Composition of Pneumocystis carinii Neutral Lipids and Identification of Coenzyme Q10 as the Major Ubiquinone Homolog

The lipids of purified preparations of Pneumocystis carinii carinii freshly isolated from infected rats were analyzed and compared with those of whole lungs from normal and methylprednisolone-immunosuppressed uninfected rats. In this study, the neutral lipid fraction was examined in detail; the relative concentrations of individual classes making up this fraction were quantified. Of particular interest was the nature of the organism's ubiquinone (coenzyme Q, CoQ) fraction because atovaquone, a hydroxynaphtho-quinone (566C80) analog of ubiquinone, is efficacious in the treatment of P. carinii pneumonia. The ubiquinone concentration in both P. carinii and lung tissues was relatively low compared to that present in rat heart and liver tissues. Two homologs were identified in the organism: CoQ₁₀ was the predominant homolog with lesser amounts of CoQ₉ present. In contrast, the lungs of normal and immunosuppressed uninfected rats had CoQ₉ and lesser amounts of CoQ₈, but no detectable CoQ₁₀. Furthermore, radiolabeled mevalonic acid was incorporated in vitro into the ubiquinone fraction of P. carinii indicating that the organism has the de novo branch of the isoprenoid biosynthetic pathway leading to polyprenyl formation. Hence, it was concluded that CoQ₁₀ (if not both CoQ1₁₀ and CoQ₉) in P. carinii as not scavenged from the host but was synthesized by the organism. Although lung tissues contained substantial free fatty acids, the organism was enriched in these lipids. The high concentration of free fatty acids and relatively low level of triglycerides in P. carinii suggest that fatty acids may represent major carbon sources for ATP production by the organism.     

Pneumocystis carinii f. sp. carinii synthesizes de novo four homologs of ubiquinone

Ubiquinone, coenzyme Q, plays a pivotal role in electron transport and is a target for chemotherapy against a number of eukaryotic infectious agents, including Pneumocystis carinii. Coenzyme Q10 was previously identified as the major ubiquinone homolog in P. carinii isolated and purified from rat lungs; CoQ9 was also present. In contrast, CoQ9 and CoQ8 (but not CoQ10) were detected in the lungs of uninfected rat controls. These observations suggested that the pathogen synthesizes CoQ10, and perhaps CoQ9 as well. In the present study, CoQ biosynthesis in P. carinii was examined in greater detail. Radiolabeled mevalonate, a precursor of the CoQ polyprenyl chain, was incorporated in vitro into P. carinii ubiquinones. Incorporation of radiolabeled mevalonate into P. carinii CoQ was not enhanced by treating cells with lovastatin, suggesting that the cells did not transport the drug, or that a lovastatin-insensitive pathway for de novo synthesis of isoprenoids may also function in this organism. Radiolabeled precursors of the ring moiety, including shikimic acid, p-hydroxybenzoic acid, and tyrosine were also incorporated into P. carinii CoQ. Unexpectedly, it was found that not only CoQ9 and CoQ10, but also CoQ7, and CoQ8, were metabolically radiolabeled by all the precursors tested, indicating that the organism synthesizes CoQ7, CoQ8, CoQ9, and CoQ10. Metabolic radiolabeling of ubiquinones in rat lung controls was not detected in experiments using either radioactive mevalonate or p-hydroxybenzoate. Thus the incorporations measured using purified P. carinii preparations were due to the enzymes of the organism.     

Immunization with recombinant Pneumocystis carinii p55 antigen provides partial protection against infection: characterization of epitope recognition associated with immunization

Many therapeutic options exist for the treatment of Pneumocystis carinii pneumonia, a common fungal opportunistic pulmonary pathogen, but treatment is often complicated by side effects and toxicity and, more recently, markers of drug resistance have been described. The development of immunotherapetic modalities such as active immunization or passive immunotherapy may play an increasing important role in the prevention and treatment of infection. Passive immunotherapy with polyclonal anti-P. carinii reagents, such as serum or T cells, and monospecific reagents reactive with the major surface glycoprotein (MSG or gpA), such as monoclonal antibodies or MSG primed T cells, reduce the severity or eradicate infection. Active immunization with whole P. carinii, P. carinii extracts or MSG has afforded partial protection against the subsequent development of P. carinii pneumonia in some animal models. Identification of additional antigens with protective benefits will aid in the development of vaccines or other reagents. The p55 antigen of rat-derived P. carinii is well recognized by animals following natural exposure to the organism. This 414 amino acid residue antigen found within the cell wall of P. carinii contains 7 repeats of a glutamic acid-rich motif in the carboxyl portion of the molecule. Both humoral and cellular immune responses reactive with this repeated domain are present following natural infection while, the amino terminal portion of the molecule is immunologically silent. In this study, immunization with recombinant p55 elicited significant humoral and cellular immune responses which persisted during 10 weeks of immunosupression in corticosteroid treated rats; rp55 immunization resulted in a significant reduction in organism burden, improved histological score, lower lung weight to body weight ratio (a marker of infection or lung inflammation) and improved survival (P < 0.01). Greater protection was afforded by immunization with a peptide containing amino acid residues 1-200, than by the entire rp55 molecule. Epitope recognition by serum from animals immunized with rp55 differed from that of naturally exposed animals with oligoclonal responses to residues 22-92 and residues 196-218. This study demonstrates that protection against P. carinii can be afforded by immunization with antigen preparations other than whole extracts of P. carinii or the major surface antigen, MSG. This antigen moiety will likely be most useful as a vaccine candidate in combination with other immunogens which provide similar partial protection.     

