Ultrastructural Observations on Life Cycle Stages of Pneumocystis carinii

An ultrastructural study of life cycle stages of Pneumocystis carinii in infected rat lungs in situ was undertaken utilizing 8 different modes of fixation. Three of the fixatives employed gave good fixation of cysts and intracystic bodies, but for the trophic forms fixation was only fair. Both the trophic forms and intracystic bodies have nuclear pores. The mitochondria of the organism have cristac that appear lamellar. One of the fixation modes revealed a thin, electron-dense layer on the outer surface of the cell wall, a "fuzzy coat" that had not been described previously. This material appears to mediate tight adhesion of trophic forms with other trophic forms, cysts, and with pneumocytcs of the lung alveolus.     

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.     

Serologic Changes Associated with the Encystment of Axenically Grown Hartmannella culbertsoni*

SYNOPSIS. Serologic reactions elicited by sonically ruptured trophic and cystic forms of Hartmannella culbertsoni were studied. The antigens of trophic amoebae reacted with their homologous rabbit antiserum showing multiple precipitin lines which could not be seen when the reacting antigens were treated with trypsin prior to application on the Ouchterlony plates. Antigens of trophic amoeba did not react with antiserum against cysts. Cyst antigens reacted with their homologous antiserum only after trypsin treatment. Antigens prepared from trophozoites excysting from cysts reacted positively with the antiserum against antigens of trophic amoebae. Antigens of trophic as well as cystic forms fixed guinea pig complement in presence of their homologous antisera. With the trophic form, this property was abolished after trypsin treatment. Non-specific complement fixation mediated by cyst antigens was abolished by treatment with cellulase. Antiserum against trophic amoebae immobilized trophozoites and, in the presence of guinea pig complement, led to their lysis.     

Giant Cysts and Cysts with Multiple Central Bodies in Azotobacter vinelandii

Cyst germination in Azotobacter vinelandii ATCC 12837 was studied by using phase contrast and electron microscopy. Germination in this organism was accompanied by the formation of large cyst forms of two different types: giant cysts and cysts containing multiple central bodies. Previously, these two types have been reported only when yeast extract was added to the encystment medium. In this study, we observed giant cysts and cysts with multiple central bodies in nitrogen-free liquid medium. The germination of "normal" cysts is often preceded by enlargement to the giant form and division of the central body to produce cysts with multiple central bodies. Structures similar in appearance to ribosomal aggregates were observed only in cysts undergoing pregermination transformations.     

Basic biology of Pneumocystis carinii: a mini review

Basic aspects of cell biology of Pneumocystis carinii are reviewed with major emphasis on its life cycle and the structural organization of the trophozoites and cyst forms. Initially considered as a protozoan it is now established that Pneumocystis belongs to the Fungi Kingdom. Its life cycle includes two basic forms: (a) trophozoites, which are haploid cells that divide by binary fission and may conjugate with each other forming an early procyst and (b) cysts where division takes place through a meiotic process with the formation of eight nuclei followed by cytoplasmic delimitation and formation of intracystic bodies which are subsequently released and transformed into trophozoites. Basic aspects of the structure of the two developmental stages of P. carinii are reviewed.     

Quantitation of Absolute Pneumocystis carinii Nuclear DNA Content. Trophic and Cystic Forms Isolated from Infected Rat Lungs are Haploid Organisms

The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis. The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry. Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA. It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P. carinii organisms, as well as the cell wall of S. cerevisiae, bound all fluorescent dyes tested to varying degrees. Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase. Two stains that appeared most specific for DNA, DB181 and 4′,6-diamidino-2-phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181. Haploid wild type Saccharomyces cerevisiae and cdc-28 temperature-sensitive mutant cells, accumulated at the restrictive temperature (37° C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P. carinii. Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities. The P. carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different. The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively). Since these would include 2C (G-2 phase) and S-phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181-stained nuclei. Almost all nuclei analyzed in all life cycle stages fell within this population. The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A-T binding of the dye and taking into account the G+C content of S. cerevisiae and P. carinii. A 4C (G-2-phase diploid nuclei) population was not detected in histograms of DB181- or DAPI-stained nuclei. The P. carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organism's chromosomes resolved by electrophoretic techniques. Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life-cycle stages demonstrate that P. carinii carinii isolated from infected rat lungs are haploid organisms.     

Serum prolactin levels in women with gross cystic breast disease.

Serum prolactin (PRL) concentrations at baseline and after TRH stimulation were determined in 15 healthy women and in 51 premenopausal patients suffering from Gross Cystic Breast Disease. All women were in the luteal phase of the menstrual cycle and patients were divided into three groups according to cyst type at presentation. Basal hormone levels were within the normal range in the control group and in the three cystic breast disease groups. The maximum PRL response to TRH stimulation was significantly higher (p < 0.001) in patients with type I cysts (low Na+/K+ intracystic ratio and apocrine epithelium) than in patients with type II cysts (high Na+/K+ intracystic ratio and flattened epithelium), type III cysts (intermediate Na+/K+ intracystic ratio and mixed epithelium) and in normal women. Serum PRL concentrations corresponding to samples obtained 60 and 90 minutes after stimulation remained higher in the first group of patients. These results led us to consider the existence of an altered central regulation of PRL secretion in patients with type I cysts at presentation.     

