Surface Labeling of Pneumocystis carinii from in Vitro Culture

Pneumocystis carinii is an opportunistic pathogen of man, carried as a commensal in healthy subjects. It frequently causes a fatal pneumonia in the immunosupprcssed host. It is a major complication of HIV-1 infection in man (AIDS). Using surface radioiodination of rat-denved P. carinii trophozoites obtained from in vitro culture, a major surface glycoprotein (gp120) has been identified. The glycoprotein exhibits adherent behavior similar to that of the intact organism. Purification of gp120 by conventional methods was unsuccessful as the glycoprotein irreversibly bound lo numerous column matrices. A combination of gel chromatography and hydroxyapatile chromatography in sodium dodecylsulfaie v. as utilized to purify the glycoprotein. Some preliminary characterization of the glycoprotein is presented.     

Surface labeling of Pneumocystis carinii from in vitro culture

Pneumocystis carinii is an opportunistic pathogen of man, carried as a commensal in healthy subjects. It frequently causes a fatal pneumonia in the immunosuppressed host. It is a major complication of HIV-1 infection in man (AIDS). Using surface radioiodination of rat-derived P. carinii trophozoites obtained from in vitro culture, a major surface glycoprotein (gp120) has been identified. The glycoprotein exhibits adherent behavior similar to that of the intact organism. Purification of gp120 by conventional methods was unsuccessful as the glycoprotein irreversibly bound to numerous column matrices. A combination of gel chromatography and hydroxyapatite chromatography in sodium dodecylsulfate was utilized to purify the glycoprotein. Some preliminary characterization of the glycoprotein is presented.     

Identification and purification of a soluble species of gp120 released by zymolyase treatment of Pneumocystis carinii.

Purified zymolyase containing beta-glucanase activity releases a soluble species of the gp120 component of the high molecular weight surface antigen complex of rat- and human-derived Pneumocystis carinii. We have purified the soluble gp120 from rat-derived P. carinii by concanavalin A-affinity- and hydrophobic-interaction liquid chromatography. A single band was detected in this fraction by silver staining and immunoblotting. We have also partially purified a soluble form of the corresponding high molecular weight surface antigen from human-derived P. carinii. Identification and purification of a nondenatured soluble species of gp120 will assist in the characterization of its interactions within the surface antigen complex and with host molecules.     

Specific T-cell response to a Pneumocystis carinii surface glycoprotein (gp120).

Pneumocystis carinii gp120 can elicit a specific T-cell proliferative response in mice after immunization with a gp120 preparation or with a crude P. carinii homogenate. It can also elicit a proliferative response from SCID mice after recovery from natural infection with P. carinii, implicating this glycoprotein as an important antigen in the host's response to P. carinii infection.     

Expression cloning of Pneumocystis carinii antigens.

We undertook expression cloning of Pneumocystis carinii antigens to overcome the difficulties encountered in purification of these antigens. Using monoclonal antibodies to the P. carinii gp120 antigen and polyclonal rabbit antiserum to rat-derived P. carinii, we have isolated cDNA clones encoding immunoreactive moieties. A cDNA clone encoding the 3' portion of a 45-55 kDa antigen of rat-derived P. carinii, was the most abundant clone isolated. The peptide encoded by this cDNA has a novel sequence with a repeated motif rich in glutamic acid residues. Affinity-purified antibodies to this peptide reacted with the 45-55 kDa band of rat-derived P. carinii. The fusion protein was recognized by serum antibodies from rats with natural exposure to P. carinii. The production of this recombinant protein should allow more detailed studies of the host-parasite relationship of this important opportunistic infection.     

Analysis of purified gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and tandem mass spectrometry

The stress protein gp96 exhibits a number of immunological activities, the majority of studies into which have used gp96 purified from a variety of tissues. On the basis of 1-D gel electrophoresis, the purity of these preparations has been reported to range between 70% and 99%. This study analyzed gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry (MS-MS). The procedure for purifying gp96 was reproducible, as similar protein profiles were observed in replicate gels of gp96 preparations. The purity of the preparations was typically around 70%, with minor co-purified proteins of varying molecular weights and mobilities being present. Dominant bands at 95-100 kDa in preparations from Wistar rats and C57BL/6 mice were identified as gp96 by ECL Western blotting. Multiple bands having similar, yet distinct molecular weights and differing pI mobility on ECL Western blots were confirmed as being gp96 in preparations from Wistar rats using MS-MS. The most striking feature of the 2-D gel analysis was the presence of additional dominant bands at 55 kDa in preparations from Wistar rats, and at 75-90 kDa in preparations from C57BL/6 mice. These were identified as gp96 by ECL Western blotting and, in the case of preparations from Wistar rats, by MS-MS. Although the lower molecular weight, gp96-related molecules might be partially degraded gp96, their reproducible presence, definition and characteristics suggest that they are alternative, species-specific isoforms of the molecule. A 55 kDa protein which exhibited a lower pI value than gp96 was present in all preparations and this was identified as calreticulin, another putative immunoregulatory molecule. This study confirms the reproducibility of the gp96 purification protocol and reveals the presence of multiple gp96 isoforms, some of which likely result from post-translational modifications such as differential glycosylation and phosphorylation.     

