Sterol Composition of Pneumocystis jirovecii with Blocked 14α‐Demethylase Activity

J Eukaryotic Microbiology

Johnston

Nkinin

et al. 2014

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The AIDS-associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The P. carinii S-adenosylmethionine:sterol C24-methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C-24 position of the sterol side chain producing both C28 and C29 24-alkylsterols in approximately the same proportions whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography (HPLC) and proton nuclear magnetic resonance spectroscopy (1H-NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C28 sterol ergosterol as the major sterol, and also resulted in low levels of C29 sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ24(28)-sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C29 sterols as that found in P. carinii.

Section: Methodsmentioning

confidence: 99%

“…Individual sterols were isolated from total sterol samples (> 500 μg) by a combination of thin‐layer chromatography and high performance liquid chromatography (HPLC) (Giner et al. , 2006; Nkinin et al. ) (Fig.…”

Section: Methodsmentioning

confidence: 99%

Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S‐Adenosylmethionine:Sterol C‐24 Methyltransferase

J Eukaryotic Microbiology

Johnston

Nkinin

et al. 2014

Self Cite

“…Most recently, definitive structural identities of 28 sterols were elucidated for P. jirovecii with blocked 14DM activity. Individual sterols were isolated by TLC and HPLC and analyzed by NMR (75). These analyses confirmed that the major noncholesterol components had a methyl group at C-14 of the sterol nucleus ( Table 6).…”

Section: Human-derived P Jirovecii: Possible Erg11 Mu-tant Population?mentioning

confidence: 72%

Sterol metabolism in the opportunistic pathogen Pneumocystis: Advances and new insights

2004

Lipids

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Pneumocystis can transiently colonize healthy individuals without causing adverse symptoms, and most people test positive for exposure to this organism early in life. However, it can cause Pneumocystis pneumonia (PcP) in people with impaired immune systems and is a major cause of death in HIV/AIDS. Although it has close affinities to the Ascomycetes, Pneumocystis has features unlike those of any single group of fungi. For example, Pneumocystis does not synthesize ergosterol, which is consistent with the inefficacy of amphotericin B and some triazoles in clearing PcP. Pneumocystis sterols include distinct delta7 24-alkylsterols. Metabolic radiolabeling experiments demonstrated that P. carinii synthesizes sterols de novo. Cholesterol is the most abundant sterol in Pneumocystis; most, if not all, is scavenged from the mammalian host lung by the pathogen. The P. carinii erg7, erg6, and erg11 genes have been cloned, sequenced, and expressed in heterologous systems. The recombinant P. carinii S-adenosyl-L-methionine:C-24 sterol methyl transferase (SAM:SMT) has a preference for lanosterol over zymosterol as substrate, and the enzyme can catalyze the transfer of either one or two methyl groups to the C-24 position of the sterol side chain. Two different sterol compositions were detected among human-derived P. jirovecii; one was dominated by C28 and C29 sterols, and the other had high proportions of higher molecular mass components, notably the C32 sterol pneumocysterol. The latter phenotype apparently represents organisms blocked at 14alpha-demethylation of the sterol nucleus. These studies suggest that SAM:SMT is an attractive drug target for developing new chemotherapy for PcP.

“…The sterol composition of human-derived P. jirovecii has been reported (Kaneshiro et al, 1999a;Giner et al, 2004). There appears to be two distinct P. jirovecii populations (strains) that differ in their sterol profiles.…”

Section: Recombinant Pneumocystis Sterol Biosynthesis Enzymesmentioning

confidence: 99%

“…in P. jirovecii populations with high C 31 and C 32 sterols, the lanosterol derivative pneumocysterol (Kaneshiro et al, 1999b) (24Z)-ethylidenelanost-8-en-3b-ol can accumulate often comprising about half the Pneumocystisspecific sterols. Definitive structural analyses of sterols in a population with high C 31 and C 32 sterols indicated that those P. jirovecii organisms lacked 14DM activity (14DM -) (Giner et al, 2004). 24-alkyllanosterol derivatives comprised almost 90 % of P. jirovecii 14DM -whereas P. carinii isolated from rat lungs has only 5 % of these sterols.…”

Section: Recombinant Pneumocystis Sterol Biosynthesis Enzymesmentioning

confidence: 99%

Biochemical research elucidating metabolic pathways inPneumocystis

2010

Parasite

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.