Karyotypes of Pneumocystis carinii derived from several mammals

Pneumocystis carinii is the most important opportunistic pathogen of humans in the world. Pneumocystis carinii is experimentally detected in the lungs of rats, mice, rabbits, and monkeys, however, the organisms from different mammals are identical in microscopic morphology. The present study tried to find out more mammalian hosts of P. carinii and also to differentiate the organisms from different mammals by karyotyping. Rats, mice, hamsters, rabbits, cats, and dogs were successfully infected by P. carinii, but guinea pigs and pigs were not. Karyotype of P. carinii from rabbits showed similar size range of chromosomes with that of the prototype, but in different pattern. The patterns from cats and dogs were also different from that of rats. The present study confirms that cats and dogs are infected by P. carinii and at least total three karyotype strains of P. carinii are proven in Korea.     

Decreased yield of Pneumocystis carinii from cortisonized rats

Thirty-five male Sprague-Dawley rats were divided into 3 groups, including 1 control and 2 experimental groups, in order to compare the efficacy of using cortisone acetate alone or in addition to intranasal inoculation of Pneumocystis carinii organisms for the purpose of inducing acute P. carinii pneumonia. The presence of P. carinii was monitored in nasal secretions on a weekly basis and in lungs at autopsy. Titers of IgG antibody were also monitored by enzyme-linked immunosorbent assay. No rat receiving cortisone acetate injections alone and only 2 of the rats receiving both cortisone and intranasal inoculation of P. carinii organisms showed Pneumocystis organisms in the lungs. However, Pneumocystis cysts did appear in the nasal secretions of 3 of the 5 control rats, all 8 rats receiving cortisone acetate injections only, and 12 of 18 rats receiving both cortisone acetate injections and an intranasal inoculum. IgG titers of both cortisonized groups remained less than 1:4 throughout the course of the experiment. The titer of the control group increased from negative to 13 (geometric mean).     

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.     

Genetic heterogeneity of Pneumocystis carinii from rats of several regions and strains

Pneumocystis carinii is a major opportunistic pathogen which has been found in the lungs of a wide variety of mammalian host species, and the fact suggests the possibility of intraspecific variation. Until now, P. carinii from different mammalian species are differentiated as subspecies, and the rats are known to be infected by two subspecies. The present study investigated genetic heterogeneity of P. carinii isolates from two strains of rats in Korea and China by molecular karyotyping, RFLP and sequencing analysis. Karyotypes of P. carinii were grouped into three, two from two strains of rats in Korea and one from rats in China. However RFLP of PCR product of ribosomal and MSG gene of the P. carinii isolates showed same pattern. The sequence homology rates of alpha-tubulin DNA of the P. carinii isolates were 96% in Seoul Wistar rats, 93% in Seoul Sprague-Dawley rats, and 85% in Chinese Sprague-Dawley rats. The present finding confirmed that P. carinii from rats in Korea are grouped into two karyotype strains which are different from that of P. carinii from rats in China. The Chinese isolate shows a little different sequences of alpha-tubulin DNA.     

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.     

Transmission modes of Pneumocystis carinii among rats observed by karyotype analysis.

To observe the transmission patterns of karyotype of Pneumocystis carinii (Pc) by rat colonies, three strains of rats, Sprague-Dawley(SD), Wistar(W) and Fisher(F) from various animal vendors, were suppressed of their immunity by injection of methyl prednisolone. They were kept for 5 to 13 weeks in 3 different animal rooms, A, B, and C. The purified organisms were prepared in low melting point agarose gel by embedded lysis method for pulsed field gel electrophoresis. Field inversion gel electrophoresis showed 2 patterns of the karyotype of Pc. The rooms A and C contained SD rats from the source P, and also the room A was used for F and W rats. However, Pc from all of the SD and F rats in the room A showed same karyotypes, the pattern I. The SD rats from different vendors, M and S, were reared in the room B, and shared the same Pc karyotypes, the pattern II. The rats of W strain were from the vendor M, and immune-suppressed in the animal room A. Five weeks after the experiment, the Pc showed the karyotype pattern II but the pattern became mixed with the type I after 7 to 8 weeks. The findings revealed that the animals born and reared in the same animal quarter harbored Pc with same karyotypes. If the animals were kept under immune-suppression in the same room with heavily infected hosts, they could be infected by Pc from their neighbors. The present experimental findings suggest that Pc is transmitted among rats through the air.     

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.     

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.     

Pneumocystis carinii: new separation method from lung tissue.

A new method of separating Pneumocystis carinii from infected rat, human, and mouse lung tissue has been developed, based, in part, on techniques used for the separation of lymphocytes from hematopoietic and lymphoid organs. The lungs are homogenized with a Teflon pestle and fine wire-mesh screen, digested with collagenase and hyaluronidase, and centrifuged on a discontinuous Ficoll-Hypaque density gradient. The method produces no morphologic alterations in P. carinii, as judged by light and electron microscopy. The method has been adapted for the quantitation of P. carinii in tissues and the production of antisera in rabbits.     

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.