Phylogenetic status of <Emphasis Type="Italic">Pneumocystis</Emphasis> from corticosteroid-treated gerbils

Pneumocystis spp. infect the lungs of multiple mammalian species and cause disease in immunosuppressed individuals. The Pneumocystis isolates that have been studied to date fall into two major clades, those from primates and those from rodents. Within each of these clades, different species have been described on the basis of host specificity and differences in sequence and morphology. Here, we demonstrate that dexamethasone immunosuppression consistently results in histologically apparent lung infection in gerbils (28/35 animals). Sequence analysis of the 18S, 5.8S and internal transcribed spacer regions of the rDNA and a portion of the mitochondrial large subunit rDNA demonstrated that this gerbil Pneumocystis is grouped with other rodent Pneumocystis spp., but is distinct from them. Our results suggest that gerbil Pneumocystis differs sufficiently from Pneumocystis species found in other rodents to be considered a separate species.     

Independent losses and duplications of autophagy-related genes in fungal tree of life

Autophagy is important for growth, development and pathogenesis in fungi. Although autophagic process is generally considered to be conserved, the conservation and evolution of ATG genes at kingdom-wide remains to be conducted. Here we systematically identified 41 known ATG genes in 331 species and analyzed their distribution across the fungal kingdom. In general, only 20 ATG genes are highly conserved, including most but not all the yeast core-autophagy-machinery genes. Four functional protein groups involved in autophagosome formation had conserved and non-conserved components, suggesting plasticity in autophagosome formation in fungi. All or majority of the key ATG genes were lost in several fungal groups with unique lifestyles and niches, such as Microsporidia, Pneumocystis and Malassezia. Moreover, majority of ATG genes had A-to-I RNA editing during sexual reproduction in two ascomycetes and deletion of FgATG11, the ATG gene with the most editing sites in Fusarium affected ascospore releasing. Duplication and divergence also was observed to several core ATG genes, such as highly divergent ATG8 paralogs in dermatophytes and multiple ATG15 duplications in mushrooms. Taken together, independent losses and duplications of ATG genes have occurred throughout the fungal kingdom and variations in autophagy exist among different lineages and possibly different developmental stages.     

Electron microscopy of spore germination and cell wall formation in Mucor rouxii

Summary The fine structure of ungerminated and aerobically germinated sporangiospores of Mucor rouxii was compared. The germination process may be divided into two stages: I, spherical growth; II, emergence of a germ tube. In both stages, germination is growth in its strictest sense with overall increases in cell organelles; e.g., the increase in mitochondria is commensurate with the overall increase in protoplasmic mass. Noticeable changes occurring during germination are the disappearance of electron-dense lipoid bodies, formation of a large central vacuole and, most strikingly, formation of a new cell wall. Unlike many other fungi, M. rouxii does not germinate by converting the spore wall into a vegetative wall. Instead, as in other Mucorales, a vegetative wall is formed de novo under the spore wall during germination stage I. This new wall grows out, rupturing the spore wall, to become the germ tube wall. Associated with the apical wall of the germ tube is an apical corpuscle previously described. The vegetative wall exhibits a nonlayered, uniformly microfibrillar appearance in marked distinction to the spore wall which is triple-layered, with two thin electron dense outer layers, and a thick transparent inner stratum. The lack of continuity between the spore and vegetative walls is correlated with marked differences in wall chemistry previously reported. A separate new wall is also formed under the spore wall during anaerobic germination leading to yeast cell formation. On the other hand, in the development of one vegetative cell from another, such as in the formation of hyphae from yeast cells, the cell wall is structurally continuous. This continuity is correlated with a similarity in chemical composition of the cell wall reported earlier.     

Genes Encoding Antigenic Surface Glycoproteins in Pneumocystis from Humans

Pneumocystis is a eukaryotic microbe that causes pneumocystosis, an AIDS-associated pneumonia. Pneumocystosis also occurs in many other mammalian species, and animal-derived organisms have been extensively utilized in Pneumocystis research. Pneumocystis from diverse hosts contain a large glycoprotein (gpA/MSG) on the surface. Antibodies elicited against gpA/MSG of Pneumocystis from humans sometimes cross-react with epitopes on proteins of similar size from Pneumocystis from other host species. Here we report the isolation and partial sequence of two presumptive gpA/MSG genes from human-derived Pneumocystis. The cloned human-derived Pneumocystis gpA/MSG genes and predicted peptides were different from those previously isolated from Pneumocystis from rats and ferrets. The genome of human-derived Pneumocystis contained multiple copies of sequences related to the two cloned gpA/MSG genes.     

Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S‐Adenosylmethionine:Sterol C‐24 Methyltransferase

The AIDS‐associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S‐adenosylmethionine:sterol C24‐methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C‐24 position of the sterol side chain producing both C₂₈ and C₂₉ 24‐alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild‐type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy (¹H‐NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C₂₈ sterol ergosterol as the major sterol, and also resulted in low levels of C₂₉ sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ²⁴⁽²⁸⁾‐sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C₂₉ sterols as that found in P. carinii.     

The Ecology of Pneumocystis: Perspectives,Personal Recollections,and Future Research Opportunities

I am honored to receive the second Lifetime Achievement Award by International Workshops on Opportunistic Protists and to give this lecture. My research involves Pneumocystis, an opportunistic pulmonary fungus that is a major cause of pneumonia (“PcP”) in the immunocompromised host. I decided to focus on Pneumocystis ecology here because it has not attracted much interest. Pneumocystis infection is acquired by inhalation, and the cyst stage appears to be the infective form. Several fungal lung infections, such as coccidiomycosis, are not communicable, but occur by inhaling < 5 μm spores from environmental sources (buildings, parks), and can be affected by environmental factors. PcP risk factors include environmental constituents (temperature, humidity, SO₂, CO) and outdoor activities (camping). Clusters of PcP have occurred, but no environmental source has been found. Pneumocystis is communicable and outbreaks of PcP, especially in renal transplant patients, are an ongoing problem. Recent evidence suggests that most viable Pneumocystis organisms detected in the air are confined to a patient's room. Further efforts are needed to define the risk of Pneumocystis transmission in health care facilities; to develop more robust preventive measures; and to characterize the effects of climatological and air pollutant factors on Pneumocystis transmission in animal models similar to those used for respiratory viruses.     

Mycogloea nipponica - the first known teleomorph in the heterobasidiomycetous yeast genus Kurtzmanomyces

A culture was obtained from a spore print of a basidiocarp of Mycogloea nipponica collected in Taiwan. A yeast stage and basidia identical to those of M. nipponica developed in laboratory media. The Taiwanese specimen of M. nipponica and its yeast anamorph were characterised in the present study. Comparative morphological, molecular, and ultrastructural studies indicated that the yeast stage can be assigned to the genus Kurtzmanomyces. The revealed connection between the sexual species Mycogloea nipponica and the asexual genus Kurtzmanomyces demonstrates the importance of anamorphic characteristics in the modern systematics of heterobasidiomycetous fungi. This revised version was published online in August 2006 with corrections to the Cover Date.     

The ultrastructure of the islets of Langerhans in normal and obese-hyperglycemic mice

Summary The islets of Langerhans in normal and obese-hyperglycemic mice were studied by electron microscopy. Two different types of islet cells with ultrastructural features well distinguished from the majority of B cells were observed. While one of them was provided with complete cell membranes, the other appeared to be arranged in a syncytium. It was postulated that these islet cells may be identified with the argyrophil A₁ cells and the non argyrophil A₂ cells demonstrated in different species with light microscopy. In the normal mouse the fine structure of the islet B cells corresponds in the main to what has been observed previously in the rat. However, in addition to the great majority of spherical B cell granules some appear rectangular, i.e. show similar morphological characteristics as described for the granules in the dog. The long termada ptation of the B cells to increased functional demands in the obese-hyperglycemic syndrome was associated with a pronounced degranulation with margination of the granules and with obvious changes of some organelles. The mechanisms for formation and liberation of insulin from the B cells are discussed in the light of the ultrastructural appearance of these cells in the obese-hyperglycemic mice.Supported by the Swedish Medical Research Council and the research grant A-5759 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.     

