Complementation and characterization of the Pneumocystis carinii MAPK,PCM

Mitogen-activated protein kinase (MAPK) pathways transfer environmental signals into intracellular events such as proliferation and differentiation. Fungi utilize a specific pheromone-induced MAPK pathway to regulate conjugation, formation of an ascus, and entry into meiosis. We have previously identified a MAPK, PCM, from the fungal opportunist Pneumocystis, responsible for causing severe pneumonia in patients with AIDS. In order to gain insight into the function of PCM, we expressed it in Saccharomyces cerevisiae deficient in pheromone signaling and tested activation and inhibition of this MAPK pathway. PCM restored pheromone signaling in S. cerevisiae fus3Delta kss1Delta mutants with alpha-factor pheromone (six-fold increase) and was not activated by osmotic stress. Signaling through this pathway decreased 2.5-fold with 10 microM U0126, and was unaffected with SB203580. We evaluated the conditions for native PCM kinase activity isolated from Pneumocystis carinii organisms and found that 0.1 mM MgCl2, pH 6.5, temperature 30-35 degrees C, and 10 microM ATP were optimal. The activity of PCM is significantly elevated in P. carinii trophic forms compared to cysts, implicating a role for PCM in the life cycle transition of P. carinii from trophic forms to cysts.     

Pneumocystis carinii infection in corticosteroid-treated cats

Corticosteroids were administered to produce Pneumocystis carinii infection in cats. Six of 10 cats, injected intramuscularly for 97-141 days with 2 mg/cat twice weekly of betamethasone sodium phosphate, developed a light infection with P. carinii. Six of 7 cats, injected intramuscularly for 11-168 days with 10-25 mg/cat weekly of prednisolone acetate, also developed a light infection with P. carinii. There was no significant difference in the infection rate between the sexes and ages of the cats. Using Giemsa staining and Gomori's methenamine silver nitrate stain, P. carinii organisms were indistinguishable morphologically from human and rat P. carinii. The cysts and trophozoites were usually present singly or in small groups, and they always were adhering to the periphery of alveoli. The inflammatory changes were inconspicuous except for the fact that alveolar macrophages often were seen. Corticosteroid-treated cats should be useful in the study of experimental P. carinii infection. This is the first reported case of experimentally induced P. carinii infection in cats.     

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).     

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.     

Biofilm formation by Pneumocystis spp

Pneumocystis spp. can cause a lethal pneumonia in hosts with debilitated immune systems. The manner in which these fungal infections spread throughout the lung, the life cycles of the organisms, and their strategies used for survival within the mammalian host are largely unknown, due in part to the lack of a continuous cultivation method. Biofilm formation is one strategy used by microbes for protection against environmental assaults, for communication and differentiation, and as foci for dissemination. We posited that the attachment and growth of Pneumocystis within the lung alveoli is akin to biofilm formation. An in vitro system comprised of insert wells suspended in multiwell plates containing supplemented RPMI 1640 medium supported biofilm formation by P. carinii (from rat) and P. murina (from mouse). Dramatic morphological changes accompanied the transition to a biofilm. Cyst and trophic forms became highly refractile and produced branching formations that anastomosed into large macroscopic clusters that spread across the insert. Confocal microscopy revealed stacking of viable organisms enmeshed in concanavalin A-staining extracellular matrix. Biofilms matured over a 3-week time period and could be passaged. These passaged organisms were able to cause infection in immunosuppressed rodents. Biofilm formation was inhibited by farnesol, a quorum-sensing molecule in Candida spp., suggesting that a similar communication system may be operational in the Pneumocystis biofilms. Intense staining with a monoclonal antibody to the major surface glycoproteins and an increase in (1,3)-beta-D-glucan content suggest that these components contributed to the refractile properties. Identification of this biofilm process provides a tractable in vitro system that should fundamentally advance the study of Pneumocystis.     

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.     

Pneumocystis: from a doubtful unique entity to a group of highly diversified fungal species

At the end of the 20th century the unique taxonomically enigmatic entity called Pneumocystis carinii was identified as a heterogeneous group of microscopic Fungi, constituted of multiple stenoxenic biological entities largely spread across ecosystems, closely adapted to, and coevolving in parallel with, mammal species. The discoveries and reasoning that led to the current conceptions about the taxonomy of Pneumocystis at the species level are examined here. The present review also focuses on the biological, morphological and phylogenetical features of Pneumocystis jirovecii, Pneumocystis oryctolagi, Pneumocystis murina, P. carinii and Pneumocystis wakefieldiae, the five Pneumocystis species described until now, mainly on the basis of the phylogenetic species concept. Interestingly, Pneumocystis organisms exhibit a successful adaptation enabling them to dwell and replicate in the lungs of both immunocompromised and healthy mammals, which can act as infection reservoirs. The role of healthy carriers in aerial disease transmission is nowadays recognized as a major contribution to Pneumocystis circulation, and Pneumocystis infection of nonimmunosuppressed hosts has emerged as a public health issue. More studies need to be undertaken both on the clinical consequences of the presence of Pneumocystis in healthy carriers and on the intricate Pneumocystis life cycle to better define its epidemiology, to adapt existing therapies to each clinical context and to discover new drug targets.     