Echinocandin Treatment of Pneumocystis Pneumonia in Rodent Models Depletes Cysts Leaving Trophic Burdens That Cannot Transmit the Infection

Fungi in the genus Pneumocystis cause pneumonia (PCP) in hosts with debilitated immune systems and are emerging as co-morbidity factors associated with chronic diseases such as COPD. Limited therapeutic choices and poor understanding of the life cycle are a result of the inability of these fungi to grow outside the mammalian lung. Within the alveolar lumen, Pneumocystis spp., appear to have a bi-phasic life cycle consisting of an asexual phase characterized by binary fission of trophic forms and a sexual cycle resulting in formation of cysts, but the life cycle stage that transmits the infection is not known. The cysts, but not the trophic forms, express β -1,3-D-glucan synthetase and contain abundant β -1,3-D-glucan. Here we show that therapeutic and prophylactic treatment of PCP with echinocandins, compounds which inhibit the synthesis of β -1,3-D-glucan, depleted cysts in rodent models of PCP, while sparing the trophic forms which remained in significant numbers. Survival was enhanced in the echincandin treated mice, likely due to the decreased β -1,3-D-glucan content in the lungs of treated mice and rats which coincided with reductions of cyst numbers, and dramatic remodeling of organism morphology. Strong evidence for the cyst as the agent of transmission was provided by the failure of anidulafungin-treated mice to transmit the infection. We show for the first time that withdrawal of anidulafungin treatment with continued immunosuppression permitted the repopulation of cyst forms. Treatment of PCP with an echinocandin alone will not likely result in eradication of infection and cessation of echinocandin treatment while the patient remains immunosuppressed could result in relapse. Importantly, the echinocandins provide novel and powerful chemical tools to probe the still poorly understood bi-phasic life cycle of this genus of fungal pathogens.     

Sporogony in Pneumocystis carinii: Synaptonemal Complexes and Meiotic Nuclear Divisions Observed in Precysts1

Evidence for meiosis was demonstrated electron microscopically for the first time in Pneumocystis carinii in rat alveoli by the observation of synaptonemal complexes followed by nuclear divisions. Synaptonemal complexes indicating meiotic nuclear divisions were observed in uninuclear precysts. Additionally, owing to the use of tannic acid as a fixative, spindle microtubules were also observed for the first time in the precyst. Based on these facts, a new life cycle of the organism is proposed. The precyst has generally been considered an intermediate form between the trophozoite and the cyst. The present paper proposes that the precyst is additionally defined as the cell in which eight intracystic bodies are produced through meiotic reduction. The most characteristic feature of the precyst is a clump of mitochondria in the cytoplasm. We divide the precyst phase into three forms, which are named early, intermediate, and late. Synaptonemal complexes were only observed in the early precyst, which is a uninuclear cell with a thin pellicle. In the intermediate precyst, nuclear divisions are observed as follows: meiosis I produces two haploid nuclei and each of these divides at meiosis II producing four nuclei. After that, another postmeiotic mitosis takes place, resulting in eight haploid nuclei. In the late precyst, a delimiting membrane originates from the mother plasmalemma and surrounds the daughter nuclei and a small portion of the adjacent cytoplasm. Finally, when the eight intracystic bodies are complete, the precyst changes to a cyst. Thus, we deduce that intracystic bodies resulting from meiotic nuclear division are haploid and, after excystation, they are haploid trophozoites. We consider that this process can be called sporogony. Although we could not distinguish between the haploid and the diploid trophozoite, it is quite plausible that copulation occurs, probably in host alveoli.     

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.     

Morphology and Life History of Colpoda maupasi, Bensonhurst Strain

Strains of Colpoda maupasi previously reported were found to produce only octogenic reproductive cysts and monogenic wrinkled resting cysts. The present form of C. maupasi (Bensonhurst strain), isolated by the senior author in 1949, was found to produce, in addition to the above, quadrigenic reproductive cysts, digenic corrugated (wrinkled) resting cysts, and smooth thick-walled monogenic, digenic and quadrigenic resting cysts. Some of the factors leading to the development of these cysts in the Bensonhurst strain are believed to be related to nutrition, age and size of the trophic forms. The cytological changes in encystment and excystment were followed with particular attention to aging monogenic resting cysts. The latter were observed for over 4 years.     