What Do Pneumocystis Organisms Tell Us about the Phylogeography of Their Hosts? The Case of the Woodmouse Apodemus sylvaticus in Continental Europe and Western Mediterranean Islands

Pneumocystis fungi represent a highly diversified biological group with numerous species, which display a strong host-specificity suggesting a long co-speciation process. In the present study, the presence and genetic diversity of Pneumocystis organisms was investigated in 203 lung samples from woodmice (Apodemus sylvaticus) collected on western continental Europe and Mediterranean islands. The presence of Pneumocystis DNA was assessed by nested PCR at both large and small mitochondrial subunit (mtLSU and mtSSU) rRNA loci. Direct sequencing of nested PCR products demonstrated a very high variability among woodmouse-derived Pneumocystis organisms with a total number of 30 distinct combined mtLSU and mtSSU sequence types. However, the genetic divergence among these sequence types was very low (up to 3.87%) and the presence of several Pneumocystis species within Apodemus sylvaticus was considered unlikely. The analysis of the genetic structure of woodmouse-derived Pneumocystis revealed two distinct groups. The first one comprised Pneumocystis from woodmice collected in continental Spain, France and Balearic islands. The second one included Pneumocystis from woodmice collected in continental Italy, Corsica and Sicily. These two genetic groups were in accordance with the two lineages currently described within the host species Apodemus sylvaticus. Pneumocystis organisms are emerging as powerful tools for phylogeographic studies in mammals.     

Observations Biologiques et Ultrastructurales sur les Selenidiidae et Leurs Conséquences sur la Systématique des Grégarinomorphes

SYNOPSIS. The taxonomic position of Selenidiidae Brasil in the class of gregarines is discussed in relation to the study of its life cycles, its possible schizogony and the fine structure of its trophozoites. The cycle of gregarines which belong to the genus Selenidium Giard is characterized by trophozoites with pendular or coiling movements, nuclear transformations in gamonts during syzygy just before cyst formation, anisogamy, sporocysts with 4 sporozoites. Schizogony of the Selenidiidae is not yet demonstrated. The “kystes á meérozoites” within the gut epithelium of Sabellaria alveolata, could be one of the stages of schizogony of S. hollandei. This sole example in our study and the absence of schizogony in numerous species, especially in S. pendula, the type species, shows that this criterion is uncertain, actually, in the definition of the order Archigregarinida Grassé. Ultrastructural studies of S. hollandei and S. pendula show that the cortical region in trophozoites of the genus Selenidium is different from that of Eugregarinida. In the Selenidiidae the epicyte is composed of longitudinal folds. Under the wall, consisting of 3 membranes there is a well-defined pellicular fibrillar system. In S. hollandei, the trophozoite has a fibrillar formation, corresponding to a conoid in its anterior region. The trophozoites of S. hollandei and S. pendula contain anterior dense bodies or rhoptries which are very well developed. All these characteristics conform to the ultrastructural organization of the dissemination forms (merozoites, schizozoites, sporozoites): The results allow one to give a new definition of the Order Archigregarinida: Order Archigregarinida (Grassé): Gregarines with ultrastructural organization of the trophozoites similar to that of dissemination forms. Presence of a well defined pellicular fibrillar system. Intestinal parasites of polychaete worms.     

Germination and parasitation of the resting sporangia ofSynchytrium endobioticum

Summary The cytoplasmic organization of the long-lived, thick walled resting stage of the sporangium ofSynchytrium endobioticum (Schilb.) Perc. is described. The cytoplasm of the resting sporangium contains a large number of closely packed lipid bodies and irregular electron dense bodies, which are interspaced with fine channels of cytoplasm. These ultrastructural observations are discussed in relation to the hypothesis ofBally (1912) andCurtis (1921) that zoospore primordia are already present during the resting stage. It is shown that the zoospore primordium is actually a lipid body and an osmiophilic body and the strands postulated to connect the individual zoospore primordia are actually the fine channels of cytoplasm.A new inner wall layer is laid down prior to the start of the germination. It is this wall layer which will protrude to form the vesicle in which sporogenesis takes place. The germination process observed, protrusion of a vesicle through a crack in the sporangial wall, the migration of the sporangial content into the vesicle, and the formation of a single, membrane-bound sporangium within this vesicle, is in full agreement with the recent light microscopic studies ofSharma andCammack (1976). These observations support the transfer ofS. endobioticum from the subgenusMesochytrium to the subgenusMicrosynchytrium (bothsensu Karling 1964).A major objective of the study, to obtain ultrastructural evidence for the location of the meiotic divisions in the life cycle, was not fulfilled.Three different fungi were observed to parasitize the resting sporangium ofS. endobioticum. These infections are discussed in relation to other mycoparasites of plant pathogenic fungi. The possibility of using a mycoparasite for the biological control of potato wart disease is considered to be without practical relevance.     