Structure of a Bacterial Virus DNA-Injection Protein Complex Reveals a Decameric Assembly with a Constricted Molecular Channel

The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. The assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. The gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.     

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. I. Evidence for an outer surface membrane

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 beta-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.     

Domain organization and polarity of tail needle GP26 in the portal vertex structure of bacteriophage P22

The attachment of tailed bacteriophages to the host cell wall as well as the penetration and injection of the viral genome into the host is mediated by the virion tail complex. In phage P22, a member of the Podoviridae family that infects Salmonella enterica, the tail contains an approximately 220 A elongated protein needle, previously identified as tail accessory factor gp26. Together with tail factors gp4 and gp10, gp26 is critical to close the portal protein channel and retain the viral DNA inside the capsid. By virtue of its topology and position in the virion, the tail needle gp26 is thought to function as a penetrating device to perforate the Salmonella cell wall. Here, we define the domain organization of gp26, characterize the structural determinants for its stability, and define the polarity of the gp26 assembly into the phage portal vertex structure. We have found that the N-terminal 27 residues of gp26 form a functional domain that, although not required for gp26 trimerization and overall stability, is essential for the correct attachment to gp10, which is thought to plug the portal vertex structure. The region downstream of domain I, domain II, folds into helical core, which exhibits four trimerization octad repeats with consensus Ile-xx-Leu-xxx-Val/Tyr. We demonstrate that in vitro, domain II represents the main self-assembling, highly stable trimerization core of gp26, which retains a folded conformation both in an anhydrous environment and in the presence of 10% SDS. The C terminus of gp26, immediately downstream of domain II, contains a beta-sheet-rich region, domain III, and a short coiled coil, domain IV, which, although not required for gp26 trimerization, enhance its thermodynamic stability. We propose that domains III and IV of the tail needle form the tip utilized by the phage to penetrate the host cell wall.     

Granulocyte-macrophage colony-stimulating factor in the innate immune response to Pneumocystis carinii pneumonia in mice

Innate immunity plays an important role in pulmonary host defense against Pneumocystis carinii, an important pathogen in individuals with impaired cell-mediated immunity. We investigated the role of GM-CSF in host defense in a model of P. carinii pneumonia induced by intratracheal inoculation of CD4-depleted mice. Lung GM-CSF levels increased progressively during the infection and were significantly greater than those in uninfected controls 3, 4, and 5 wk after inoculation. When GM-CSF gene-targeted mice (GM-/-) depleted of CD4+ cells were inoculated with P. carinii, the intensities of infection and inflammation were increased significantly compared with those in CD4-depleted wild-type mice. In contrast, transgenic expression of GM-CSF directed solely in the lungs of GM-/- mice (using the surfactant protein C promoter) dramatically decreased the intensity of infection and inflammation 4 wk after inoculation. The concentrations of surfactant proteins A and D were greater in both uninfected and infected GM-/- mice compared with those in wild-type controls, suggesting that this component of the innate response was preserved in the GM-/- mice. However, alveolar macrophages (AM) from GM-/- mice demonstrated impaired phagocytosis of purified murine P. carinii organisms in vitro compared with AM from wild-type mice. Similarly, AM production of TNF-alpha in response to P. carinii in vitro was totally absent in AM from GM-/- mice, while GM-CSF-replete mice produced abundant TNF in this setting. Thus, GM-CSF plays a critical role in the inflammatory response to P. carinii in the setting of impaired cell-mediated immunity through effects on AM activation.     