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.     

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.     

The effects of extracellular matrix (ECM) proteins on the attachment of Pneumocystis carinii to lung cell lines in vitro.

Pneumocystis carinii cells labeled with fluorescein isothiocyanate were co-cultured with tissue culture cells. Measurements of attachment was determined by the tissue culture cell fluorescence after washing out the P. carinii organisms. The effects of the extracellular matrix proteins, laminin and fibronectin, on the binding of P. carinii onto the monolayer of cultured cells were investigated for better understanding of organism-cell interactions. The internalization of P. carinii by MRC5 cells was observed.     

The β-tubulin gene from rat and human isolates of Pneumocystis carinii

The development of new drugs for treating Pneumocystis carinii infections in AIDS patients is hampered by the lack of long-term culture systems, and by our generally limited knowledge of this organism. Recently, however, we observed significant activity of various benzimidazoles against growth of this organism in short-term cultures. Benzimidazoles inhibit microtubule polymerization; there is strong evidence that the primary target is the beta-tubulin subunit. To understand the basis for benzimidazole activity against P. carinii, and to examine the apparent relatedness of this organism to fungi, we have cloned and sequenced the single beta-tubulin gene from a rat P. carinii isolate. There was 89-91% identity at the amino acid level to beta-tubulins from filamentous fungi, but only 79-82% identity to yeast and protozoal beta-tubulins. Also, eight introns were distributed throughout the P. carinii beta-tubulin gene in a pattern characteristic of filamentous fungi. Specific residues previously implicated in benzimidazole sensitivity were conserved in P. carinii beta-tubulin. The polymerase chain reaction was used to amplify a segment of P. carinii beta-tubulin DNA from bronchoalveolar lavages obtained from two patients with AIDS. There was considerable divergence at the DNA level between the human and rat sequences, but 100% identity at the amino-acid level.     

Diagnosis of Phytophthora infections in raspberry and strawberry plants by ELISA tests

Plant diseases caused by Phytophthora spp. may be reproducibly diagnosed by DAS ELISA techniques, but this type of analysis has long been hampered by the presence of phenolic and related compounds in plants to be tested, not least in strawberry and raspberry plants. The compounds will interfere with the ELISA test procedure, leading to high non-specific optical density readings. To overcome this, a series of experiments was performed. Phenolic and related compounds in the samples were first absorbed to polymers during antigen extraction and thereafter separated by filtration at slow rate. To inhibit non-specific binding of enzyme-conjugated antibodies, the plastic wells were preincubated with non-sensitized wells, equally high background values of optical density were seen under untreated conditions and after the use of polymeric adsorbents. A marked reduction in optical density was, however, seen after blocking with non-fat dry milk, but the optimal conditions for all concentrations ofantigen were seen first after combined pre-treatment with polymeric adsorbents and non-fat dry milk. When the threshold absorbance for positive detection was calculated, the low optical density values from healthy plants, at all antigen concentrations studied, contributed to an excellent discrimination between samples from diseased and healthy plants.     

Non-specific adsorption of fish immunoglobulin M (IgM) to blocking reagents on ELISA plate wells

Enzyme-linked immunosorbent assay (ELISA) is a popular technique for quantifiable detection of specific antibodies in warm-blooded animals, but it has not been accepted for detection of fish antibodies because of its low reproducibility, which is due in part to high background optical density (OD) measurements. In the present study, we report that the high background of a fish antibody-detection ELISA resulted from non-specific adsorption of fish immunoglobulin M (IgM) to blocking reagents on the ELISA plate wells. Four fish sera (from rainbow trout Oncorhynchus mykiss, masu salmon O. masou, Japanese flounder Paralichthys olivaceus and koi Cyprinus carpio) were poured into ELISA plate wells pre-blocked with several blocking reagents (skim milk, soybean milk, bovine serum albumin, fetal bovine serum, gelatin and Roche BlockingReagent) and then washed out in order to measure the remaining fish IgM on the ELISA plate wells. Significant amounts of fish IgMs (OD absorbance at 492 nm: 0.3 to 1.1) remained on the ELISA plate wells with no antigenic protein except blocking reagents. The amount of remaining fish IgMs on the ELISA plate wells decreased significantly following treatment of fish sera with skim milk. However, the specific immuno-reactivity of fish IgM was not reduced by such treatment. Thus, we conclude that treatment of fish sera with skim milk is useful in reducing the high background OD often observed in fish IgM detection ELISA.     

