production was catalysed by the supernatant was examined by mass spectrometry and found to be an alpha 1----4 glucan, which is either unbranched or has relatively few branches. Polymer formation was completely inhibited by the addition of alpha amyloglucohydrolase to the supernatant. Polymer formation in the pellet of deoxycholate P. carinii preparations, unlike that in the supernatant, was partially resistant to alpha amyloglucohydrolase. The soluble glucan synthase activity in the supernatant was stable for more than 30 h at room temperature and was approximately 50 times more active on a cell-to-cell basis than the supernatant from deoxycholate preparations of the yeast Saccharomyces cerevisae.     

Fine analysis of the Pneumocystis carinii f. sp. carinii genome by two-dimensional pulsed-field gel electrophoresis

Pneumocystis carinii is a general designation for a group of unusual unicellular fungal parasites responsible of pneumopathy in animal hosts. Divided into several subgroups termed the 'special forms', P. carinii is prone to an extensive karyotype variation. In previous studies, the nuclear genome of these organisms has been considered to be haploid and a set of 16 chromosomes has been assigned to P. carinii f. sp. carinii, a special form known to infect rats. We report the analysis of the genome of an isolate representative of the karyotype 1 of this special form, using two-dimensional pulsed-field gel electrophoresis procedures. The 'karyotype and restriction display' (KARD) fingerprints indicated the presence of 17 different chromosomes. The haploid genome size was estimated to be 8.4 Mbp. Some homologous chromosomes were distinguished on the basis of a single restriction fragment length polymorphism, which raises the possibility of a diploid nucleus. A restriction map of the chromosome 15, characterized by two homologues with a size difference of 7 kb, was constructed. Hybridization data indicated that insertion/deletion events may have occurred within subtelomeric regions which carry genes encoding the major surface glycoprotein (MSG) of Pneumocystis.     

Expression cloning of Pneumocystis carinii antigens.

We undertook expression cloning of Pneumocystis carinii antigens to overcome the difficulties encountered in purification of these antigens. Using monoclonal antibodies to the P. carinii gp120 antigen and polyclonal rabbit antiserum to rat-derived P. carinii, we have isolated cDNA clones encoding immunoreactive moieties. A cDNA clone encoding the 3' portion of a 45-55 kDa antigen of rat-derived P. carinii, was the most abundant clone isolated. The peptide encoded by this cDNA has a novel sequence with a repeated motif rich in glutamic acid residues. Affinity-purified antibodies to this peptide reacted with the 45-55 kDa band of rat-derived P. carinii. The fusion protein was recognized by serum antibodies from rats with natural exposure to P. carinii. The production of this recombinant protein should allow more detailed studies of the host-parasite relationship of this important opportunistic infection.     

Videomicroscopic recording of Pneumocystis carinii motion.

Videomicroscopy in combination with differential-contrast optics was used to study fresh preparations of Pneumocystis carinii from immunosuppressed rats. Certain spherical intracystic bodies appeared to move freely within the cyst wall. Flexing type movement was observed in intracystic ellipsoidal forms attached at a common point in the inner margin of the cyst wall. Greater movement was seen in non-attached thinner elongated forms. Possible extracellular trophic forms and movement were also identified. The movement of the morphological forms of P. carinii has been recorded in real time onto videotape. These initial observations suggest P. carinii is capable of movement and additional studies are under way to substantiate this possibility.     

Assays for testing Pneumocystis carinii viability.

A series of classical vital stains and fluorescent indicator compounds were evaluated as viability assays of P. carinii. The combination of the acetoxymethyl ester of calcein with either ethidium homodimer or propidium iodide distinguished between live, dead and moribund organisms and provided high fluorescence intensity and low bleaching enabling photodocumentation at high magnifications.     

Karyotypes of Pneumocystis carinii from Korean rats.

Molecular karyotyping was applied to Pneumocystis carinii(Pc) from two strains of experimental rats, Sprague Dawley(SD) and Fisher(F), in Korea. Field inversion gel electrophoresis and contour clamped homogeneous electric field electrophoresis resolved 15 chromosomal bands from the Pc. The size of the bands was estimated 270kb to 684kb from SD rats, and 273kb to 713 kb from F rats. The bands of 283 kb from SD rats and of 273 kb from F rats stained more brightly suggesting duplicated bands. Total number of chromosomes was at least 16, and total genomic size was estimated 7 x 10(6) bp. All of the bands from F rats hybridized to the probe of repeated DNA sequences of Pc and the band of 448 kb size was proved to contain rDNA sequences, but Pc. chromosome bands from SD rats showed no reactions to the probes. The 2 different karyotypes of P. carinii from 2 strains of rats were maintained consistently for 2 years.     

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.