The Pneumocystis life cycle.

First recognised as 'schizonts' of Trypanosoma cruzi, Pneumocystis organisms are now considered as part of an early-diverging lineage of Ascomycetes. As no robust long-term culture model is available, most data on the Pneumocystis cell cycle have stemmed from ultrastructural images of infected mammalian lungs. Although most fungi developing in animals do not complete a sexual cycle in vivo, Pneumocystis species constitute one of a few exceptions. Recently, the molecular identification of several key players in the fungal mating pathway has provided further evidence for the existence of conjugation and meiosis in Pneumocystisorganisms. Dynamic follow-up of stage-to-stage transition as well as studies of stage-specific proteins and/or genes would provide a better understanding of the still hypothetical Pneumocystislife cycle. Although difficult to achieve, stage purification seems a reasonable way forward in the absence of efficient culture systems. This mini-review provides a comprehensive overview of the historical milestones leading to the current knowledge available on the Pneumocystis life cycle.

The Pneumocystis life cycle

First recognised as "schizonts" of Trypanosoma cruzi, Pneumocystis organisms are now considered as part of an early-diverging lineage of Ascomycetes. As no robust long-term culture model is available, most data on the Pneumocystis cell cycle have stemmed from ultrastructural images of infected mammalian lungs. Although most fungi developing in animals do not complete a sexual cycle in vivo, Pneumocystis species constitute one of a few exceptions. Recently, the molecular identification of several key players in the fungal mating pathway has provided further evidence for the existence of conjugation and meiosis in Pneumocystisorganisms. Dynamic follow-up of stage-to-stage transition as well as studies of stage-specific proteins and/or genes would provide a better understanding of the still hypothetical Pneumocystislife cycle. Although difficult to achieve, stage purification seems a reasonable way forward in the absence of efficient culture systems. This mini-review provides a comprehensive overview of the historical milestones leading to the current knowledge available on the Pneumocystis life cycle.

Pneumocystis oryctolagi sp. nov., an uncultured fungus causing pneumonia in rabbits at weaning: review of current knowledge, and description of a new taxon on genotypic, phylogenetic and phenotypic bases.

The genus Pneumocystis comprises noncultivable, highly diversified fungal pathogens dwelling in the lungs of mammals. The genus includes numerous host-species-specific species that are able to induce severe pneumonitis, especially in severely immunocompromised hosts. Pneumocystis organisms attach specifically to type-1 epithelial alveolar cells, showing a high level of subtle and efficient adaptation to the alveolar microenvironment. Pneumocystis species show little difference at the light microscopy level but DNA sequences of Pneumocystis from humans, other primates, rodents, rabbits, insectivores and other mammals present a host-species-related marked divergence. Consistently, selective infectivity could be proven by cross-infection experiments. Furthermore, phylogeny among primate Pneumocystis species was correlated with the phylogeny of their hosts. This observation suggested that cophylogeny could explain both the current distribution of pathogens in their hosts and the speciation. Thus, molecular, ultrastructural and biological differences among organisms from different mammals strengthen the view of multiple species existing within the genus Pneumocystis. The following species were subsequently described: Pneumocystis jirovecii in humans, Pneumocystis carinii and Pneumocystis wakefieldiae in rats, and Pneumocystis murina in mice. The present work focuses on Pneumocystis oryctolagi sp. nov. from Old-World rabbits. This new species has been described on the basis of both biological and phylogenetic species concepts.

Diagnosis of pulmonary pneumocystosis by microscopy on wet mount preparations.

We have compared the searching of the presence of "honeycomb" structures by direct microscopy on wet mount preparations with the direct immunofluorescence (DIF) for the diagnosis of Pneumocystis carinii pneumonia (PCP) in 115 bronchoalveolar (BAL) fluids. The samples belonged to 115 AIDS patients; 87 with presumptive diagnosis of PCP and 28 with presumptive diagnosis other than PCP. The obtained results were coincident in 114 out of 115 studied samples (27 were positive and 87 negative) with both techniques. A higher percentage of positive results (32.18%) among patients with presumptive diagnosis of PCP with respect to those with presumptive diagnosis other than PCP (3.57%) was observed. One BAL fluid was positive only with DIF, showed scarce and isolated P. carinii elements and absence of typical "honeycomb" structures. The searching for "honeycomb" structures by direct microscopy on wet mount preparations could be considered as a cheap and rapid alternative for diagnosis of PCP when other techniques are not available or as screening test for DIF. This method showed a sensitivity close to DIF when it was applied to BAL fluids of AIDS patients with poor clinical condition and it was performed by an experienced microscopist.