A hypothesis on the preferential destruction of melanized tissues

Based on existing biological data and the known amorphous semi-conductor properties of the melanins, a hypothesis to explain the preferential death of melanized cells is presented. This hypothesis suggests that melanized cell death associated with Parkinson's disease and other syndromes involving non-illuminated areas occurs as a result of non-radiative transfer of energy to and from the melanins. This mechanism is also consistent with the observation that melanized cells are preferentially killed by high intensity ultraviolet light in illuminated areas, but the melanins appear to be cytoprotective at low intensities.     

Structure of Major Surface Determinants and DNA Diagnosis of Pneumocystis carinii

Pneumocystis carinii is a pathogen which, causes fatal pneumonia in patients with the acquired immune deficiency syndrome (AIDS). To facilitate the basic study of P. carinii , we have analyzed its major surface proteins by both immunochemical and biochemical methods. The major protein components of both cysts and trophozoites are a group of proteins called "P115" with apparent masses of 105–120 kd. It includes 6 isoelcclric variants. A monoclonal antibody raised against cysts recognizes all 6 variants and reacts with epitopes located in the cell wall indicating that P115 is an immunorcactive surface component. The isoelectric variants contain identical or closely related protein components and they are mannose-rich glycoproteins. The isoelectric variation may be due primarily to differences in glycosylation. The majority of sera from humans with diagnosed pneumocystosis that were tested reacted strongly with the P115 proteins. To develop probes for DNA diagnosis and to facilitate molecular studies, a genomic DNA library of P. carinii has been constructed. Some of these clones were used for DNA hybridization analysis of rat and human lungs.     

Structure of major surface determinants and DNA diagnosis of Pneumocystis carinii

Pneumocystis carinii is a pathogen which causes fatal pneumonia in patients with the acquired immune deficiency syndrome (AIDS). To facilitate the basic study of P. carinii, we have analyzed its major surface proteins by both immunochemical and biochemical methods. The major protein components of both cysts and trophozoites are a group of proteins called "P115" with apparent masses of 105-120 kd. It includes 6 isoelectric variants. A monoclonal antibody raised against cysts recognizes all 6 variants and reacts with epitopes located in the cell wall indicating that P115 is an immunoreactive surface component. The isoelectric variants contain identical or closely related protein components and they are mannose-rich glycoproteins. The isoelectric variation may be due primarily to differences in glycosylation. The majority of sera from humans with diagnosed pneumocystosis that were tested reacted strongly with the P115 proteins. To develop probes for DNA diagnosis and to facilitate molecular studies, a genomic DNA library of P. carinii has been constructed. Some of these clones were used for DNA hybridization analysis of rat and human lungs.     

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.     

Development of an RT-PCR on the Heat Shock Protein 70 Gene for Viability Detection of Pneumocystis carinii f. sp. hominis in Patients with Pneumocystosis and in Air Sample

SUMMARY. To test the viability of Pneumocystis carinii f. sp. hominis , an RT-PCR assay that employs specific primers from the Heat Shock Protein 70 gene was developed. Using this method, the viability of P.c. hominis in bronchoalveolar lavage fluids from patients developing PCP and in the environment of PCP patients was established.     

Parallel Phylogenies of Pneumocystis Species and their Mammalian Hosts

ABSTRACT. The single name Pneumocystis carinii consists of an heterogeneous group of specific fungal organisms that colonize a very wide range of mammalian hosts. In the present study, mitochondrial large subunit (mtLSU) and small subunit (mtSSU) rRNA sequences of P. carinii organisms from 24 different mammalian species were compared. The mammals were included in six major groups: Primates (12 species). Rodents (5 species). Carnivores (3 species). Bats (1 species), Lagomorphs (1 species), Marsupials (1 species) and Ungulates (1 species). Direct sequencing of PCR products demonstrated that specific mtSSU and mtLSU rRNA Pneumocystis sequence could be attributed to each mammalian species. No animal harbored P. carinii f. sp. hominis. Comparison of combined mtLSU and mtSSU aligned sequences confirmed cospeciation of P. carinii and corresponding mammalian hosts. P. carinii organisms isolated from mammals of the same zoological group systematically clustered together. Within each cluster, the genetic divergence between P. carinii organisms varied in terms of the phylogenetic divergence existing among the corresponding host species. However, the relative position of P. carinii groups (rodent, carnivore or primate-derived P. carinii) could not be clearly determined. Further resolution will require the integration of additional sequence data.     