Uptake and Metabolism of L-Serine by Pneumocystis carinii carinii

ABSTRACT. Serine is an important amino acid that is utilized in the biosyntheses of proteins and lipids. It is directly incorporated into the head group of phosphatidylserine, which in turn can be converted to other phospholipids. Also, it is required for the formation of long chain bases, precursors of sphingolipids. Uptake and incorporation of radiolabeled serine into both lipids and acid-precipitable material were demonstrated in Pneumocystis carinii carinii organism preparations freshly isolated from infected rat lungs. Radioactivity in proteins was about double that observed in lipids. Liquid scintillation spectrometry of metabolically radiolabeled lipids separated by thin-layer chromatography showed 53% of the total radioactivity were in phosphatidylserine, 12% in phosphatidylethanolamine, 24% in ceramides, and 11% in long chain bases and other compounds. Four long chain bases were detected by thin-layer chromatography in hydrolyzed P. carinii ceramides metabolically labeled with radioactive serine. Phytosphingosine and dihydrosphingosine were tentatively identified by their migrations on thin-layer plates. Radiolabeled ethanolamine was incorporated into P. carinii phosphatidylethanolamine, but relatively low incorporation of radiolabeled choline into phosphatidylcholine occurred. The observations made in this study indicated that P. carinii has the biosynthetic capacity to metabolize phospholipid head groups and to de novo synthesize sphingolipids. L-Cycloserine and β-CI-D-alanine, inhibitors of long chain base synthesis, reduced the incorporation of serine into P. carinii long chain bases and ceramides, which supported the conclusion that the pathogen synthesizes sphingolipids.     

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.     

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.     

Quantitation of the blocking effect of tween 20 and bovine serum albumin in ELISA microwells

ELISA provides a highly sensitive procedure for quantitating antigens and antibodies. In that assay, microwells are coated initially with a specific ligand and then saturated with inert molecules to minimize nonspecific background. Coating can be improved by pretreating the microwells with poly-l-lysine (PLL). Proteins and Tween 20 are most often used to block vacant binding sites in enzyme-linked immunosorbent assay (ELISA). In the present study the blocking effects of Tween 20 and bovine serum albumin (BSA) were estimated using an original novel approach. In the assay the magnitude of saturation of the microwells was quantitated by measuring the enzymatic activity of alkaline phosphatase adsorbed to residual vacant sites in the microwell. Tween 20 completely saturated ELISA microwells at concentrations higher than 2 microg/ml. If the microwells were pretreated with PLL, even high concentrations of the detergent did not completely saturate the wells. In contrast, BSA completely saturated both PLL-treated and nontreated microwells at 5 microg/ml. Complementation of Tween 20-induced saturation of PLL-treated microwells was achieved only by addition of BSA at concentration required for BSA alone to reach complete saturation. This approach is applicable for assessing binding to ELISA microwells of any reagent of choice either as a ligand or as a blocking reagent.     

Detergents as selective inhibitors and inactivators of enzymes

In order to study the detergent-enzyme interaction and to clarify whether such an interaction produces specific or non-specific effects, we investigated the action of natural and synthetic detergents on enzymatic systems of different levels of complexity (crystalline enzymes, crude homogenates, organ preparations, organisms in toto i.e. rats and germinating seeds). The enzyme-detergent interaction was examined both as a time-independent phenomenon (inhibition) and as a time-dependent phenomenon (inactivation). In in vitro experiments a clear inhibition of pyridine-dependent dehydrogenases by long-chain anionic detergents was found. Cationic detergents have their greatest effect on lipase, LDH, MDH and ICDH from rat liver homogenates. At low concentrations SDS inactivates all the dehydrogenase enzymes studied. With high concentrations (10 mM) of SDS and dodecyltrimethylammonium bromide (C12), there was a sharp and non-specific decrease of enzymatic activities. In the in vivo studies, rats were given detergents to drink; the cationic detergent (C12) was far more effective than SDS with enzymes from both intestine and liver homogenates. SDS and C12 do not seem to interfere with enzyme activities at the beginning of the germination of Pinus pinea and Triticum durum seeds. However a marked reduction of activities does occur at the respective maximum germination times of these seeds. The nonionic detergent is ineffective both as inhibitor and as inactivator.     

