Analysis of Pneumocystis carinii Cyst Wall I. Evidence for an Outer Surface Membrane

ABSTRACT. It has long been thought that the cyst form of Pneumocystis carinii , which can resist host defenses and antimicrobial drugs, is responsible for relapses of P. carinii pneumonia. The thick wall of the cyst is immunogenic and rich in glucosyl/mannosyl and N-acetyl-D-glucosamine residues. In this study we have demonstrated the presence of a hitherto unreported outer membrane in the cyst wall of P. carinii . This membrane was detected by a combination of techniques, including transmission electron microscopy, freeze-fracture electron microscopy, and membrane labeling with fluorescent lipid analogs following treatment of P. carinii cysts from infected rats for 30 min with Zymolyase, a β-1–3 glucanase. As in gram-negative bacteria and blue-green algae, this 2nd membrane may have an important role in osmoregulation and nutrient utilization; it may also mediate the interaction of P. carinii with its host and serve as a target for drug therapy.     

Analysis of Pneumocystis carinii Cyst Wall. II. Sugar Composition

ABSTRACT. Pneumocystis carinii cysts are capable of resisting host defenses and antimicrobial drugs and are therefore thought to be responsible for relapses of P. carinii pneumonia in AIDS and other immunocompromised patients. The interaction of P. carinii with its host, and other P. carinii , might be mediated by molecules which form the outer surfaces of this organism. Carbohydrates are known to play many roles in cell-cell adhesion, and have been detected on the surface of P. carinii by lectin labeling experiments. In this study P. carinii cyst wall material was obtained from Zymolyase treatment. Alditol acetate derivatives of neutral and amino sugars or trimethylsilyl derivatives of methyl glycosides were prepared from the monosaccharides released from the sample by acid hydrolysis. Analyses were done by a combination of gas chromatography and mass spectrometry. Glucose was found to be the major sugar constituent. Mannose and galactose were present in equal ratios. A lesser amount of N-acetyl-D-glucosamine, and trace amounts of ribose and sialic acid were present in the cyst wall samples analyzed. These sugars may mediate P. carinii -host interaction and play an important protective role by creating a permeability barrier around the cyst.     

Analysis of Pneumocystis carinii cyst wall. II. Sugar composition

Pneumocystis carinii cysts are capable of resisting host defenses and antimicrobial drugs and are therefore thought to be responsible for relapses of P. carinii pneumonia in AIDS and other immunocompromised patients. The interaction of P. carinii with its host, and other P. carinii, might be mediated by molecules which form the outer surfaces of this organism. Carbohydrates are known to play many roles in cell-cell adhesion, and have been detected on the surface of P. carinii by lectin labeling experiments. In this study P. carinii cyst wall material was obtained from Zymolyase treatment. Alditol acetate derivatives of neutral and amino sugars or trimethylsilyl derivatives of methyl glycosides were prepared from the monosaccharides released from the sample by acid hydrolysis. Analyses were done by a combination of gas chromatography and mass spectrometry. Glucose was found to be the major sugar constituent. Mannose and galactose were present in equal ratios. A lesser amount of N-acetyl-D-glucosamine, and trace amounts of ribose and sialic acid were present in the cyst wall samples analyzed. These sugars may mediate P. carinii-host interaction and play an important protective role by creating a permeability barrier around the cyst.     

Cell wall assembly by Pneumocystis carinii. Evidence for a unique gsc-1 subunit mediating beta -1,3-glucan deposition