Electron microscopy as a tool for identifying new pathogens.

Electron microscopy (EM) is still an important tool for the investigation of infectious diseases, despite the introduction of powerful new methods, mainly involving the polymerase chain reaction. Particularly in the field of parasitic protozoology associated with AIDS, where many new species of human pathogens have been recognized in tissue biopsies, EM remains an essential 'catch-all' diagnostic method. The resolved ultrastructural details of these newly recognized parasites allows a unique insight into the biology of these organisms. The information produced by EM is different, but complementary, to that provided by alternative methods.

Ultrastructural and molecular characterization of Pneumocystis carinii isolated from a rhesus monkey (Macaca mulatta).

High levels of heterogeneity have been observed among isolates of Pneumocystis carinii derived from different mammalian host species. We report the characterization of P. carinii isolated from a rhesus monkey (Macaca mulatta), which was immunosuppressed as a result of infection with a chimeric simian-human immunodeficiency virus (SHIVsbg). Histopathological examination showed evidence of severe P. carinii pneumonia with a large predominance of trophozoite forms. Alveolitis consisted of typical foamy, honeycomb exudate, with only a few alveolar macrophages. The lung inflammatory response was rather moderate without type-2 pneumocyte hyperplasia or collagenosis. P. carinii organisms were sometimes observed in the bronchiolar lumen. Ultrastructurally, macaque-derived P. carinii was more similar to human- or rabbit-derived parasites than to mouse-derived P. carinii. Molecular studies were carried out on the macaque-derived P. carinii DNA at two genetic loci: the genes encoding the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) and the mitochondrial small subunit ribosomal RNA (mt SSU rRNA). Comparison of the DNA sequences with those from P. carinii isolated from eight other host species demonstrated that the macaque-derived P. carinii was genetically distinct at both loci, and was more closely related to human-derived P. carinii than to P. carinii derived from non-primate sources. We propose that macaque-derived P. carinii be named Pneumocystis carinii f.sp. macacae.

Pneumocystis carinii pneumonia in a Yorkshire terrier dog.

A 14-month-old male Yorkshire terrier was presented to the Autonomous University of Barcelona Veterinary Teaching Hospital because of a history of chronic non-productive cough and acute dyspnea. A follow-up radiograph revealed a diffuse, bilaterally interstitial-alveolar lung disease with presence of air bronchograms. The dog died 5 h after admission with severe dyspnea. Histological sections of the necropsy specimens revealed the presence of characteristic Pneumocystis carinii cysts within alveolar spaces. A diagnosis of P. carinii pneumonia (PCP) was made on the basis of these results. To our knowledge, PCP has not been described in a Yorkshire terrier dog.

Continuous axenic cultivation of Pneumocystis carinii.

Continuous axenic culture of Pneumocystis carinii has been achieved. A culture vessel is used that allows for frequent medium exchange without disturbance of organisms that grow attached to a collagen-coated porous membrane. The growth medium is based on Minimal Essential Medium with Earle's salt supplemented with S-adenosyl-L-methionine, putrescine, ferric pyrophosphate, N-acetyl glucosamine, putrescine, p-aminobenzoic acid, L-cysteine and L-glutamine, and horse serum. Incubation is in room air at 31 degrees C. The pH of the medium begins at 8.8 and rises to approximately 9 as the cells grow. Doubling times calculated from growth curves obtained from cultures inoculated at moderate densities ranged from 35 to 65 hours. With a low-density inoculum, the doubling time is reduced to 19 hours. The morphology of cultured organisms in stained smears and in transmission electron micrographs is that of P. carinii, and P. carinii-specific mAbs label the cultured material. Cultured organisms are infective for immunosuppressed rats and can be stored frozen and used to reinitiate culture.

