C27 to C32 sterols found in Pneumocystis, an opportunistic pathogen of immunocompromised mammals.

Pneumocystis carinii is the paradigm of opportunistic infections in immunocompromised mammals. Prior to the acquired immunodeficiency syndrome (AIDS) pandemic and the use of immunosuppressive therapy in organ transplant and cancer patients, P. carinii was regarded as a curiosity, rarely observed clinically. Interest in this organism exploded when it was identified as the agent of P. carinii pneumonia (PcP), the direct cause of death among many AIDS patients. Aggressive prophylaxis has decreased the number of acute PcP cases, but it remains among the most prevalent opportunistic infections found within this patient population. The taxonomic assignment of P. carinii has long been argued; molecular genetics data now demonstrate that it is a fungus. Several antimycotic drugs are targeted against ergosterol or its biosynthesis, but these are not as effective against PcP as they are against other fungal infections. This can now be explained in part by the identification of the sterols of P. carinii. The organism lacks ergosterol but contains distinct C28 and C29 delta7 24-alkylsterols. Also, 24-methylenelanost-8-en-3beta-ol (C31) and pneumocysterol, (24Z)-ethylidenelanost-8-en-3beta-ol (C32) were recently identified in organisms infecting humans. Together, the delta7 24-alkylsterols and pneumocysterol are regarded as signature lipids of the pathogen that can be useful for the diagnosis of PcP, since no other lung pathogen is known to contain them. Cholesterol (C27), the dominant sterol component in P. carinii, is probably totally scavenged from the host. De novo synthesis of sterols has been demonstrated by the presence of lovastatin-sensitive 3-hydroxy-3-methylglutaryl-CoA reductase activity, the incorporation of radiolabeled mevalonate and squalene into P. carinii sterols, and the reduction in cellular ATP in cells treated with inhibitors of enzymes in sterol biosynthesis.

The fatty acid and monosaccharide compositions of three neutral and three phosphorylated glycolipids isolated from Leishmania donovani promastigotes grown in a chemically defined medium.

Several lipids and macromolecular lipoconjugates of Leishmania spp. have now been well characterized; however, the glycolipids of L. donovani have not been thoroughly examined. In the present study, 3 neutral and 3 phosphorylated glycolipids were detected in promastigote forms of the organism grown in a chemically defined medium. The fatty acid and sugar compositions of these glycolipids, isolated and purified by adsorption column chromatography and thin-layer chromatographic procedures, were identified and quantified by gas-liquid chromatography and mass spectrometry. Myristate (14:0), palmitate (16:0), palmitoleate (16:1), stearate (18:0), oleate (18:1), and linoleate (18:2) were the major fatty acids in all 6 glycolipids. Arabinose, mannose, glucose, and galactose were detected in the glycolipids. The biochemical nature of these lipids suggested that the major components in the isolated preparations of the 6 glycolipids are diacylglycerophospholipids, distinct from the major precursors of macromolecular lipoconjugates such as the lipid anchors of cell surface antigens that have been reported. These appear to be terminal products of lipid biosynthesis in this parasite.

Quantitation of absolute Pneumocystis carinii nuclear DNA content. Trophic and cystic forms isolated from infected rat lungs are haploid organisms.

The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis. The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry. Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA. It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P. carinii organisms, as well as the cell wall of S. cerevisiae, bound all fluorescent dyes tested to varying degrees. Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase. Two stains that appeared most specific for DNA, DB181 and 4',6-diamidino-2-phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181. Haploid wild type Saccharomyces cerevisiae and cdc-28 temperature-sensitive mutant cells, accumulated at the restrictive temperature (37 degrees C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P. carinii. Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities. The P. carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different. The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively). Since these would include 2C (G-2 phase) and S-phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181-stained nuclei. Almost all nuclei analyzed in all life cycle stages fell within this population. The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A-T binding of the dye and taking into account the G + C content of S. cerevisiae and P. carinii. A 4C (G-2-phase diploid nuclei) population was not detected in histograms of DB181- or DAPI-stained nuclei. The P. carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organism's chromosomes resolved by electrophoretic techniques. Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life-cycle stages demonstrate that P. carini carinii isolated from infected rat lungs are haploid organisms.

Effects of steroidal allenic phosphonic acid derivatives on the parasitic protists Leishmania donovani, Leishmania mexicana mexicana, and Pneumocystis carinii carinii.