Pneumocystis carinii remains a persistent cause of severe pneumonia in immune compromised patients. Recent studies indicate that P. carinii is a fungal species possessing a glucan-rich cyst wall. Pneumocandin antagonists of beta-1,3-glucan synthesis rapidly suppress infection in animal models of P. carinii pneumonia. We, therefore, sought to define the molecular mechanisms of beta-glucan cell wall assembly by P. carinii. Membrane extracts derived from freshly purified P. carinii incorporate uridine 5'-diphosphoglucose into insoluble carbohydrate, in a manner that was completely inhibited by the pneumocandin L733-560, an antagonist of Gsc-1-type beta-glucan synthetases. Using degenerative polymerase chain reaction and library screening, the P. carinii Gsc-1 catalytic subunit of beta-1,3-glucan synthetase was cloned and characterized. P. carinii gsc1 exhibited homology to phylogenetically related fungal beta-1,3-glucan synthetases, encoding a predicted 214-kDa integral membrane protein with 12 transmembrane domain structure. Immunoprecipitation of P. carinii extracts, with a synthetic peptide anti-Gsc-1 antibody, specifically yielded a protein of 219.4 kDa, which was also capable of incorporating 5'-diphosphoglucose into insoluble glucan carbohydrate. As opposed to other fungi, the expression of gsc-1 mRNA is uniquely regulated over P. carinii's life cycle, having minimal expression in trophic forms, but substantial expression in the thick-walled cystic form of the organism. These results indicate that P. carinii contains a unique catalytic subunit of beta-1,3-glucan synthetase utilized in cyst wall formation. Because synthesis of beta-1,3-glucan is absent in mammalian cells, inhibition of the P. carinii Gsc-1 represents an attractive molecular target for therapeutic exploitation.     

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.     

Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracellular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4-6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (-15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.     

Ultrastructure of Frenkelia sp. from a Norwegian lemming in Finland

An apparently healthy Norwegian lemming (Lemmus lemmus) caught in northern Finland was observed to have a whitish body 0.5 to 1.0 mm in diameter in the external layer of the cerebral cortex. By light microscopy a highly lobulated cyst of Frenkelia sp. was observed. By transmission electron microscopy lemmus) collected in the cyst wall was seen consisting of a parasitophorous vacuolar membrane, an underlying electron-dense layer and a granular layer. The membrane was only slightly convoluted. The protrusions of the cyst wall appeared round but were often not distinctive. A very thin septum divided the interior of the cyst into compartments packed with bradyzoites and maturing zoites. The bradyzoites were elongate measuring 5-8 x 1.5-2 microm. This is the first electron microscopical study of Frenkelia sp. from L. lemmus.     

Yeast glucan in the cyst wall of Pneumocystis carinii

Ultrastructurally, the cyst wall of Pneumocystis carinii consists of an electron-dense outer layer, an electron-lucent middle layer, and an innermost plasmalemma. This is similar in appearance to the cell wall of some yeasts, e.g. Saccharomyces cerevisiae, which consists of an outer dense layer of mannan, a middle lucent layer of beta-1,3-glucan (yeast glucan) and an innermost plasmalemma. The cyst wall of P. carinii, as well as the cell wall of S. cerevisiae, can be labeled by a variety of methods which stain polysaccharides, such as Gomori's methenamine silver (GMS) and by Aniline blue, a dye which selectively stains beta-1,3-glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is beta-1,3-glucan laminaripentaohydrolase, results in lysis of the outer 2 layers of the cyst wall and the loss of positive staining by both GMS and Aniline blue. The lysis of elements of the cyst wall of P. carinii is achieved under the same conditions and concentration at which Zymolyase lyses the outer 2 layers of the cell wall of viable cells of S. cerevisiae. These observations indicate that a major component of the cyst wall of P. carinii is beta-1,3-glucan.     

Electron microscopy of stages of Isospora felis of the cat in the mesenteric lymph node of the mouse.

Stages of Isospora felis of the cat in the mesenteric lymph node of the mouse 25 days after oral inoculation with oocysts, have been described at the ultrastructural level. The organisms occurred singly within parasitophorous vacuoles in host cell cytoplasm and were sporozoite-like, having a large crystalloid body up to 5.5 mum in length posterior to the nucleus. The size and appearance of the parasitophorous vacuole varied. Some vacuoles contained numerous, small, electron dense granules about 30 nm in diameter. Because of the aggregation of granules and their arrangement within the parasitophorous vacuole, the impression was sometimes gained by light microscopy that parasites were surrounded by a sheath or cyst wall. However, a cyst wall was not present. In host cells, spherical, membrane-bound bodies with a homogeneous, electron dense core and a maximum diameter of 0.25 mum were filed along the limiting membrane of the parasitophorous vacuole. These extra-intestinal parasites were considered to be waiting stages, with a biological function similar to that of the tissue cyst stage of other general of isosporan coccidia.     

