Characterization of <i>Pneumocystis carinii</i> Preparations Developed for Lipid Analysis

Kaneshiro, Edna S.; Wyder, Michael; Zhou, Linda; Ellis, Jayne E.; Voelker, Dennis R.; Langreth, Susan G.

doi:10.1111/j.1550-7408.1993.tb04479.x

Cited by 50 publications

(89 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Results from the present study are in general agreement with those from previous studies from other laboratories (19,21,26). Our analyses showed that the major lipids in P. carinii include phosphatidylcholine, phosphatidylethanolamine (26), and cholesterol (19).…”

Section: Resultssupporting

confidence: 83%

“…We found no evidence for the presence of either phosphonolipids or DGTS (data not shown). Cholesterol was the major sterol present in all of our preparations examined as described previously (21), and the percentage of cholesterol observed (approximately 80%) agrees in general terms with that reported previously (21). Thus, while this is the first report that identifies sterols other than cholesterol in P. carinii preparations, other studies have reported that cholesterol does not constitute the entire sterol content of the organism.…”

Section: Resultssupporting

confidence: 75%

See 1 more Smart Citation

Phytosterols are present in Pneumocystis carinii

Furlong

Samia

Rose

et al. 1994

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Although originally classified as a protozoan, Pneumocystis carinii is now considered to have fungal characteristics. Drugs typically used for the treatment of fungal infections target ergosterol. Because P. carinii is an important pathogen in AIDS and other immunocompromised patients, knowledge of the sterol content of this organism may be useful as a basis for developing new treatment strategies or for improving diagnosis. P. carinii organisms were harvested from infected rat lungs and were purified by filtration. Control preparations from uninfected animals were identically prepared. Lipids were extracted from the organisms and control preparations and were separated into neutral lipid, glycolipid, and phospholipid fractions by silicic acid chromatography. The neutral lipid fraction was further treated by alkaline hydrolysis and was analyzed by reversed-phase high-pressure liquid chromatography (HPLC), gas chromatography (GC), and GC-mass spectrometry (GC-MS). As shown by HPLC, the neutral lipid fraction from infected rats contained a minimum of six peaks, while in control preparations a single peak with a retention time identical to that of cholesterol was observed. The predominant sterol in these preparations was positively identified by GC-MS as cholesterol and constituted 80 to 90% of the total. The remaining peaks had relative retention times similar to those of phytosterols by both HPLC and GC, and the similarity of these sterols to those derived from plants and fungi was confirmed by MS. Ergosterol, however, was not present. These results provide further evidence for a close phylogenetic relationship between P. carinii and fungi and suggest that these sterols could be used as targets for drug development and for improving diagnosis.Pneumocystis carinii is one of the leading causes of opportunistic infection in AIDS patients (30). Despite the importance of this microorganism as an opportunistic pathogen, much basic information about the parasite is lacking. While originally classified phylogenetically as a protozoan, analyses of P. carinii DNA and mitochondrial and rRNA sequences have generally shown a higher degree of homology to the mitochondrial and rRNA sequences of fungi (yeasts) than to those of protozoa (9,13,27,31,34,35). The exact relationship remains to be determined. Analyses of 5S RNA have suggested a somewhat close phylogenetic relationship to members of the Rhizopoda-Myxomycota-Zygomycota group of "protista fungi." Other studies have suggested similarities to Saccharomyces, Candida, and Neurospora spp. and "red yeast" fungi. In general, the arguments in favor of classifying P. carinii as a protozoan are the amoeboid appearance of the trophic form, susceptibility to antiprotozoal drugs such as pentamidine isethionate or trimethoprim-sulfamethoxazole, insusceptibility to antifungal drugs such as amphotericin B, and the lack of ergosterol in Pneumocystis cell membranes. Arguments in favor of including P. carinii among the fungi include similarities of the sporogenous state to ascospore format...

show abstract

Section: Resultssupporting

confidence: 83%

Section: Resultssupporting

confidence: 75%

Phytosterols are present in Pneumocystis carinii

Furlong

Samia

Rose

et al. 1994

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…Furthermore, no evidence of direct ergosterol precursors were detected; trienes, tetraenes and ⌬ 5,7 sterol nuclei were not detected. The inability to detect ergosterol by all highly sensitive analytical methods and instrumentation used in this study further verifies that P. carinii prepared from our rat model and purified by our protocol (18) are pure and free from other fungi that synthesize this sterol. If ergosterol were present in this large pooled P. carinii total sterol sample, it should have been detected in this study or in previous studies using GLC-MS techniques.…”