AN ULTRASTRUCTURAL STUDY OF CELL DIVISION IN THE CAMBIUM

The fine structure of dividing cambial cells of Ulmus americana and Tilia americana has been studied in material fixed in glutaraldehyde followed by osmium tetroxide. The cambia examined consisted of 7–9 rows of unexpanded fusiform cells, all of which had similar ultrastructural components. The fine structure and sequence of events of mitosis and cytokinesis in the dividing cambial cells apparently are similar to those of dividing cells in root tips and leaves. Of special interest was the observation that during cytokinesis, a broad cytoplasmic plate or phragmosome precedes the developing phragmoplast and cell plate through the dividing cambial cell. Smooth and coated vesicles derived from dictyosomes are associated with cell plate formation in these cells, smooth vesicles primarily with earlier stages of plate formation, and coated vesicles in later stages.     

Molecular Phylogeny and Ultrastructure of Aphelidium desmodesmi,a New Species in Aphelida (Opisthosporidia)

Aphelids are a diverse group of intracellular parasitoids of algae and diatoms, and are sister to true fungi. Included in four genera, the 14 described species utilize phagocytosis as their mode of nutrition, and the life cycles of these taxa are remarkably similar. However, their putative specificity of host, morphological and ultrastructural features, and genetic divergence have been considered in taxon delineation. Here, we examine the host specificity, morphology, ultrastructure, and molecular 18S gene sequence of a new species in Aphelida, Aphelidium desmodesmi sp. nov. This taxon is in a well‐supported clade with two other species of Aphelidium, and this lineage is sister to Amoeboaphelidium and Paraphelidium. Of interest, the mitochondrial structure of Aph. desmodesmi is more like that of Paraphelidium than that of Aphelidium aff. melosirae, the only other species of Aphelidium to have been examined ultrastructurally. This research examines and expands our understanding of host range, morphological diversity, and genetic divergence of the aphelids.     

MUC1 mediates Pneumocystis murina binding to airway epithelial cells

Previous studies have shown that Pneumocystis binds to pneumocytes, but the proteins responsible for binding have not been well defined. Mucins are the major glycoproteins present in mucus, which serves as the first line of defence during airway infection. MUC1 is the best characterised membrane‐tethered mucin and is expressed on the surface of most airway epithelial cells. Although by electron microscopy Pneumocystis primarily binds to type I pneumocytes, it can also bind to type II pneumocytes. We hypothesized that Pneumocystis organisms can bind to MUC1 expressed by type II pneumocytes. Overexpression of MUC1 in human embryonic kidney HEK293 cells increased Pneumocystis binding, while knockdown of MUC1 expression by siRNA in A549 cells, a human adenocarcinoma‐derived alveolar type II epithelial cell line, decreased Pneumocystis binding. Immunofluorescence labelling indicated that MUC1 and Pneumocystis were co‐localised in infected mouse lung tissue. Incubation of A549 cells with Pneumocystis led to phosphorylation of ERK1/2 that increased with knockdown of MUC1 expression by siRNA. Pneumocystis caused increased IL‐6 and IL‐8 secretion by A549 cells, and knockdown of MUC1 further increased their secretion in A549 cells. Taken together, these results suggest that binding of Pneumocystis to MUC1 expressed by airway epithelial cells may facilitate establishment of productive infection.     

