University of Cincinnati

UNIVERSITY OF CINCINNATI Date: 17-Dec-2009 I, Tiffany Michelle Joffrion , hereby submit this original work as part of the requirements for the degree of: Doctor of Philosophy in Pathobiology & Molecular Medicine It is entitled: Sterol biosynthesis and sterol uptake in the fungal pathogen Pneumocystis carinii Student Signature: Tiffany Michelle Joffrion This work and its defense approved by: Committee Chair: Melanie Cushion, PhD Melanie Cushion, PhD Gary Dean, PhD Gary Dean, PhD Alan Smulian, MD Alan Smulian, MD Sean Davidson, PhD Sean Davidson, PhD Laura Woollett, PhD Laura Woollett, PhD David Askew, PhD David Askew, PhD 3/2/2010 418 Sterol biosynthesis and sterol uptake in the fungal pathogen Pneumocystis carinii A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathobiology and Molecular Medicine of the College of Medicine by Tiffany M. Joffrion B.S. Dillard University, 2000 Committee Chair: Melanie T. Cushion, PhD Abstract Fungi in the genus Pneumocystis are the cause of a potentially life threatening pneumonia, Pneumocystis pneumonia (PCP). The understanding of the lifecycle, metabolism, and drug development has been hindered due to a lack of a long term in vitro culture system. Unlike most other fungi, members of the genus Pneumocystis do not appear to synthesize the major fungal sterol, ergosterol. However, genome scans and in vitro assays suggest the presence of functional genes involved in a sterol pathway. One of the goals of this work was to characterize the P. carinii sterol enzyme, lanosterol synthase (Erg7p), an essential enzyme of the sterol pathway. The activity of P. carinii Erg7p was assessed by heterologous expression of P. carinii Erg7p in a Saccharomyces cerevisiae Erg7p null mutant. Growth rates and lanosterol production were similar between S. cerevisiae expressing the P. carinii enzyme and S. cerevisiae expressing its own Erg7p under the same conditions, indicating that not only does P. carinii produce a functional Erg7p, but also that the enzyme functionally complements the S. cerevisiae enzyme. Western blotting and fluorescent localization studies revealed that P. carinii Erg7p localizes to lipid particles in S. cerevisiae as does S. cerevisiae Erg7p. A novel finding of these studies, was that P. carinii contains lipid particles, and that P. carinii Erg7p localizes to lipid particles in P. carinii. These studies indicate that P. carinii Erg7p functions similar to the S. cerevisiae enzyme, and may perform a similar function in P. carinii. Biochemical analyses of sterols within the membranes of P. carinii have shown that it utilizes cholesterol rather than ergosterol as its bulk sterol. However, P. carinii does not appear to synthesize cholesterol from a de novo pathway, but rather scavenges iii exogenous sterols from its mammalian host. S. cerevisiae is induced to undergo sterol scavenging under anaerobic conditions. Consequently, another goal of this work was to provide information on the effect of O2 on sterol biosynthesis and sterol scavenging by P. carinii. ATP measurements revealed that the viability of P. carinii is severely decreased when maintained under hypoxic conditions, and this decrease correlated with an increase in drug susceptibility. We show that uptake of exogenous cholesterol by P. carinii occurred under normal O2 tensions, indicating that sterol scavenging is not limited to anaerobic conditions. Microarray analysis indicated that hypoxic maintenance of P. carinii resulted in decreased transcription of several genes involved in sterol and lipid biosynthesis suggesting that while hypoxic conditions down-regulated genes involved in sterol biosynthesis, down-regulation of sterol biosynthesis is not a requirement for sterol scavenging in P. carinii. The ability of P. carinii to scavenge exogenous sterols under normal O2 tensions at which the sterol pathway is unaffected provides evidence that sterol scavenging may be the primary means that P. carinii utilizes to obtain its sterols. iv v Acknowledgments: The Pathobiology and Molecular Medicine Program has helped me to recognize my long-standing dream of becoming a doctor. The faculty, staff and students of the department have shared knowledge and insight that has helped to foster my growth and maturity as a research scientist, and for that I am eternally grateful. I would like to extend a special thanks both Regina Sewell and Heather Anderson, who provided not only information to help me succeed in this program, but provided moral support and encouragement throughout my career as student. My lab and extended lab have been a tremendous help to me and excellent sources of all facets of support. I would like to thank my mentor, Dr. Melanie T. Cushion for her invaluable support