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Studies on Sterol Metabolism in the Opportunistic Pathogen Pneumocystis carinii

Wright, Edward A
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1367934810

Abstract Details

2013, MS, University of Cincinnati, Arts and Sciences: Biological Sciences.
Pneumocystis is a genus of opportunistic fungi responsible for a type of pneumonia in immunocompromised hosts. The pathways by which these organisms synthesize sterols have been found to be unique amongst fungi. While most fungi have ergosterol as their main sterol, its de novo synthesis does not occur in Pneumocystis. However, Pneumocystis does produce unique Δ7 C28 and C29 24-alkyl sterols, while using cholesterol scavenged from the mammalian host as its main sterol. This thesis presents several distinct lines of research, all of which address sterol biosynthesis in Pneumocystis. The first aim was to elucidate the sterol biosynthesis pathways utilized by the organism. For this, cryopreserved P. carinii were intratracheally administered to corticosteroid-immunosuppressed rats to initiate infection. When moribund, the rats were sacrificed and P. carinii organisms were isolated and purified from the infected lungs. These organisms were incubated with labeled sterol precursors, namely [2-13C] leucine to determine whether this amino acid is a precursor of the organism's sterols. Sterols were then isolated from the organisms and analyzed by Nuclear Magnetic Resonance (NMR) spectroscopy. The second aim was to continue studies initiated in our laboratory on a critical P. carinii enzyme S-adenosylmethionine: sterol C24-methyltransferase (SAM:SMT) expressed in the budding yeast Saccharomyces cerevisiae. This protein, coded by the erg6 gene, has been previously expressed in the bacterium Escherichia coli and the ciliate Tetrahymena thermophila, but these systems were shown to be of limited utility. In addition to S. cerevisiae, fission yeast Schizosaccharomyces pombe was studied as a potential expression system. Work previously done in the laboratory initiated studies on the expression of the P. carinii SAM:SMT in the S. cerevisiae erg6 null mutant. My contribution to this project was to provide gas-liquid chromatography (GLC) analyses of the sterol profiles of wild type, the null mutant and the null mutant transformed with the P. carinii erg6 gene. The sterol profiles of the three strains were characterized by both GLC analysis and by isolation of individual sterols by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) followed by definitive structural analysis by Nuclear Magnetic Resonance (NMR) spectroscopy. These studies verified complementation of the recombinant enzyme protein. The goals of the third project were to initiate studies on the expression of the P. carinii erg6 gene in the fission yeast Schizosaccharomyces pombe. Confirmation of recombinant protein production was to be demonstrated by Western blot analysis. Also, whether or not complementation was achieved was to be demonstrated by GLC sterol profile analyses of S. pombe erg6 null mutant transformed with the P. carinii erg6 gene. While significant insights were made into developing the protocol, time did not permit the completion of the project. However, the sterol profiles of the parental and knockout strains were both characterized by GLC analysis.
Edna Sayomi Kaneshiro, Ph.D. (Committee Chair)
Charlotte Paquin, Ph.D. (Committee Member)
Jodi Shann, Ph.D. (Committee Member)
72 p.
Biology
Pneumocystis; sterol; opportunistic pathogen; recombinant protein; metabolism; lipid;

Recommended Citations

Citations

  • Wright, E. A. (2013). Studies on Sterol Metabolism in the Opportunistic Pathogen Pneumocystis carinii [Master's thesis, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1367934810

    APA Style (7th edition)

  • Wright, Edward. Studies on Sterol Metabolism in the Opportunistic Pathogen Pneumocystis carinii. 2013. University of Cincinnati, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1367934810.

    MLA Style (8th edition)

  • Wright, Edward. " Studies on Sterol Metabolism in the Opportunistic Pathogen Pneumocystis carinii." Master's thesis, University of Cincinnati, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1367934810

    Chicago Manual of Style (17th edition)

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This open access ETD is published by University of Cincinnati and OhioLINK.