Yeast Glucan in the Cyst Wall of Pneumocystis carinii

Ultrastructurally, the cyst wall of Pneumocystis carinii consists of an electron-dense outer layer, an electron-lucent middle layer, and an innermost plasmalemma. This is similar in appearance to the cell wall of some yeasts, e.g. Saccharomyces cerevisiae , which consists of an outer dense layer of mannan, a middle lucent layer of β−1,3-glucan (yeast glucan) and an innermost plasmalemma. The cyst wall P. carinii , as well as the cell wall of S. cerevisiae , can be labeled by a variety of methods which stain polysaccharides, such as Gomori's methenamine silver (GMS) and by Aniline blue, a dye which selectively stains β-1,3-glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is β,3-gIucan laminari-pentaohydrolase, results in lysis of the outer 2 layers of the cyst wall and the loss of positive staining by both GMS and Aniline blue. The lysis of elements of the cyst wall of P. carinii is achieved under the same conditions and concentration at which Zymolyase lyses the outer 2 layers of the cell wall of viable cells of S. cerevisiae . These observations indicate that a major component of the cyst wall of P. carinii is β-1,3-glucan.     

Role of the zymolyase-sensitive cyst wall of Pneumocystis carinii in the oxidative burst of macrophages.

Alveolar macrophages are thought to participate in clearing Pneumocystis carinii (Pc) from the lungs. We have recently demonstrated that Pc cysts and trophozoites induce an oxidative burst in a cell line of rat alveolar macrophages (NR8383). In order to investigate the mechanism of this response, we examined the effect that disruption of the Pc cyst wall with zymolyase had on the cyst's ability to elicit H2O2 from NR8383 macrophages and correlated these results with the electron microscopic appearance of the cyst wall.     

Morphological evaluation of Pneumocystis carinii after extraction from infected lung

Changes in Pneumocystis carinii induced by the extraction of the parasite from rabbit lung have been investigated. Samples obtained using 4 extraction methods were evaluated by light and transmission electron microscopy. Light microscopic evaluation was insufficient to give a measure of the P. carinii viability or to detect parasitic cellular alterations. In contrast, ultrastructural evaluation provided information on host and P. carinii cell integrity, which is a critical condition for viability. None of the tested methods was ideal. How thoroughly and in what shape P. carinii need to be extracted from tissues will determine which extraction technique is of best use.     

Morphologic and biochemical studies of chitin expression in Pneumocystis carinii.

Tomato lectin, which binds oligosaccharides of N-acetyl-D-glucosamine, and an antiserum against macromolecular chitin were used to probe sections of human and murine lungs infected with Pneumocystis carinii. By light, fluorescence and electron microscopy, lectin and antiserum binding patterns indicated that both human and murine strains of P. carinii express chitin at all identifiable stages of their life cycles. Light microscopic autoradiographs of murine P. carinii cultured in vitro with 3H-glucosamine revealed dense incorporation of the radiolabel into the cell walls in a pattern analogous to those of the antiserum and lectin binding studies. These investigations offer further evidence that chitin is an integral part of the cell wall of P. carinii trophozoites and cysts.     

Zymolyase Treatment Exposes p55 Antigen of Pneumocystis carinii

Rats exposed to Pneumocystis carinii mount antibody responses to a broad band migrating on western blot with an apparent molecular weight of 45-55 kDa. One antigen within this band, designated p55, is uniformly recognized by P. carinii exposed rats. Although the gene encoding the p55 antigen had been previously cloned, the location of this antigen within the organism was unknown. Prior attempts to localize the protein were unsuccessful. A monospecific polyclonal antiserum raised against a carboxyl-terminai 15-oligomer peptide yielded specific reactivity with a single 55 kDa band on a western blot of P. carinii. Using this antiserum, little to no reactivity could be detected with P. carinii organisms by immunofluorescence assay (IIFA). However, zymolyase treatment of P. carinii dramatically increased the intensity and proportion of organisms reactive by IFA. Zymolyase, an enzyme with β-1,3 glucanase activity, has previously been shown to remove the electron dense outer layer of the P. carinii cell wall, exposing an electron lucent layer. Immunoelectron microscopy performed on zymolyase treated organisms showed the majority of labeling occurs within the cell wall.     

