Studies on Sterol Metabolism in the Opportunistic Pathogen Pneumocystis carinii A thesis submitted to the Division of Research and Advanced Studies at the University of Cincinnati in partial fulfillment of the requirements for the degree of Master of Science In the Department of Biological Sciences McMicken College of Arts and Sciences 2012 By Edward Allen Wright B.S., Centre College, 2009 Research Advisory Committee Dr. Edna S. Kaneshiro, Ph.D., Chair Dr. Jodi R. Shann, Ph.D. Dr. Charlotte Paquin, Ph.D. 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 7 unique Δ 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 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. ii iii Acknowledgments First and foremost, I would like to thank my advisor Dr. Edna Kaneshiro for believing in 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: Dr. Jodi Shann, Dr. Charlotte Paquin, Dr. Apryll Stalcup, and Dr. Julio Urbina. I would also like to thank my lab mates: Laura Johnston, Jenny Custer, Dr. Stephenson Nkinin, Mamadou Niang, Nathan Siegel, Alexander Gainey, and Joshua Olsen, without whom this research would not have been possible. I would like to thank Julie Stacey and Catherine Hayward for their advice and use of laboratory equipment and supplies. I also want to thank Dr. Scott Keely for his assistance in getting me started in learning molecular techniques. I also owe a debt of gratitude to Ashley Dorworth for seeing me through the writing process and assistance with revisions. Finally, I would like to thank my family and friends for their support and encouragement through these past few years. I especially wish to thank my parents for all the help they’ve given me, and to thank my brother Tom for providing constant comic relief. This research was supported in part by an NIH grand ROI106084 to Dr. Kaneshiro. iv TABLE OF CONTENTS Title Page Abstract i Acknowledgments iii Table of Contents iv List of Figures viii List of Tables ix List of Abbreviations x I. Background and literature review 1 A. General overview of Pneumocystis 1 B. History of Pneumocystis 2 C. Taxonomy of Pneumocystis 3 D. Life cycle of Pneumocystis 6 E. Transmission and infection 11 F. Treatment 12 G. Sterols 14 II. Pneumocystis carinii sterol biosynthesis 16 A. Background 16 1. Pneumocystis sterols 16 2. Sterol metabolic pathways 18 B. Materials and methods 21 1. Animals 21 v 2. Preparation of P. carinii inoculum for infecting rats 23 3. Pneumocystis carinii organism preparations used for sterol analyses 24 4. Harvesting organisms for precursor incorporation assays 24 5. Incorporation of radiolabeled lanosterol 29 6. Incorporation of heavy leucine 29 6. Extraction and purification of lipids 29 8. Fractionation of lipids 30 9. Mild alkaline hydrolysis 30 10. Radioactivity measurements 31 C. Results 31 1. Incorporation of lanosterol into P. carinii sterols 31 2. Incorporation of leucine into P. carinii sterols 33 D. Discussion 34 1. Lanosterol incorporation 34 2. Leucine incorporation 35 III. Studies of the P. carinii erg6 gene expressed in Saccharomyces cerevisiae 35 A. Background 35 1. Saccharomyces cerevisiae as a model organism 35 2. S-Adenosylmethionine:sterol C-24 methyltransferaese (SAM:SMT) 36 B. Materials and methods 41 1. Transformation procedure 38 2. Saccharomyces cerevisisae cultures 41 3. Detection of recombinant protein 43 vi 4. Column clean-up and derivatization 44 5. Gas-Liquid Chromatography 45 C. Results 45 1. Genetic analysis of S. cerevisiae strains 45 2. Detection of recombinant SAM:SMT enzyme protein 46 3. GLC analysis of sterols in S. cerevisiae strains 46 4. NMR spectroscopy of isolated S. cerevisiae sterols 50 D. Discussion 50 IV. Studies on the transformation of Schizosaccharomyces pombe with the P. carinii erg6 gene 52 A. Background 52 B. Materials and Methods 53 1. Schizosaccharomyces pombe organisms 53 2. Plasmid 53 3. Amplification of gene 54 4. Construction of pREP41X-erg6 for expression in Schizosaccharomyces 57 pombe 5. Transformation procedure of Schizosaccharomyces pombe with 59 P. carinii erg6 gene 6. Verification of gene sequence integrity 61 C. Results 61 1. Transformation of Schizosaccharomyces pombe with P. carinii erg6 gene 61 2. GLC analysis of Schizosaccharomyces pombe sterols 62 vii D. Discussion 64 1. Transformation of Schizosaccharomyces pombe 64 2. GLC of sterols of ARC039 strains 67 V. Future directions 68 A. Pneumocystis carinii biochemical studies 68 B. Studies of the P. carinii erg6 gene expressed in Saccharomyces cerevisisae 69 C. Studies on the transformation of Schizosaccharomyces pombe 71 D. Summary of Work 72 VI. References 73 viii List of Figures Chapter I 1. Proposed Pneumocystis life cycle 8 2. Electron micrograph of Pneumocystis carinii 9 Chapter II 3. Pneumocystis carinii sterols 17 4. Proposed scheme for leucine incorporation into Leishmania mexicana sterols 19 5. Labeling patterns for [2-13C] leucine incorporation into Leishmania mexicana sterols 20 6. Inoculation stand and box for rat infection 25 7. Diff-Quick stain of lung smears (rat #29-77) 27 Chapter III 8. The sequence of the P. carinii erg6 gene 37 9. SAM:SMT enzyme mechanism of action 39 10. Diagram of the pYES2.1 plasmid 40 11. PCR verification of S. cerevisiae strain identities 47 12. GLC tracing of S. cerevisiae sterols 48 Chapter IV 13. Diagram of the pREP41X-pcerg6 plasmid 55 14. Restriction enzyme digest on pREP41X-pcerg6 plasmid 60 15. Colonies of the ARC039pcerg6 transformant grown on an agar plate 63 16. GLC tracings of Schizosaccharomyces pombe sterols 65 ix List of Tables Chapter I 1. Taxonomy of the genus Pneumocystis 4 2. Nomenclature of Pneumocystis organisms with their host 5 3. Table of drugs currently used against Pneumocystis infections 15 Chapter II 4. Incorporation of [U-14C] L-leucine into P. carinii sterol TLC fraction in the presence of lovastatin 22 5. Pneumocystis carinii infection score system 26 6. Incorporation of radiolabeled lanosterol into Pneumocystis carinii whole cells 32 Chapter III 7. Primers used in molecular analysis of S. cerevisiae strains 42 8. GLC analyses of Saccharomyces cerevisiae sterols 49 9. NMR analysis of isolated Saccharomyces cerevisiae sterols 51 Chapter IV 10. Primers used in creating P. carinii erg6 for insert into pREP41X plasmid 56 11. PCR program utilized for amplifying the erg6 gene for pREP41X plasmid 58 12. GLC analyses of Schizosaccharomyces pombe sterols 66 x List of abbreviations AIDS Acquired Immune Deficiency Syndrome AP Alkaline