Section: Resultsmentioning

confidence: 99%

Comprehensive and definitive structural identities of Pneumocystis carinii sterols

Giner

Zhao

Beach

et al. 2002

Journal of Lipid Research

Self Cite

View full text Add to dashboard Cite

Pneumocystis causes a type of pneumonia in immunodeficient mammals, such as AIDS patients. Mammals cannot alkylate the C-24 position of the sterol side chain, nor can they desaturate C-22. Thus, the reactions leading to these sterol modifications are particularly attractive targets for the development of drugs against fungal and protozoan pathogens that make them. In the present study, the definitive structures of 43 sterol molecular species in rat-derived Pneumocystis carinii were elucidated by nuclear magnetic resonance spectroscopy. Ergosterol, ⌬ 5,7 sterols, trienes, and tetraenes were not among them. Most (32 of the 43) were 24-alkylsterols, products of S -adenosyl-L -methionine:C-24 sterol methyl transferase (SAM:SMT) enzyme activity. Their abundance is consistent with the suggestion that SAM:SMT is highly active in this organism and that the enzyme is an excellent anti-Pneumocystis drug target. In contrast, the comprehensive analysis strongly suggest that P. carinii does not form ⌬ 22 sterols, thus C-22 desaturation does not appear to be a drug target in this pathogen. The lanosterol derivatives, 24-methylenelanost-8-en-3 ␤ -ol and ( Z )-24-ethylidenelanost-8-en-3 ␤ -ol (pneumocysterol), previously identified in human-derived Pneumocystis jiroveci , were also detected among the sterols of the rat-derived P. carinii organisms. Pneumocystis pneumonia (PcP) remains among the most prevalent opportunistic infections in immunocompromised individuals such as AIDS patients. It has becoming evident that the high incidence of PcP in AIDS patients is global and that the organism can infect other immunodeficient people such as patients undergoing chemotherapy for cancer or solid organ transplant (1, 2), children prior to becoming fully immunocompetent, and the elderly with diminished immune systems. Pneumocystis can also transiently colonize normal, healthy people and animals without causing overt symptoms of respiratory disorder. The combination of trimethoprim and sulfamethoxazole and other agents (e.g., pentamidine, atovaquone) used for prophylaxis and for clearing PcP has successfully reduced the number of deaths directly attributed to PcP. However, AIDS and other patients with prolonged immunodeficiency experience recurrent Pneumocystis jiroveci ( Pneumocystis carinii f. sp. hominis ) (3, 4) infections. Also, some individuals cannot tolerate these drugs and suffer undesirable side effects. The development of drug-resistant pathogen populations is of serious concern, making it imperative to develop a larger armamentarium of diverse drugs to circumvent these problems (5 Ϫ 7). Although the organism can be maintained in long-term, small-volume axenic cultures (8), growth is very slow and insufficient numbers of organisms are obtained for most biochemical studies. Thus, Pneumocystis remains an organism considered difficult to manipulate for experimental work. Despite this difficulty, much is now known about the organism's lipids from analyses performed on preparations purified from infected rat lungs (9 Ϫ 11...

show abstract

“…carinii organisms were isolated from the lungs of athymic mice using a modification of the method of Kaneshiro et al (36). Athymic mice infected with P. carinii were killed during pentobarbital anesthesia, and the lungs were perfused via the right ventricle with NaCaHEPES buffer (150 mM NaCl, 1.8 mM CaCl 2 , and 25 mM HEPES, pH 7.4) containing 0.5% glutathione and then were removed aseptically.…”

Section: Isolation Of P Carinii Organisms For In Vitro Studymentioning

confidence: 99%

Granulocyte-Macrophage Colony-Stimulating Factor in the Innate Immune Response toPneumocystis cariniiPneumonia in Mice

Paine

Preston

Wilcoxen

et al. 2000

The Journal of Immunology

138

111

View full text Add to dashboard Cite

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-␣ 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.

show abstract

Characterization of Pneumocystis carinii Preparations Developed for Lipid Analysis

Cited by 50 publications

References 22 publications

Phytosterols are present in Pneumocystis carinii

Phytosterols are present in Pneumocystis carinii

Comprehensive and definitive structural identities of Pneumocystis carinii sterols

Granulocyte-Macrophage Colony-Stimulating Factor in the Innate Immune Response toPneumocystis cariniiPneumonia in Mice

Contact Info

Product

Resources

About