Freeze-fracture studies on Pneumocystis carinii. I. Structural alteration of the pellicle during the development from trophozoite to cyst

Pneumocystis carinii has generally been distinguished in three developmental stages, namely, trophozoite, precyst and cyst. The fine structure of the pellicle--the plasma membrane and the outer layer existing outside this plasma membrane--of each stage was studied by freeze-fracture technique. By this technique, P. carinii was cleaved through the cytoplasm or through the hydrophobic region of the plasma membrane, and the cross-fractured face of the outer layer was revealed on the replicas. The outer layer, which is electron-dense in the thin section, consisted of numerous fine granules about 15 nm in diameter in freeze-fracture images, whereas the electron-lucent middle layer which appeared in the precyst and cyst was less granular. Measurement of the intramembranous particles (IMP) also was carried out. The number of IMP per square micrometer of the plasma membrane of the trophozoite was 1,512 +/- 125 on the P face and 417 +/- 44 on the E face. In the precyst, the IMP density decreased, and 1,037 +/- 56 on the P face and 262 +/- 22 on the E face. In the cyst, it further decreased, nd 875 +/- 59 and 150 +/- 20 respectively. It is generally assumed that the density of IMP is related to the physiological activity of the cell membrane, so that the present results obtained in P. carinii suggest that the trophozoite is the most active stage, and that metabolic activity of the pellicle gradually decreases with the progress of development to the precyst then to the cyst.     

The release of secretory vesicle in encysting Giardia lamblia

Giardia is an intestinal parasite that undergoes adaptation for survival outside the host. It secretes an extracellular cyst wall using a poorly understood process. An encystation-specific secretory vesicle (ESV) was previously described containing cyst wall proteins. The process of release of these vesicles has been suggested to occur after fragmentation of large ESV in small secretory vesicles, followed by exocytosis, but it was not demonstrated. The release of the ESV was studied by transmission electron microscopy. It was observed: (1) the moment of vesicle release; (2) that a large vesicle is exocytosed and does not fragment into small vesicles; (3) membrane fusion is distinct from traditional exocytosis since it is incomplete; (4) the occurrence of membrane fragmentation and that those membranes reseal to form ghosts; (5) these membrane ghosts may be endocytosed, adhered to flagellar surface or/and form empty vesicles in the extracellular medium.     

High-resolution electron microscopical study of cyst walls of Entamoeba spp

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.     

Electron microscopy of Giardia lamblia cysts.

The flagellated protozoan Giardia lamblia is a recognized public health problem. Intestinal infection can result in acute or chronic diarrhea with associated symptoms in humans. As part of a study to evaluate removal of G. lamblia cysts from drinking water by the processes of coagulation and dual-media filtration, we developed a methodology by using 5.0-microns-porosity membrane filters to evaluate the filtration efficiency. We found that recovery rates of G. lamblia cysts by membrane filtration varied depending upon the type and diameter of the membrane filter. Examination of membrane-filtered samples by scanning electron microscopy revealed flexible and flattened G. lamblia cysts on the filter surface. This feature may be responsible for the low recovery rates with certain filters and, moreover, may have implications in water treatment technology. Formation of the cyst wall is discussed. Electron micrographs of cysts apparently undergoing binary fission and cysts exhibiting a possible bacterial association are shown.     

Attempts at in vitro cultivation of Pneumocystis carinii from human bronchoalveolar lavage fluids without feeder cells.

We attempted to cultivate Pneumocystis carinii obtained from two bronchoalveolar lavage fluids of AIDS patients with P. carinii pneumonia, in a system wherein cysteine and 2-mercaptoethanol were substituted for the feeder cells. The presence of P. carinii cysts was monitored for 11 days under conditions of continuous culture. Moderate increase in cyst forms was observed until day 11. Further study with this system would be required to determine if the observed increase in cyst numbers is reproducible and whether the cyst form is a response to adverse in vitro conditions or is a manifestation of growth.     

Ultrastructural Changes During Encystment of Schizopyrenus russelli*

Ultrastructural changes associated with the encystment of Schizopyrenus russelli have been studied by electron microscopy. Before encystment small “black bodies” appear in the cytoplasm and later migrate toward the periphery. The outer cyst wall is secreted at this stage as a thin discontinuous layer which thickens and subsequently becomes continuous. Concomitant with this, the endoplasmic reticulum surrounds the mitochondria. The inner cyst wall later appears as a multilayered structure which presumably is cast off from the plasma membrane. Between the inner and outer layers of the cyst wall, there is a middle, less electron-dense layer wherein extruded cytoplasmic material is found embedded at certain places.