Pneumocystis carinii in pleural fluid. The cytologic appearance

We describe the cytologic appearance of Pneumocystis carinii in pleural fluid of a patient with acquired immunodeficiency syndrome and a rapidly accumulating pleural effusion. The diagnosis of P carinii infection was made by examination of air-dried, Diff-Quik-stained Cytospin preparations of the pleural fluid. The diagnostic appearances of P carinii organisms stained by this method and by the Papanicolaou stain are reviewed. The unusual predominance of the trophozoite forms of the organism in this case made Diff-Quik an ideal special stain for identifying the organisms. Furthermore, this case illustrates a novel presentation of P carinii infection and suggests that P carinii should be considered an etiologic agent in the differential diagnosis of pleural effusion in an immunocompromised host.     

Characterization of the beta-tubulin gene of Pneumocystis carinii.

To examine the potential of antimicrotubule drugs for treating Pneumocystis carinii infections, and to learn more about this unusual organism on a molecular level, we are studying its tubulin genes. A 0.3 kbp fragment of the P. carinii beta-tubulin gene was amplified by the polymerase chain reaction. Sequence analysis of this DNA revealed that P. carinii beta-tubulin is most closely related to those of the fungal molds. Consistent with these results, P. carinii growth in vitro was sensitive to the antifungal benzimidazoles benomyl and carbendazim.     

Identification of antigens and antibodies specific for Pneumocystis carinii

To increase understanding of Pneumocystis carinii and its interaction with its hosts, Ag specific for rodent and human P. carinii were identified by the immunoblot method after PAGE of P. carinii organism extracts. The m.w. of the major Ag of rat P. carinii were 45,000, 110,000, and a broad band of 49,000 to 64,000, and of human P. carinii were 22,000, 24,000, and a broad band of 35,000 to 45,000 daltons. Human and rat pneumocystis were not antigenically identical. Specific antibodies against rat P. carinii Ag were found in 18 of 79 rats by the immunoblot method. Specific antibodies against human P. carinii Ag were found in 32 of 33 adult human sera, but in only 1 of 8 sera from infants and children. Specific antibodies were found in sera of 13 of the 14 adults with no history of P. carinii pneumonia, and all 19 patients with recently diagnosed P. carinii pneumonia, including 9 patients with P. carinii pneumonia associated with AIDS. The results of this study support previous suggestions that a large proportion of adults have been exposed to P. carinii and provide a basis for the further investigations of host-P. carinii interactions.     

Effects of sterol alterations on nystatin sensitivity in Saccharomyces cerevisiae

A systematic study of the qualitative and quantitative effects of sterol on nystatin sensitivity has been made in a single organism. The use of a sterol auxotroph of Saccharomyces cerevisiae offered a convenient way to control the sterol content of the yeast cell. There was a correlation between the ergosterol content of the cell and sensitivity to nystatin, as monitored by both potassium leakage from the cell and viability. When the sterol auxotroph contained high levels of ergosterol, the cells were sensitive to the effects of nystatin. When the ergosterol content was low or when ergosterol was replaced by cholesterol or cholestanol, sensitivity to nystatin was markedly decreased. Although resistant to nystatin, cholestanol enriched cells showed an enhanced background of potassium ion loss.     

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.     

Bronchoalveolar lavage fluid cytology in patients with Pneumocystis carinii pneumonia

OBJECTIVE: To evaluate bronchoalveolar lavage (BAL) cytology and organism burden in patients with Pneumocystis carinii pneumonia (PCP) who were infected with the human immunodeficiency virus (HIV) and in those with other immunodeficiencies. STUDY DESIGN: BAL fluid samples from patients with PCP were selected (HIV-infected patients, n = 15; patients with other immunodeficiencies, n = 11). May-Grünwald-Giemsa-stained cytocentrifuge preparations were evaluated. Foamy alveolar casts (FACs) and P carinii clusters were counted. RESULTS: The numbers of FACs and P carinii clusters in BAL fluid samples of HIV-infected patients were significantly higher as compared to those in samples from patients with other immunodeficiencies. Striking cytologic findings observed in half the samples from both patient groups included the presence of foamy alveolar macrophages, activated lymphocytes, plasma cells and reactive type II pneumocytes. Furthermore, a peculiar cell type, "nonidentified cell" (NIC), was observed almost exclusively in BAL fluid samples from HIV-infected patients. CONCLUSION: BAL fluid samples from HIV-infected patients with PCP displayed higher organism burdens as compared to those from patients with other immunodeficiencies. Moreover, cytologic findings suggestive of noninfectious lung conditions were common in BAL fluid samples obtained from patients with PCP. Further study is required to elucidate the identity of the NIC cell type.