Clemson University TigerPrints All Dissertations Dissertations December 2019 Role of Cryptococcus neoformans Pyruvate Decarboxylase and Aldehyde Dehydrogenase Enzymes in Acetate Production and Virulence Mufida Ahmed Nagi Ammar Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Recommended Citation Ammar, Mufida Ahmed Nagi, "Role of Cryptococcus neoformans Pyruvate Decarboxylase and Aldehyde Dehydrogenase Enzymes in Acetate Production and Virulence" (2019). All Dissertations. 2488. https://tigerprints.clemson.edu/all_dissertations/2488 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. Role of Cryptococcus neoformans Pyruvate Decarboxylase and Aldehyde Dehydrogenase Enzymes in Acetate Production and Virulence A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Biochemistry and Molecular Biology by Mufida Ahmed Nagi Ammar December 2019 Accepted by: Dr. Kerry Smith, Committee Chair Dr. Julia Frugoli Dr. Cheryl Ingram-Smith Dr. Lukasz Kozubowski ABSTRACT The basidiomycete Cryptococcus neoformans is is an invasive opportunistic pathogen of the central nervous system and the most frequent cause of fungal meningitis. C. neoformans enters the host by inhalation and protects itself from immune assault in the lungs by producing hydrolytic enzymes, immunosuppressants, and other virulence factors. C. neoformans also adapts to the environment inside the host, including producing metabolites that may confer survival advantages. One of these, acetate, can be kept in reserve as a carbon source or can be used to weaken the immune response by lowering local pH or as a key part of immunomodulatory molecules. Thus, cryptococcosis could be treated by targeting acetate production. The Smith laboratory has identified two potential pathways for acetate production. The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) - acetate kinase (Ack) pathway, previously thought to be present only in bacteria, converts phosphoketose sugars to acetate through acetyl- phosphate. The pyruvate decarboxylase (Pdc) and acetaldehyde dehydrogenase (Ald) pathway, found in other fungi, converts pyruvate to acetate through acetaldehyde. The genes encoding enzymes from these pathways have been shown to be upregulated during infection, suggesting that acetate production may be a required part of cryptococcal infection. In Saccharomyces cerevisiae, Pdc works with one or more Alds to produce acetate. Eight of the nine C. neoformans Alds and the sole Pdc all contributed to the cellular acetate pool, and loss of some of these enzymes reduced cell survival during growth on various carbon sources, ii under oxidative or nitrosative stress, under pseudo-hypoxia conditions, and when the cell wall integrity was disrupted. In addition, deletion mutants of of some of these enzymes affected capsule formation and melanization, two primary determinants of Cryptococcus, and led to decreased virulence in macrophages and Galleria mellonella, an invertebrate model of infection. Metabolic adaptability is an important attribute for fungal pathogens to successfully infect and cause disease. Carbon metabolism is critical for virulence in C. neoformans, but little is known about which carbon sources are utilized during infection. Lung alveolar macrophages, the first line of host defense against C. neoformans infection, provide a glucose- and amino acid-poor environment, and nonpreferred carbon sources such as lactate and acetate are likely important early in establishment of a pulmonary infection. A global screening was undertaken to identify C. neoformans proteins necessary in acetate utilization, as possible drug targets. From two libraries, together comprising 3936 gene knockouts, 41 mutants failed to grow on media with either glucose or acetate as the carbon source, or on both media. Of the known proteins lacking in these mutants, most function in gluconeogenesis, arginine biosynthesis, or mitochondrial transmembrane transport. Overall, this work elucidated the roles of C. neoformans acetate production and utilization pathways in virulence. iii DEDICATION It is with my genuine gratefulness and warmest regard that I dedicate this dissertation to my family: my beloved mom, who has always been there for me when I needed emotional support, my dad, whose dream, before he passed away, was for me to obtain my Ph.D. in the United States, and whose encouraging voice I still hear whenever I am struggling, my husband, Akram, who always puts me first, my daughter Mariam, my bright star, and my brothers Mohamed, Adel, Salah, and Nuri. Without these people in my life, this dissertation would not have been completed. I love you all! iv ACKNOWLEDGMENTS I would like to thank my advisor and committee chairman, Dr. Kerry Smith, for his continuous support of my studies and research over the years, as well as my dissertation committee members, Dr. Julia Frugoli, Dr. Cheryl Ingram-Smith, and Dr. Lukasz Kozubowski for their patience, motivation, enthusiasm, and guidance throughout my time in graduate school. The completion of my project would not have been possible without the support of everyone, including the professors, staff, and graduate students, of the Eukaryotic Pathogens Innovation Center and of the Genetics and Biochemistry Department at large. I would like to especially express my gratitude to Dr. Meredith Morris, Dr. Lesly Temesvari, and Dr. Kimberly Paul for making my day and life more cheerful and happier in hard times. I am also extremely grateful to all my fellow lab members, past and present, who helped me with my experiments and assisted me in myriad ways throughout my time at Clemson. I would like to thank my Libyan family: my husband Akram and my sweet daughter Mariam for their constant love and support, my beloved mother Salma, and my brothers, who have been my source of inspiration and who have given me strength and their continuous moral, spiritual, and emotional support when I thought of giving up. Finally, last but by no means least, I am also grateful to my American family and friends: Cheryl Jones, Nicole Clark-Mount, Hollie H. Smith, Sylvia Cole, v Sophia Altamirano, and Archana Nalin Mankad for their love and for supporting me along the way. Julia Frugoli and Cheryl Ingram Smith, who, in addition to agreeing to be on my committee and giving me technical advice, also acted as a personal friend and extended family member. vi TABLE OF CONTENTS Page TITLE PAGE ........................................................................................................ i ABSTRACT ........................................................................................................ ii ACKNOWLEDGMENTS. .................................................................................... v TABLE OF CONTENTS ................................................................................... vii LIST OF FIGURES ............................................................................................ xi LIST OF TABLES ............................................................................................. xv CHAPTER ONE LITERATURE REVIEW ................................................................................. 1 Cryptococcus neoformans: Epidemiology and Sources of Exposure ....... 1 Ecological niche of Cryptococcus ............................................................ 4 Mechanism of infection and host response .............................................. 5 Cryptococcal response to immune attack and dissemination .................. 8 Virulence Factors ................................................................................... 13 Resistance to Hypoxia ........................................................................... 29 Response to Oxidative and Nitrosative stress ....................................... 31 Degradative enzymes involved in Cryptococcus virulence .................... 33 Metabolites important for C. neoformans infection ................................. 39 vii Table of Contents (Continued) Page Four eukaryotic acetate production pathways in protists ....................... 44 Acetate production pathways in fungi .................................................... 45 Pyruvate Decarboxylase (PDC) ............................................................. 50 Aldehyde dehydrogenases (ALDs) ........................................................ 56 Aldehyde dehydrogenases in fungi ........................................................ 57 Summary and Study Objective .............................................................. 63 References ............................................................................................ 68 CHAPTER TWO THE ROLE OF CRYPTOCOCCOUS NEOFORMANS ALDEHYDE DEHYDROGENASE FAMILY MEMBERS UNDER STRESS AND IN VIRULENCE ......................................................................................... 109 Abstract................................................................................................ 109 Introduction .......................................................................................... 112 Materials and methods ........................................................................ 116