Properties of the bubble protein, a defensin and an abundant component of a fungal exudate

Colonies of the ascomycete fungus Penicillium brevicompactum Dierckx produce bright yellow-green fluorescent exudate bubbles on its surface when grown on standard plant cell culture medium. According to SDS-PAGE analysis, the exudate is enriched in one protein, named bubble protein (BP). Detailed characteristics of BP are described, and also its corresponding genomic promoter and terminator sequences that flank sequences encoding signal peptide and a precursor sequence upstream of that of the mature protein. Following on previous work, the protein is now biochemically characterized. BP, the structure of which mainly consists of beta sheets, has four very stable disulfide bridges that resist standard procedures for reduction. With such traits, BP can now be categorized as a new member of the ever growing class of defensins. Indeed, the protein revealed anti-fungal effects as it inhibits growth of the yeast Saccharomyces cerevisiae in a dose-dependent manner. Structural classification places BP into the group of proteins with a knottin fold, founding the BP superfamily. Based on genomic alignments that revealed very high homology to four proteins of related fungi, a 3D structure prediction of the corresponding proteins was made. In addition, it was discovered that the closely related fungus Penicillium chrysogenum encodes a BP homolog – in addition to its PAF protein, which also is similar to BP – further suggesting that fungi may possess more than one defensin.     

ULTRASTRUCTURAL INVESTIGATIONS ON THE TWO-NUCLEATE POLLEN GRAIN OF TILLANDSIA CAPUT-MEDUSAE MORR. (BROMELIACEAE)

The fine structure of the pollen grain of Tillandsia caput-medusae Morr. (Bromeliaceae) prior to germination has been studied. The development, after the first mitosis, is here schematized in three stages which are in accordance with the main steps described in angiosperms. The ultrastructural modifications occurring in the generative and vegetative cells are discussed in view of their different destiny. The results obtained are compared with the data known about tropical orchids and epiphyteic plants like Tillandsia. The following differences have been observed: a large vacuole in the vegetative cell; rapid thinning of the wall between the generative and vegetative cells; great quantity of ribosomes and rough endoplasmic reticulum surrounding the vacuoles in the generative cell. The above-listed ultrastructural features may have a meaning, considering the peculiar environmental conditions in which the epiphytism of Tillandsia is achieved.     

Ultrastructure of Cocconeis diminuta pantocsek

Summary The ultrastructural morphology of Cocconeis diminuta, a small benthic diatom, is described. It has been found to possess a typical naviculoid organisation, and does not differ significantly from other species examined with the electron microscope. The pseudoraphe, frequently regarded as an important taxonomic criterion, has been found to be a variable feature in C. diminuta and its significance in this regard appears doubtful. Special reference is made to the substructure of the pyrenoid, since it appears to possess a crystal lattice that is similar in structure to the one described in the dinoflagellate Prorocentrum micans.Contribution no. 1445 from the Rosenstiel School of Marine and Atmospheric Science.     

Elastoidin actinotrichia in coelacanth fins: A comparison with teleosts

In this work, we present the first ultrastructural evidences of actinotrichia in the Coelacanth Latimeria. We describe its actinotrichia with the electron microscope (SEM and TEM) and compare their structure to Teleost actinotrichia. Both elements present similar fine structure, i.e. a periodic cross-striation of 60–65 nm; the plesiomorphic character of actinotrichia is discussed in Osteichthyes.     

Fine Structure of Differentiating Oocysts and Mature Sporozoites of Haemoproteus metchnikovi in its Intermediate Host Chrysops callidus*

The fine structure of the sporogonic stages of Haemoproteus metchnikovi has been investigated by electron microscopy. Young oocysts are found beneath the basement membrane of midgut epithelial cells. These eventually protrude outward into the haemocoel space and are surrounded by a distinct oocyst capsule. Sporozoite formation begins with a subcapsular vacuolation. Evagination of the oocyst cytoplasm occurs in regions of membrane thickenings and 100–200 sporozoites are formed about a single sporoblastoid body. Remnants of the ookinete pellicle can be observed in maturing oocysts and always are found in the residual body. The fine structure of the mature sporozoite is essentially similar to that which has been described for other haemosporidia and a spherical body is described in association with the mitochondrion of the sporozoite. The sporogonic stages of H. metchnikovi have features common to the sporogonic stages of Plasmodium and Leucocytozoon that are not held in common by the latter 2 genera, including pattern of sporozoite formation and number of sporozoites formed, the presence of a cytostome and of “crystalloid” in the sporozoite.