Study on the therapeutic effects of interferon and gamma-globulin in experimental Pneumocystis carinii pneumonia]

This study was performed to observe the therapeutic effects of interferon-gamma(IFN-gamma) and gamma-globulin(gamma-globulin) in experimental Pneumocystis carinii pneumonia of immune suppressed mice. After 9 weeks, trimethoprim-sulfamethoxazole(TMP-SMZ; 10-50 mg/mouse/day), mouse IFN-gamma(5 x 10(4) units/mouse/day) and mouse gamma-globulin(20 mg/mouse/day) were administered to the mice for 3 weeks by the experimental group. The therapeutic efficacy was evaluated by body weights, histopathologic and electron microscopic findings of the lungs, and number of P. carinii cysts by Gomori's methenamine silver stain. Body weights of the mice were significantly increased in the group of combination therapy of TMP-SMZ with IFN-gamma or gamma-globulin, and in the group of TMP-SMZ treatment (p < 0.05), however, little effect was found in the group of gamma-globulin alone. Histopathologic findings of P. carinii pneumonia were much improved in the group of combination therapy of TMP-SMZ with IFN-gamma. Treatment with either TMP-SMZ or IFN-gamma significantly reduced the number of cysts in the P. carinii pneumonia, but gamma-globulin alone was ineffective. In electron microscopic findings of P. carinii pneumonia, the number of trophozoites and cysts were reduced by treatment with either TMP-SMZ or IFN-gamma, and most of the cysts were empty or containing one or two intracystic bodies. The present results suggested, that combination therapy of TMP-SMZ with IFN-gamma had synergistic effects in treatment of P. carinii pneumonia in experimental mice.     

A novel betaproteobacterial agent of gill epitheliocystis in seawater farmed Atlantic salmon (Salmo salar)

Epitheliocystis, a disease characterised by cytoplasmic bacterial inclusions (cysts) in the gill and less commonly skin epithelial cells, has been reported in many marine and freshwater fish species and may be associated with mortality. Previously, molecular and ultrastructural analyses have exclusively associated members of the Chlamydiae with such inclusions. Here we investigated a population of farmed Atlantic salmon from the west coast of Norway displaying gill epitheliocystis. Although 'Candidatus Piscichlamydia salmonis', previously reported to be present in such cysts, was detected by PCR in most of the gill samples analysed, this bacterium was found to be a rare member of the gill microbiota, and not associated with the observed cysts as demonstrated by fluorescence in situ hybridization assays. The application of a broad range 16 S rRNA targeted PCR assay instead identified a novel betaproteobacterium as an abundant member of the gill microbiota. Fluorescence in situ hybridization demonstrated that this bacterium, tentatively classified as 'Candidatus Branchiomonas cysticola', was the cyst-forming agent in these samples. While histology and ultrastructure of 'Ca. B. cysticola' cysts revealed forms similar to the reticulate and intermediate bodies described in earlier reports from salmon in seawater, no elementary bodies typical of the chlamydial developmental cycle were observed. In conclusion, this study identified a novel agent of epitheliocystis in sea-farmed Atlantic salmon and demonstrated that these cysts can be caused by bacteria phylogenetically distinct from the Chlamydiae.     

The chrysophycean paleocyst flora of the bottom sediments of Kortowskie Lake (Poland) and its ecological significance

The postglacial sediments of Kortowskie Lake contain well preserved chrysophycean cysts. Forty-six morphotypes were identified by scanning electron microscopy (SEM) and light microscopy (LM). Ten new morphotypes of fossil chrysophycean cysts are described. Some of the most abundant taxa show clear trends associated with the increase in lake trophic status. The cyst flora is divided into four groups: eutrophic forms (Cysta globata, C. cortoviensis, C. vermicularis); oligotrophic forms (Cysta curvicollis); cool-water forms (Cysta carinifera, C. crassicollis, C. microspinosa, C. modica and Cysta stellata), and indifferent forms (all other morphotypes which did not show a clear tendency). The percentage abundance of chrysophycean cysts wa related to climate changes, showing a considerable increase during the Subboreal period. Two factors, lake fertility and water level changes affected the abundance of the fossil cyst flora in the sediments of Kortowskie Lake.     

Ploidy of cell-sorted trophic and cystic forms of Pneumocystis carinii

Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP) is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i) mating/fusion leading to a diploid status or (ii) asexual mitotic division or (iii) both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the complexity of its modes of proliferation.     

Ribonucleoprotein-Containing Vesicles in Cysts of Naegleria gruberi*

SYNOPSIS. Ultraviolet microscopy and electron microscope autoradiography were used to study ribonucleoprotein in cysts of Naegleria gruberi. The absorption maximum for cysts is at 265 nm with little detectable absorption occurring at 295 nm. Pre-cystic trophozoites absorb less strongly than the cysts at 265 mm. Acridine orange staining indicated concentrations of ribose nucleic acid or ribonucleoprotein in the cytoplasm of young cysts. The dye stained discrete vesicles in the cytoplasm. Tritiated uridine and tritiated proline were used to follow changes in RN-protein at encystment. Label was incorporated into vesicles filled with ribosome-like particles. These are presumably the sites of acridine orange staining. Relatively little label was associated with the cyst cytoplasmic matrix; most of the silver grains lay over the nucleus and cytoplasmic organelles. The vesicles are believed to represent autophagosomal-type vacuoles with the contents derived from breakdown of organelles such as mitochondria. The path of label into the vesicles is via lysis of labeled cytoplasmic organelles. The RN-protein vesicles of Naegleria gruberi cysts are compared to the chromatoid bodies of Entamoeba invadens. It is concluded that, tho differences in detail are present, the role of the structures in the cysts is probably the same. They are a ready source of amino acids and ribosomes in a dedifferentiated or pool state to be used for synthetic reactions that accompany resumption of trophic existence.