Isolation of Cholesterol by Egg Yolk

The degradation of cholesterol by Pseudomonas sp. NCIB 10590 under aerobic conditions. 1. R W Owen, 2. A N Mason and 3. R F Bilton ABSTRACT The metabolic pathway of cholesterol degradation by bacteria has not been completely established. Several possible intermediates have not been identified and many pathway delineations have not involved the use of the cholesterol molecule per se and just one bacterial species. The bacterial degradation of cholesterol by Pseudomonas sp. NCIB has been studied.

Major biotransformation products included cholest-5-en-3-one, cholest-4-en-3-one, 26-hydroxycholest-4-en-3-one, androsta-1, 4-dien-3-17-dione, cholest-4-en-3-one-26-oic acid, chol-4-en-3-one-24-oic acid, pregn-4-en-3-one-20-carboxylic acid, and pregna-1, 4-dien-3-one-20-carboxylic acid. Studies with selected intermediates have enabled the elucidation of a comprehensive pathway of cholesterol degradation by bacteria. November 1983 The Journal of Lipid Research, 24, 1500-1511. http://www. jlr. org/content/24/11/1500. short

Mycobacterial persistence requires the utilization of host cholesterol 1. Amit K. Pandey and 2. Christopher M. Sassetti * +Author Affiliations 1. Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 1. Edited by Barry R. Bloom, Harvard School of Public Health, Boston, MA, and approved January 22, 2008 (received for review November 26, 2007) Abstract A hallmark of tuberculosis is the ability of the causative agent, Mycobacterium tuberculosis, to persist for decades despite a vigorous host immune response.

Previously, we identified a mycobacterial gene cluster, mce4, that was specifically required for bacterial survival during this prolonged infection. We now show thatmce4 encodes a cholesterol import system that enables M. tuberculosis to derive both carbon and energy from this ubiquitous component of host membranes. Cholesterol import is not required for establishing infection in mice or for growth in resting macrophages. However, this function is essential for persistence in the lungs of chronically infected animals and for growth within the IFN-? -activated macrophages that predominate at this stage of infection.

This finding indicates that a major effect of IFN-? stimulation may be to sequester potential pathogens in a compartment devoid of more commonly used nutrients. The unusual capacity to catabolize sterols allows M. tuberculosis to circumvent this defense and thereby sustain a persistent infection. Published online before print March 11, 2008, doi:10. 1073/pnas. 0711159105 http://www. pnas. org/content/105/11/4376. abstract Initial Steps in the Anoxic Metabolism of Cholesterol by the DenitrifyingSterolibacterium denitrificans*¦ 1. Yin-Ru Chiang ‡ , 2. Wael Ismail ‡ , 3. Michael Muller § and 4. Georg Fuchs ‡ 1 +Author Affiliations 1. Mikrobiologie, Fakultat fur Biologie, Universitat Freiburg, Schanzlestrasse 1, D-79104 Freiburg, Germany and the §Institut fur Pharmazeutische Wissenschaften, Universitat Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany 1. 1 To whom correspondence should be addressed. Tel. : 497612032649; Fax: 497612032626; E-mail: georg. [email protected] uni-freiburg. de. Abstract The anoxic metabolism of the ubiquitous triterpene cholesterol is challenging because of its complex chemical structure, low solubility in water, low number of active functional groups, and the presence of four alicyclic rings and two quaternary carbon atoms.

Consequently, the aerobic metabolism depends on oxygenase catalyzed reactions requiring molecular oxygen as co-substrate. Sterolibacterium denitrificans is shown to metabolize cholesterol anoxically via the oxidation of ring A, followed by an oxygen-independent hydroxylation of the terminal C-25 of the side chain. The anaerobic hydroxylation of a tertiary carbon using water as oxygen donor is unprecedented and may be catalyzed by a novel molybdenum containing enzyme. First Published onFebruary 16, 2007, doi:10. 1074/jbc. M610963200May 4, 2007 The Journal of Biological Chemistry, 282,13240-13249. http://www. bc. org/content/282/18/13240. abstract Coxiella burnetii Expresses a Functional [pic]24 Sterol Reductase[pic] Stacey D. Gilk, Paul A. Beare, and Robert A. Heinzen* Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840 Received 12 July 2010/ Accepted 10 September 2010 Coxiella burnetii, the etiological agent of human Q fever, occupies a unique niche inside the host cell, where it replicates in a modified acidic phagolysosome or parasitophorous vacuole (PV).