Lung parasites of least weasels in Finland.

Because of their constant exposure to normal rodent definitive hosts, least weasels (Mustela nivalis) were trapped in southern Finland in late fall 1994 and examined for lung parasites. Histological examination showed that 46% of the weasels (n = 46) were infected with adiaspores identified as Chrysosporium sp. Granulomas surrounding the adiaspores consisted of mostly unorganized layers of mononuclear cells. The adiaspores from least weasels were much smaller than those reported from their prey animals. Infection with Pneumocystis carinii also was found in two weasels.

Morphological and ultrastructural methods for Pneumocystis.

Pneumocystis is a eukaryotic unicellular microorganism with marked fungal affinities. All known life cycle stages of this parasite were observed in the lung of mammals. The cystic forms of this microorganism may be observed microscopically by using stains with affinity for the components of their relatively thick cell wall. However, about 100 years ago they were observed for the first time thanks to panoptic stains which do not stain their cell wall. Methanol-Giemsa technique as well as Giemsa-like rapid stainings are often used to reveal vegetative or cystic forms of this parasite on air dried smears of clinical or experimental samples. For many years, hypotheses on its life cycle, which remains unknown, were based on transmission electron microscopy (TEM) studies. However, only for the last years progresses in the quality of fixation for TEM led to a better understanding of the Pneumocystis cell structure. In this chapter, strategies to reveal Pneumocystis organisms in clinical or experimental specimens by using light microscopy, as well as techniques allowing a good preparation of parasitic samples for TEM, are given and shortly discussed.

Different ultrastructural morphology of Pneumocystis carinii derived from mice, rats, and rabbits.

Pneumocystis carinii (PC) is a fungus present in the lungs of many mammal species. Even though studies of the genome, the isoenzymes, and the antigens have proved some host-species-linked heterogeneity, the existence of distinct Pneumocystis species or subspecies has still not been accepted. Comparative studies of the ultrastructural morphology of pneumocysts derived from several host species may support evidence of host-species-linked heterogeneity. We have compared the ultrastructural morphology of pneumocysts derived from mice, rats, and rabbits. The density of membrane-limited electron-dense cytoplasmic granules was found to be higher in mouse-derived pneumocysts than in rabbit-derived pneumocysts, and furthermore the average diameter of the granules from mouse pneumocysts was larger than that of granules from rabbit-derived pneumocysts. The average diameter of the filopodia of mouse-derived pneumocysts was smaller than that of filopodia from rat-derived pneumocysts, which was smaller than that of filopodia from rabbit-derived pneumocysts. Globular electron-dense bulbous dilatations at the tip of the filopodia were described for the first time and they were only found on filopodia of mouse-derived pneumocysts. These distinct host-species-linked morphological differences of pneumocysts from mouse, rat, and rabbit may support previous biochemical data indicating the existence of different Pneumocystis species or subspecies.

Detection of parasites with DNA-binding bisbenzimide H33258 in Pneumocystis carinii- and Leishmania-containing materials.

Even for routine purposes, standard staining of Pneumocystis- or Leishmania-containing materials, e.g., with Giemsa or Diff-Quik, is often unsatisfactory due to poor contrast and to staining of irrelevant structures. In comparison, the bisbenzimide dye Hoechst 33258, a DNA-binding fluorochrome, allows a more precise analysis of such materials. Bisbenzimide stained all stages of these fungal or protozoal organisms with brilliant contrast against a uniformly dark background. The level of background luminescence and staining of detritus or non-DNA structures was very low. Organisms were stained both outside of and within phagocytic cells with equal intensity. Counting of individual microorganisms, e.g., in macrophages heavily parasitized with Leishmania or in Pneumocystis-infected bronchoalveolar lavage, was simplified and more precise. Air-dried cell suspensions, cytocentrifuge preparations, impression smears, or cryocut micrographs showed the advantages of bisbenzimide staining over Diff-Quik. Staining with bisbenzimide could be a valuable auxiliary technique for the analysis of material infected with a variety of microorganisms.

Recognition of Pneumocystis carinii antigen on its surface by immunohistochemistry and immunoelectron microscopy.