Several pathogenic fungi and protozoa are known to have sterols distinct from those of their mammalian hosts. Of particular interest as targets for drug development are the biosyntheses of the sterols of important parasites such as the kinetoplastid flagellates and the AIDS-associated opportunistic protist Pneumocystis carinii. These pathogens synthesize sterols with an alkyl group at C-24, and some have a double bond at C-22 of the side chain. Humans and other mammalian hosts are incapable of C-24 alkylation and C-22 desaturation. In the present study, three steroidal compounds with side chains substituted by phosphonyl-linked groups were synthesized and tested for their effects on Leishmania donovani and L. mexicana mexicana culture growth. The compounds inhibited organism proliferation at concentrations in micrograms per milliliter. The most potent inhibitors of this group of compounds were characterized by two ethyl groups at the phosphate function. Leishmania organisms treated with 17-[2-(diethylphosphonato) ethylidienyl]3-methoxy-19-norpregna-1,3,5-triene exhibited reduced growth after transfer into inhibitor-free medium. Because there are currently no axenic methods available for the continuous subcultivation of P. carinii, the effects of these drugs on this organism were evaluated by two alternative screening methods. The same two diethyl phosphonosteroid compounds that inhibited Leishmania proliferation were also the most active against P. carinii as determined by the potent effect they had on reducing cellular ATP content. Cystic as well as trophic forms responded to the drug treatments, as evaluated by a dual fluorescent staining live-dead assay. Other modifications of steroidal phosphonates may lead to the development of related drugs with increased activity and specificity for the pathogens.

Changes in lipid composition during in vitro encystation and fatty acid desaturase activity of Giardia lamblia.

Lipids of axenically-cultured Giardia lamblia trophozoites were compared with those of cells undergoing in vitro encystation. Although the lipid composition of the organisms grossly resembled those of low-bile or high-bile culture media, differences were clearly detected. Encysting trophozoites incubated in a high-bile medium for 24 h had a higher concentration of unsaturated fatty acids in the total cellular lipids than did nonencysting trophozoites. The organism, but not the medium, contained linoleate and linolenate, suggesting that G. lamblia desaturates oleate. The presence of a fatty acid desaturase activity in the organism was demonstrated by the conversion of a radiolabeled monounsaturated fatty acid (oleate) to radiolabeled polyunsaturated fatty acids. Triglycerides, a common form of storage lipids, were unusually low in G. lamblia, but steryl esters (which can also serve as reserves) were abundant. Steryl esters increased during encystation of G. lamblia. The changes observed in G. lamblia lipids (increased fatty acid unsaturation and the accumulation of storage lipids) are consistent with parasite differentiation into a cyst stage that is able to survive outside the host at reduced temperatures and reduced available nutrient resources. This study also demonstrated that G. lamblia not only has the capacity to de novo synthesize isoprenoid lipids (ubiquinone, prenylated proteins), but it can also metabolize fatty acids by the addition of double bonds.

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 CoQ10 and CoQ9) in P. carinii was 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.

Characterization of Pneumocystis carinii preparations developed for lipid analysis.

Pneumocystis carinii organisms were isolated from viral antibody-negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was > 5 (100-1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (< 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated > 99.5% purity. The total non-P. carinii protein in the final preparation (< 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 10(8)-10(9) organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester-propidium iodide assay, was 80-95%.

Lipophosphoglycan antigen shedding by Leishmania donovani.

The biochemical characterizations of lipophosphoglycans from various Leishmania species reported by other workers may or may not contain several types of lipophosphoglycan molecules. This is the first report in which a specific lipophosphoglycan has been defined by both its antigenic and electrophoretic properties. Furthermore, a purification procedure for this specific lipophosphoglycan is described and some biochemical characterizations are presented. Phospholipase C and the so-called phosphatidylinositol-specific phospholipase C of Bacillus cereus convert the amphipathic form of the lipophosphoglycan antigen to the hydrophilic form. Under equivalent incubation conditions, other phospholipases tested were not effective in conversion of the amphipathic to the hydrophilic form. Since the amphipathic form is present in conditioned media, antigen shedding cannot be explained by phospholipase C digestion of the amphipathic form, which would result in the release of only the hydrophilic form into the medium. Both the pellet and the supernatant fractions of conditioned media contained both forms of the antigen and did not differ in the relative amounts of the two. This observation rules out membrane blebbing as the major mechanism for the release of the amphipathic form.