The PV membrane is cholesterol-rich, and inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. The precise source(s) of PV membrane cholesterol is unknown, as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. C. burnetii lacks enzymes for de novo cholesterol biosynthesis; however, the organism encodes a eukaryote-like [pic]24 sterol reductase homolog, CBU1206. Absent in other prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis.

In the present study, we examined the functional activity of CBU1206. Amino acid alignments revealed the greatest sequence identity (51. 7%) with a [pic]24 sterol reductase from the soil amoeba Naegleria gruberi. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under the control of a galactose-inducible promoter. Erg4 is a yeast [pic]24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-green fluorescent protein (GFP), a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum.

Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during C. burnetii intracellular growth. Journal of Bacteriology, December 2010, p. 6154-6159, Vol. 192, No. 23 0021-9193/10/$12. 00+0     doi:10. 1128/JB. 00818-10 Copyright © 2010, American Society for Microbiology. http://jb. asm. org/cgi/content/abstract/192/23/6154

Study of Anoxic and Oxic Cholesterol Metabolism bySterolibacterium denitrificans[pic] Yin-Ru Chiang,1,[pic] Wael Ismail,1,[pic] Dimitri Heintz,2 Christine Schaeffer,2Alain Van Dorsselaer,2 and Georg Fuchs1* Mikrobiologie, Fakultat Biologie, Albert-Ludwigs-Universitat Freiburg, Freiburg, Germany,1 Laboratoire de spectrometrie de masse Bio-Organique, CRNS, ECPM, Universite Louis Pasteur, Strasbourg, France2 Received 21 September 2007/ Accepted 12 November 2007 The initial enzymes and genes involved in the anoxic metabolism of cholesterolwere studied in the denitrifying bacterium Sterolibacterium denitrificans Chol-1ST.

The second enzyme of the proposed pathway, cholest-4-en-3-one-[pic]1-dehydrogenase (AcmB), was partially purified. Based on amino acid sequence analysis, a gene probe was derived to screen a cosmid library of chromosomal DNA for the acmB gene. A positive clone comprising a 43-kbp DNA insert was sequenced. In addition to the acmB gene, the DNA fragment harbored theacmA gene, which encodes the first enzyme of the pathway, cholesteroldehydrogenase/isomerase. The acmA gene was overexpressed, and the recombinant dehydrogenase/isomerase was purified.

This enzyme catalyzes the predicted transformation of cholesterol to cholest-4-en-3-one. S. denitrificanscells grown aerobically with cholesterol exhibited the same pattern of soluble proteins and cell extracts formed the same 14C-labeled products from [14C]cholesterol as cells that were grown under anoxic, denitrifying conditions. This is especially remarkable for the late products that are formedby anaerobic hydroxylation of the cholesterol side chain with water as the oxygen donor.

Hence, this facultative anaerobic bacterium may use the anoxic pathway lacking any oxygenase-dependent reaction also under oxic conditions. This confers metabolic flexibility to such facultative anaerobic bacteria.

Journal of Bacteriology, February 2008, p. 905-914, Vol. 190, No. 3 0021-9193/08/$08. 00+0     doi:10. 1128/JB. 01525-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved. http://jb. asm. org/cgi/content/abstract/190/3/905? maxtoshow=&hits=10&RESULTFORMAT=&fulltext=cholesterol+degradation+by+bacteria&searchid=1&FIRSTINDEX=0&resourcetype=HWCIT

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