Magnetic bead capture eliminates PCR inhibitors in samples collected from the airborne environment, permitting detection of Pneumocystis carinii DNA

PCR detection methods are useful in studies of organisms not amenable to culture. Inhibitors in environmental samples can interfere with such assays. We describe a magnetic bead DNA capture protocol that removes inhibitors from outdoor air samples, maintaining the sensitivity of a 16S Pneumocystis carinii mitochondrial rRNA gene-based PCR.     

Development and Evaluation of a Direct Fluorescent Antibody Method for the Diagnosis of Pneumocystis carinii Infections in Experimental Animals

Because no fully satisfactory diagnostic method has been available for use in pneumocystis infection, an attempt was made to apply the fluorescent antibody technique in the identification of Pneumocystis carinii. Hyperimmune sera were prepared in rabbits against P. carinii from human and rat sources. After proper adsorption, these antisera were conjugated with fluorescein isothiocyanate and used as reagents in a direct fluorescent antibody procedure. Each of the two reagents was found to stain trypsin-treated P. carinii organisms from either human or rat sources, indicating the presence of common antigens. Stained organisms were demonstrated in the hypopharyngeal material from rats in which pneumocystis infection had been activated by the administration of corticosteroid. From the results reported here, the procedure outlined is considered sufficiently sensitive and specific to justify tests on pneumocystis infections in man. The findings in a series of specimens from human subjects will be reported separately. The method also provides an extended approach to related research problems. The need for controls of the procedure at all points is emphasized.     

Population structure of rat-derived Pneumocystis carinii in Danish wild rats

The rat model of Pneumocystis carinii pneumonia is frequently used to study human P. carinii infection, but there are many differences between the rat and human infections. We studied naturally acquired P. carinii in wild rats to examine the relevance of the rat model for human infection. P. carinii DNA was detected in 47 of 51 wild rats and in 10 of 12 nonimmunosuppressed laboratory rats. Evidence for three novel formae speciales of rat-derived P. carinii was found, and these were provisionally named Pneumocystis carinii f. sp. rattus-secundi, Pneumocystis carinii f. sp. rattus-tertii, and Pneumocystis carinii f. sp. rattus-quarti. Our data suggest that low-level carriage of P. carinii in wild rats and nonimmunosuppressed laboratory rats is common and that wild rats are frequently coinfected with more than one forma specialis of P. carinii. We also examined the diversity in the internally transcribed spacer (ITS) regions of the nuclear rRNA operon of Pneumocystis carinii f. sp. carinii by using samples from wild rats and laboratory rats and spore trap samples. We report a lack of variation in the ITS1 and ITS2 regions that is consistent with an evolutionary bottleneck in the P. carinii f. sp. carinii population. This study shows that human- and rat-derived P. carinii organisms are very different, not only in genetic composition but also in population structure and natural history.     

Pneumocystis carinii of the Common Shrew, Sorex araneus, Shows a Discrete Phenotype

ABSTRACT. We carried out an immunohistological and morphological study on Pneumocystis carinii originating from the common shrew, Sorex araneus . Immunologic properties were studied by applying two commercially available immunofluorescence staining kits with differing developmental form specificity to a lung homogenate. The cyst form-specific staining kit reacted with cysts originating from S. araneus . Ultrastructurally this particular antigen epitope specifically deposited on the electron-lucent middle layer of the cyst pellicle. The immunohistochemical staining kit reacting with both cyst and trophozoite forms from human and rat origin did not react with any developmental forms of P. carinii originating from S. araneus . Both kits demonstrated P. carinii in the lung homogenate of a field vole, Microtus agrestis. In morphologic examination, the methenamine silver-stained cyst forms of P. carinii from S. araneus and from M. agrestis differed in size from each other and from those originating from laboratory rats. Ultrastructurally P. carinii from S. aruneus did not differ from organisms of rat origin.     

Application of ATP measurement to evaluation of the growth of parasitic protozoa in vitro with a special reference to Pneumocystis carinii.

1. There was a significant correlation between the increase in the number of Entamoeba histolytica, Trichomonas vaginalis, Giardia lamblia and Leishmania donovani in culture, and their ATP contents determined by luciferase reaction. 2. The similar correlation was also demonstrated between the decreased number of E. histolytica in the presence of an anti-amebic quassinoid and the nucleotide content in vitro. 3. In the case of Pneumocystis carinii, the numbers of the organism remained relatively constant in culture for at least 7 days without growth; however, the ATP content dropped rapidly in 1 to 3 days except in RPMI 1640. 4. The possibility that ATP determination of P. carinii is complicated by the host cell nucleotide seemed to be excluded, since the concentration of this nucleotide in normal lung was almost negligible. These observations suggest that the present procedure is useful for evaluating the growth and viability of these organisms in vitro.