Differential regulation of growth and checkpoint control mediated by a Cdc25 mitotic phosphatase from Pneumocystis carinii

Pneumocystis carinii is an opportunistic fungal pathogen phylogenetically related to the fission yeast Schizosaccharomyces pombe. P. carinii causes severe pneumonia in immunocompromised patients with AIDS and malignancies. Although the life cycle of P. carinii remains poorly characterized, morphologic studies of infected lung tissue indicate that P. carinii alternates between numerous small trophic forms and fewer large cystic forms. To understand further the molecular mechanisms that regulate progression of the cell cycle of P. carinii, we have sought to identify and characterize genes in P. carinii that are important regulators of eukaryotic cell cycle progression. In this study, we have isolated a cDNA from P. carinii that exhibits significant homology, but unique functional characteristics, to the mitotic phosphatase Cdc25 found in S. pombe. P. carinii Cdc25 was shown to rescue growth of the temperature-sensitive S. pombe cdc25-22 strain and thus provides an additional tool to investigate the unique P. carinii life cycle. Although P. carinii Cdc25 could also restore the DNA damage checkpoint in cdc25-22 cells, it was unable to restore fully the DNA replication checkpoint. The dissociation of checkpoint control at the level of Cdc25 indicates that Cdc25 may be under distinct regulatory control in mediating checkpoint signaling.     

Roles of genes 44, 50, and 51 in regulating gene expression and host takeover during infection of Bacillus subtilis by bacteriophage SPO1

We show that the products of SPO1 genes 44, 50, and 51 are required for the normal transition from early to middle gene expression during infection of Bacillus subtilis by bacteriophage SPO1; that they are also required for control of the shutoff of host DNA, RNA, and protein synthesis; and that their effects on host shutoff could be accounted for by their effects on the regulation of gene expression. These three gene products had four distinguishable effects in regulating SPO1 gene expression: (i) gp44-50-51 acted to restrain expression of all SPO1 genes tested, (ii) gp44 and/or gp50-51 caused additional specific repression of immediate-early genes, (iii) gp44 and/or gp50-51 stimulated expression of middle genes, and (iv) gp44 and/or gp50-51 stimulated expression of some delayed-early genes. Shutoff of immediate-early gene expression also required the activity of gp28, the middle-gene-specific sigma factor. Shutoff of host RNA and protein synthesis was accelerated by either the 44- single mutant or the 50(-)51(-) double mutant and more so by the 44(-)50(-)51(-) triple mutant. Shutoff of host DNA synthesis was accelerated by the mutants early in infection but delayed by the 44(-)50(-)51(-) triple mutant at later times. Although gp50 is a very small protein, consisting almost entirely of an apparent membrane-spanning domain, it contributed significantly to each activity tested. We identify SPO1 genes 41 to 51 and 53 to 60 as immediate-early genes; genes 27, 28, and 37 to 40 as delayed-early genes; and gene 52 as a middle gene.     

Carboxypeptidase D (gp180), a Golgi-Resident Protein, Functions in the Attachment and Entry of Avian Hepatitis B Viruses

Carboxypeptidase D (gp180), one of many candidate receptors proposed for hepatitis B viruses (HBVs), was examined and found to be the actual cellular receptor for avian HBVs. This conclusion was based on the following observations: (i) gp180 was the only host protein that bound with high affinity to the pre-S ectodomain of the large duck hepatitis B virus (DHBV) envelope protein, which is known to be essential for virus infection; (ii) a pre-S subdomain which determines physical binding to gp180 was found to coincide with a domain functionally defined in infection competition experiments as a receptor binding domain; (iii) soluble gp180, lacking the membrane anchor, efficiently inhibited DHBV infection; (iv) efficient interspecies gp180–pre-S interaction was limited to the natural hosts of avian hepadnaviruses; and (v) expression of gp180 in a heterologous hepatoma cell line mediated cellular attachment and subsequent internalization of fluorescently labeled viral particles into vesicular structures. However, gp180 expression did not render transfected heterologous cells permissive for productive infection, suggesting that a species-specific coreceptor is required for fusion to complete viral entry. In contrast to the case for known virus receptors, gp180 was not detected on the hepatocyte cell surface but was found to be concentrated in the Golgi apparatus, from where it functions by cycling to and from the plasma membrane.     

Survival and Infectivity of Pneumocystis carinii Outside the Mammalian Host.

SUMMARY Air blower prefilters servicing HEPA-filtered Biobubble housing Pneumocystis carinii -infected rats were stored for up to five months at -80°C to room temperature. After storage, 76% of immunosuppressed rats exposed to these prefilters developed P. carinii infections. In contrast, only 4% of control immunosuppressed rats exposed to autoclaved filters had P. carinii infections. These observations indicate that the organism has a dormant form that remains infective for at least several months outside the mammalian host.     

