Year: 2013
Type: article
Abstract: 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 Δ 7 C 28 and C 29 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 [2-13 C] 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 C 24 -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 i 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.me and providing guidance both in research and outside.I would also like to thank the members of my committee, both past and present for new ideas for getting past problems and assistance with techniques and procedures:
Author: Edward Allen Wright
Institution: University of Cincinnati
Language: English
Subfield: Epidemiology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W359632450