Characterization of the gene encoding a histidine and aspartic acid-rich protein from Pneumocystis carinii

A cDNA clone derived from Pneumocystis carinii contained an unusual sequence (GTGATG)2(ATGGTG)4(ATG)4 and many GAT repeats. It was found to encode a histidine and aspartic acid-rich protein (HARP). The complete cDNA contained an 888-bp open reading frame encoding a putative protein of 32.6 kDa. The deduced HARP protein contained 39 aspartic acid and 22 histidine residues. The genomic copy of the HARP gene (1203 bp in length) was found to contain 3 small introns of 46, 44, and 38 bp, respectively. HARP was predicted by computer programs to be a plasma membrane protein with nickel-binding activity.     

Characterization and Cloning of Pneumocystis carinii Nucleic Acid

Large numbers of Pneumocystis carinii (2 × 10¹⁰ nuclei) were isolated and separated from the lungs of immunosupprcsscd rats by an enzymaric (collagcnasc, hyaluronidasc and DN'asc) digestion procedure. The nucleic acid isolated from this P. cartnii-cnnchcd preparation was characterized by melting point analysis and RNA-sizing gels. The GC content of P. carinii DNA was approximately 33% while the rat DNA was 41.4%. In addition, RNA isolated from the P. curmii-enrichcd preparation showed unique ribosomal RNA bands of 3.4 kb and 1.8 kb as compared with uninfected rat lung ribosoma! RNA. which banded at 4.8 and 1.9 kb. Following isolation and fragmentation by sonicaüon, the P. carinii D.VA fragments were inserted into the vector, λ gt-11. The resultant library contained 1.1 × 10⁵ phage, of which 40–45% hybridized to P. carinii DNA but not to rat DNA.     

Characterization and cloning of Pneumocystis carinii nucleic acid

Large numbers of Pneumocystis carinii (2 X 10(10) nuclei) were isolated and separated from the lungs of immunosuppressed rats by an enzymatic (collagenase, hyaluronidase and DNase) digestion procedure. The nucleic acid isolated from this P. carinii-enriched preparation was characterized by melting point analysis and RNA-sizing gels. The GC content of P. carinii DNA was approximately 33% while the rat DNA was 41.4%. In addition, RNA isolated from the P. carinii-enriched preparation showed unique ribosomal RNA bands of 3.4 kb and 1.8 kb as compared with uninfected rat lung ribosomal RNA which banded at 4.8 and 1.9 kb. Following isolation and fragmentation by sonication, the P. carinii DNA fragments were inserted into the vector, lambda gt-11. The resultant library contained 1.1 X 10(5) phage, of which 40-45% hybridized to P. carinii DNA but not to rat DNA.     

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.     

Characterization of Pneumocystis carinii Preparations Developed for Lipid Analysis

Pneumocystis carinii organisms were isolated from viral antibody-negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was > 5 (100-1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (< 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated > 99.5% purity. The total non-P. carinii protein in the final preparation (< 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 10⁸-10⁹ organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester-propidium iodide assay, was 80–95%.     

Characterization of dihydrofolate reductase of Pneumocystis carinii and Toxoplasma gondii

Pneumocystis carinii and Toxoplasma gondii are opportunistic pathogens of immunosuppressed patients that are susceptible to therapy with inhibitors of dihydrofolate reductase (DHFR). The DHFR of these two organisms was characterized to facilitate the identification of more selective inhibitors. Similar to all reported protozoa, T. gondii has a bifunctional enzyme, of 120,000 Da, that possesses both DHFR and thymidylate synthase (TS) activity. Unexpectedly, P. carinii DHFR activity was present on a small molecule, of 26,000 Da. T. gondii DHFR and TS activity coeluted during affinity chromatography using a methotrexate-Sepharose column, whereas P. carinii DHFR and TS activity could be separated by affinity chromatography using the same column. P. carinii DHFR could be easily distinguished from rat DHFR, which is similar in size, by the differences in Km for dihydrofolate (P. carinii, 17.6 +/- 3.9 microM; rat, 4.0 +/- 2.2 microM). Since all protozoa reported have a large molecular weight, bifunctional DHFR, these studies support the classification of P. carinii as a fungus. These studies also provide a basis for the development of more effective therapeutic agents for these pathogens.     

Determination of the copy number of the nuclear rDNA and beta-tubulin genes of Pneumocystis carinii f. sp. hominis using PCR multicompetitors

Multiple copies of a gene may lead to difficulty in the interpretation of typing results because polymorphism of the copies may wrongly lead to the conclusion that different types are present in a specimen. To determine the copy number per genome of the nuclear rDNA and beta-tubulin genes analyzed for the typing of Pneumocystis carinii f. sp. hominis, we developed a strategy based on the use of the same multicompetitor molecule in two different quantitative-competitive PCRs, one for the gene under study and the other for a reference single copy gene, allowing direct comparison of the results of both PCRs. Control experiments showed that the strategy was sensitive enough to detect duplication of a gene. The copy number of the nuclear rDNA operon was determined by amplification of the intron of the 26S rDNA gene and that of the beta-tubulin by amplification of the region surrounding the intron no. 6. The method was first tested on P. c. carinii, the special form commonly infecting rats. Pneumocystis c. carinii was found to contain a single copy of the rDNA operon. The method was then applied to P. c. hominis. The results confirmed that P. c. hominis genome contains a single copy of the nuclear rDNA and beta-tubulin genes.