The aim of this study was to detect the surface antigens in different stages of experimental induced Pneumocystis carinii in Sprague-Dawley rats. Immunohistochemical staining with monoclonal (900, 902 and 904) and polyclonal (SP-D) antibodies demonstrated that the P. carinii organisms were mostly in the alveolar lumina. The binding sites of the monoclonal (900, 902 and 904) and polyclonal (SP-D) antibodies developed against P. carinii were examined at the ultrastructural level by using a post-embedding immunogold labeling. The gold particles were observed evenly on the surface of precyst and cyst stages of the P. carinii. In the trophozoite stage, scattered gold particles were seen on the pellicles and tubular expansions. The monoclonal antibodies reacted mainly with pellicles of P. carinii, whereas SP-D labeled pellicles, intracystic bodies, cytoplasms of alveolar macrophages, free floating surfactant material in the alveolar spaces, and adjacent type II epithelial cells. In the immunogold labeling, basically no significant differences were found in the precyst, cyst, and ruptured cyst stages. These results indicate that the gold particles were observed adhering to every stage of P. carinii, mostly concentrated on the pellicles, and more concentrated in the precyst or cyst stage than trophozoite stage which may be due to an increase in antigen accumulation during development from the trophozoite to the cyst.

Pneumocystis carinii interactions with respiratory epithelium.

Ultrastructural studies indicate that the attachment of Pneumocystis carinii to alveolar epithelial cells is a central feature during development of this pneumonia. P. carinii attaches preferentially to type I alveolar epithelial cells, and rarely binds to type II cells. Extensive in vitro investigations support roles for both fibronectin and vitronectin, which bind to the organism, and mediate P. carinii attachment to respiratory epithelial cells through corresponding integrin receptors. Additional studies indicate that P. carinii cytoskeletal rearrangement occurs after initial binding of the organism to lung cells. Further, the attachment of P. carinii to host epithelial cells is believed to induce ultrastructural changes in these cells and to result in enhanced epithelial cell permeability. Further studies will be required to determine the mechanisms and consequences of P. carinii attachment to host epithelial cells in the intact host.

[An ultrastructural evaluation of Pneumocystis carinii]. / Evaluación ultraestructural del Pneumocystis carinii.

The pneumocystis carinii (PNC) is a challenge, because its taxonomy has not been completely explained. PNC seems to be a chimera with fungal and parasitic characteristics. The objective of the present study is to analyze the different stages observed in PNC using transmission electron microscopy in order to correlate them with bibliographic reports regarding a possible taxonomic classification. The trophic stage was the form most frequently encountered in alveolar spaces and their organisms varying in size and shape from 6 to 20 microns. Cyst were round or oval, with diameters ranging between 4 and 12 microns, showing four intracystic bodies were individually limited by a membrane. Connections between the membrane of intracystic bodies and junctions between them and the inner cell membrane of the cyst wall were also observed. Our ultrastructural findings appear to be in agreement with several reports which considered PNC among the fungi, most likely related them to the non-gemating Ascomycetes.

Pneumocystis carinii of the common shrew, Sorex araneus, shows a discrete phenotype.

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. araneus did not differ from organisms of rat origin.

Light and electron microscopy study of carbohydrate antigens found in the electron-lucent layer of Pneumocystis carinii cysts.

The localization and biochemical nature of antigens found in the electron-lucent layer (ELL) of Pneumocystis carinii cysts using polyclonal rabbit antibodies are described. These antigens, specific for the cystic stages of the parasite, were shared by organisms from different hosts, suggesting that they represent functionally important components of the cyst cell wall. The binding sites were situated on an interwoven net of fibrils in the ELL produced by mild to strong proteolysis. Degradation of this residue by glucanase and chitinase confirms that this layer contains branched glucan and chitin. In contrast, the prompt susceptibility of the polysaccharide-rich ELL to proteolysis reveals that proteins are also relevant in building up the cyst-wall glucan skeleton. It is therefore concluded that the formation of the Pneumocystis cyst wall shows differences to the typical fungal cell-wall architecture. The taxonomical debate regarding this unique protist is ongoing, and consideration of these immunological and morphological findings may be useful for the study of the biology and phylogeny of Pneumocystis.