Evidence for the Presence of “Metabolic Sterols” in <i>Pneumocystis</i>: Identification and Initial Characterization of <i>Pneumocystis carinii</i> Sterols

Kaneshiro, Edna S.; Ellis, Jayne E.; Jayasimhulu, Koka; Beach, David

doi:10.1111/j.1550-7408.1994.tb05938.x

Cited by 64 publications

(109 citation statements)

References 17 publications

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“…Since the manuscript was submitted for publication another report of a study (20) that used a somewhat different methodology from that used in the present study provided similar but not identical results to those described in the present report.…”

Section: Acknowledgmentssupporting

confidence: 75%

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Phytosterols are present in Pneumocystis carinii

Furlong

Samia

Rose

et al. 1994

Antimicrob Agents Chemother

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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...

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Section: Acknowledgmentssupporting

confidence: 75%

“…To further identify the sterols contained in P. carinii, acetate-derivatized E ;l 20 samples were analyzed by GC. On a nonpolar capillary column with flame ionization detection, multiple sterol peaks were observed, similar to the results observed by HPLC (Fig.…”

Section: Resultsmentioning

confidence: 99%

Phytosterols are present in Pneumocystis carinii

Furlong

Samia

Rose

et al. 1994

Antimicrob Agents Chemother

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“…This pooled sample, containing 35 mg total sterols, made it possible to fractionate the components by nondestructive HPLC and to analyze the isolated fractions by NMR. Here we present a comprehensive NMR analysis providing unambiguous structural assignments for 43 P. carinii sterols and correlate their identities with those previously characterized by GLC and GLC-MS (9,11,13). Most of the sterol molecular species were 24-alkylsterols, consistent with the suggestion that the Pneumocystis S -adenosyl-l -methionine:C-24 sterol methyl transferase (SAM: SMT) activity and other sterol biosynthesis reactions in this pathogen are excellent targets for the development of new therapies against PcP (2, 9 Ϫ 22).…”

mentioning

confidence: 99%

“…carinii ) organisms isolated and purified from infected lungs of corticosteroid-immunmosuppressed rats contained sterols distinct from those in the rat lung (12), and more than 20 sterol components were resolved by gas-liquid chromatography (GLC) using capillary columns (13).…”

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confidence: 99%

Comprehensive and definitive structural identities of Pneumocystis carinii sterols

Giner

Zhao

Beach

et al. 2002

Journal of Lipid Research

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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...

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“…These sterols have, for example, a D 5 double bond in the sterol nucleus but there is no evidence that Pneumocystis can form a D 5 double bond . although Pneumocystis is a fungus it does not contain ergosterol but synthesizes unique 24-alkylsterols with a single double bond in the sterol nucleus, which is at the D 7 position (Kaneshiro et al, 1994;Urbina et al, 1997;Giner et al, 2002). as is common in other animal parasites, there is abundant cholesterol in Pneumocystis, which is synthesized by the host and scavenged by the pathogen (Worsham et al, 2003).…”

Section: S-adenosylmethionine (Sam Adomet)mentioning

confidence: 99%

Biochemical research elucidating metabolic pathways inPneumocystis

Kaneshiro

2010

Parasite

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Mots clés : S-adénosyl-L-méthionine, atovaquone, buparvaquone, chorismate, erg6, erg7, erg11, sam1, gènes, lanostérol synthase, poumon, microsomes, mitochondrie, nicotine, pneumocystérol, shikimate, stigmatellin, triazoles, ubiquinones (Coenzyme Q, CoQ). Summary:Advances in sequencing the Pneumocystis carinii genome have helped identify potential metabolic pathways operative in the organism. Also, data from characterizing the biochemical and physiological nature of these organisms now allow elucidation of metabolic pathways as well as pose new challenges and questions that require additional experiments. These experiments are being performed despite the difficulty in doing experiments directly on this pathogen that has yet to be subcultured indefinitely and produce mass numbers of cells in vitro. This article reviews biochemical approaches that have provided insights into several Pneumocystis metabolic pathways. It focuses on 1) S-adenosyl-L-methionine (AdoMet; SAM), which is a ubiquitous participant in numerous cellular reactions; 2) sterols: focusing on oxidosqualene cyclase that forms lanosterol in P. carinii; SAM:sterol C-24 methyltransferase that adds methyl groups at the C-24 position of the sterol side chain; and sterol 14a-demethylase that removes a methyl group at the C-14 position of the sterol nucleus; and 3) synthesis of ubiquinone homologs, which play a pivotal role in mitochondrial inner membrane and other cellular membrane electron transport.

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Evidence for the Presence of “Metabolic Sterols” in Pneumocystis: Identification and Initial Characterization of Pneumocystis carinii Sterols

Cited by 64 publications

References 17 publications

Phytosterols are present in Pneumocystis carinii

Phytosterols are present in Pneumocystis carinii

Comprehensive and definitive structural identities of Pneumocystis carinii sterols

Biochemical research elucidating metabolic pathways inPneumocystis

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