A comparison of the antigenic characteristics of rat and human Pneumocystis carinii by immunoblotting

The antigenic characteristics of rat Pneumocystis carinii obtained from infected lungs and grown in tissue culture were compared with the properties of human P. carinii obtained from the lungs of AIDS and non-AIDS patients by the immunoblotting technique, using different sources of antibody. Major immunoreactive bands of 45, 50, and 116 kd were found in both lung and tissue culture-derived rat P. carinii, suggesting the organism retains its antigenic characteristics in short-term culture. The principal immunoreactive bands in human P. carinii included a band of 40 kd, and to a lesser extent, a band of 66 kd; these antigens were found in the lungs of six and seven AIDS patients but in only one of eight non-AIDS patients with pneumocystosis. The rat and human P. carinii antigens reacted with sera from immunized rabbits, from rats with pneumocystosis and prolonged environmental exposure to the organism, from AIDS and non-AIDS P. carinii patients, and from healthy blood donors. Reactivity of these antigens could be removed by adsorption of antisera with P. carinii-infected lungs but not with normal lungs or lungs infected with bacteria and fungi. We conclude that rat and human P. carinii have shared, as well as species-specific, antigenic determinants, which should be useful for a variety of studies with this organism.     

Isolation and characterization of rat lung Pneumocystis carinii gp120

The principal glycoprotein (gp120) of Pneumocystis carinii obtained from infected rat lung was isolated by differential extraction and size-exclusion chromatography. The purified glycoprotein was cleaved with CNBr to two peptides of approximately 27 and 33 kDa. Amino acid sequences were obtained from both peptides. Proteolytic digestion with V8 protease yielded several peptides and sequences were obtained from peptides of 10 and 19 kDa. The cyanogen bromide cleavage results led to the conclusion that gp120 exists as a homodimer.     

Energy Dependency of Glucocorticoid Exacerbation of gp120 Neurotoxicity

Abstract: The HIV envelope glycoprotein, gp120, a well documented neurotoxin, may be involved in AIDS-related dementia complex. gp120 works through an NMDA receptor- and calcium-dependent mechanism to damage neurons. We have previously demonstrated that both natural and synthetic glucocorticoids (GCs) exacerbate gp120-induced neurotoxicity and calcium mobilization in hippocampal mixed cultures. GCs, steroid hormones secreted during stress, are now shown to work in conjunction with gp120 to decrease ATP levels and to work synergistically with gp120 to decrease the mitochondrial potential in hippocampal cultures. Furthermore, energy supplementation blocked the ability of GCs to worsen gp120's effects on neuronal survival and calcium mobilization. A GC-induced reduction in glucose transport in hippocampal neurons, as previously documented, may contribute to this energetic dependency. These results may have clinical significance, considering the common treatment of severe cases of Pneumocystis carinii pneumonia, typical of HIV infection, with large doses of synthetic GCs.     

Pneumocystis carinii polyamine catabolism.

DL-alpha-Difluoromethylornithine (DFMO) causes polyamines of the AIDS-associated opportunistic pathogen Pneumocystis carinii to diminish 15 times more rapidly than mammalian host cells. The proposed mechanism was that, unlike mammalian cells, P. carinii is unable to regulate polyamine catabolism when synthesis is blocked. To test this, the responses of the polyamine catabolic enzymes spermidine/spermine acetyltransferase (SSAT) and polyamine oxidase (PAO) were determined using a new high-performance liquid chromatography assay to measure the products of these enzymes. The specific activities in untreated Pneumocystis carinii were 1.78 +/- 0.5 pmol min(-1) mg protein(-1) for SSAT, similar to mammalian cells, and 6.42 +/- 0.8 pmol min(-1) mg protein(-1) for PAO, 19% of that of mammalian cells. DFMO treatment for 12 h caused reductions of only 11 and 4% in SSAT and PAO, respectively, despite polyamine reductions of 94, 96, and 90% for putrescine, spermidine, and spermine, respectively. The P. carinii SSAT K(m) value of 25 microM spermidine is 20% of that of mammalian cells, and the PAO K(m) value of 14 nM N(1)-acetylspermidine is 0.01% of that of mammalian cells. Acetylated polyamines continue to be lost from P. carinii even when exposed to DFMO. Collectively, these results support the hypothesis that P. carinii is unable to regulate polyamine catabolism.     

New rapid method for the study of Pneumocystis carinii interaction with alveolar macrophages.

Understanding the pathophysiology of Pneumocystis carinii infection has been limited by the availability of methods for precisely measuring the interaction of P. carinii with host cells. Here we describe a new method which allows for the rapid assessment of P. carinii binding to, and internalization by, adherent alveolar macrophages. The method is based on the detection of fluorescein-labelled P. carinii by an automated fluorescence measurement system.