throughout this process. I appreciate her willingness to work with me despite her busy schedule, and her ability to teach me how think to critically and to become an independent scientist. In addition to my primary mentor, I have been blessed with two other mentors, Drs. Michael Linke and George Smulian who have helped me enumerable times over the years, and have been wonderful sources of support and advice. I would like to extend a special thank you to my parents and my siblings, particularly my sister, for their unconditional love, support, and encouragement that has allowed me to continue despite challenges. In edition I would like to thank my extended family at the COTLG Temple 51, and the Grant family for making Cincinnati, a home away from home. Lastly, I would be remiss if I did not honor God for giving me the endurance to have lasted this long. All things are possible with Him, but without Him, I am nothing. vi Table of Contents: Table of Contents vi List of Tables and Figures viii-ix List of Abbreviations x-xi Chapter 1: Introduction 1-69 Chapter 2: Functional characterization and localization 70-114 of Pneumocystis carinii lanosterol synthase Chapter 3: The effect of oxygen on viability, sterol uptake, 115-158 and transcriptional responses in Pneumocystis carinii Chapter 4: Conclusions and Future Directions 159-172 vii Lists of Tables and Figures: Putative Pneumocystis lifecycle 66 Molecular structures of cholesterol and ergosterol 67 Committed ergosterol biosynthetic pathway 68 Committed cholesterol biosynthetic pathway 69 in silico transmembrane helix predictions of PcErg7 and 101 ScErg7 (Table) CHEF Blot of P. carinii chromosomal localization 106 Multiple sequence alignment of fungal lanosterol synthases 107 Detection of P. carinii and S. cerevisiae lanosterol synthase 108 in yeast and P. carinii cell lysates Detection of P. carinii, and S. cerevisiae lanosterol synthase 109 in yeast cell lysates Growth curve comparing growth rates of yeast expressing 110 wild type ERG7, pYES2.1/PcERG7, and pYES2.1/ScERG7, and pYES2.1 Lanosterol production by yeast expressing pYES2.1/PcERG7, 111 pYES2.1/ScERG7 and wild type ERG7 PcErg7 localization to lipid particles in yeast 112 Fluorescent localization of PcErg7 to lipid particles in yeast 113 and in P. carinii Putative P. carinii sterol biosynthetic pathway 114 viii Effect of oxygen on P. carinii viability 117 Putative P. carinii genes up-regulated in response to anaerobic conditions (Table) 151 Putative P. carinii genes down-regulated in response to 152-153 anaerobic conditions (Table) Effect of oxygen tension on P. carinii viability 156 P. carinii NBD-cholesterol uptake 157 P. carinii NBD-cholesterol localization 158 Working model of Pneumocystis exposure to low oxygen and 172 high carbon dioxide concentrations in the lung ix Table of Abbreviations: PCP Pneumocystis pneumonia HAART Highly Active Antiretroviral Therapy TMP-SMX Trimethoprim Sulfamethoxazole DHPS Dihydropteroate Synthase AIDS Acquired Immune Deficiency Syndrome HIV Human Immunodeficiency Virus CDC Centers for Disease Control rRNA ribosomal Ribonucleic Acid ICBN International Code of Nomenclature MSG Major Surface Glycoprotein PFGE Pulse Field Gel Electrophoresis SREBP Sterol Regulatory Element Binding Protein LDL Low Density Lipoprotein LDLR Low Density Lipoprotein Receptor UPC2 Uptake Control Protein ECM22 Extracellular Mutant SRE Sterol Response Element ER Endoplasmic Reticulum ERG11 Lanosterol 14 demethylase ERG6 Sterol C-24 methyltransferase ERG7 Lanosterol synthase/oxidosqualene cyclase ERG3 Sterol C-5 Desaturase x ERG1 Squalene epoxidase ACAT Acyl-Coenzyme Cholesterol Acylransferase HAP1 Heme Activator Protein ROX1 Repressor of Hypoxic Genes SUT1 Sterol Uptake Gene ARS1 Anaerobic Response Element DAN1 Delayed Anaerobic Gene CYC8 Cytochrome C TUP1 Deoxythymidine Monophosphate Uptake Gene AUS1 ABC protein involved in Uptake of Sterols NMR Nuclear Magnetic Resonance DAVID Database for Annotation, Visualization and Integrated Discovery ER Endoplasmic reticulum ERAD ER Associated Degradation xi Chapter 1: Introduction Members of the genus Pneumocystis are opportunistic fungi capable of causing a lethal pneumonia in mammalian hosts. Pneumocystis colonization of immunocompetent hosts appears to have minimal clinical consequences, but colonization in hosts with debilitated or compromised immune systems may result in the development of Pneumocystis pneumonia (PCP) (139,210). Prior to the AIDS epidemic in the early 1980s, PCP was a rare occurrence seen only in malnourished children, transplant recipients, cancer patients and those with immune deficiencies (65). Today, despite highly active anti-retroviral therapy (HAART),

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