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Clemson University TigerPrints All Dissertations Dissertations 5-2017 Acetate Transport Is Essential for Survival and Virulence of Cryptococcus neoformans Grace Naanyu Kisirkoi Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Recommended Citation Kisirkoi, Grace Naanyu, "Acetate Transport Is Essential for Survival and Virulence of Cryptococcus neoformans" (2017). All Dissertations. 1915. https://tigerprints.clemson.edu/all_dissertations/1915 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]. ACETATE TRANSPORT IS ESSENTIAL FOR SURVIVAL AND VIRULENCE OF CRYPTOCOCCUS NEOFORMANS _________________________________________________________________ A Dissertation Presented to the Graduate School of Clemson University _________________________________________________________________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Genetics _________________________________________________________________ by Grace Naanyu Kisirkoi May 2017 _________________________________________________________________ Accepted by: Kerry S. Smith, Committee Chair Cheryl Ingram-Smith Lukasz Kozubowski Julia Frugoli Meredith Morris Abstract Cryptococcus neoformans is a basidiomycetous fungal pathogen that claims 625,000 lives annually worldwide. Particularly among the immunocompromised, it is the leading cause of fungal meningitis following pulmonary colonization and dissemination to the central nervous system via the blood-brain barrier. The success of C. neoformans as a pathogen is largely attributable to metabolic scavenging during starvation conditions within the host’s environment. Lung alveolar macrophages, which are among the first immune effectors to combat an initial pulmonary infection, present a glucose- and amino acid-poor environment that likely necessitates metabolism of nonpreferred carbon sources such as lactate and acetate for establishment of a pulmonary infection. While acetate transporters have been characterized in ascomycetous fungi such as Saccharomyces cerevisiae, Candida albicans, Aspergillus nidulans and Yarrowia lipolytica the role of acetate production and transport in C. neoformans is not well understood and its importance in infection has not been established. Putative acetate transporter genes, designated as ADY2 and ATO2, from the GPR1/FUN34/YAAH family have been identified in C. neoformans and are highly expressed during infection in the lung and during growth on acetate as sole carbon source. Studies have identified acetate as one of the metabolites in brain tissue biopsies of infected rats and in culture supernatant. We propose that Ady2 and Ato2 have a role in acetate transport as a metabolic adaptation during starvation and stressful environmental conditions. Growth of the ady2 single mutant, and the ady2ato2 double mutant, but not ato2 single mutant, was severely impaired in 1 mM versus 10 mM acetate plates, suggesting that Ady2 is the ii essential acetate importer during growth on low acetate. Intensity of growth defects was emphasized by a 137-fold overexpression of ADY2 during growth on acetate in relation to growth on glucose. The role of Ato2 during prolonged starvation was highlighted by diminished acetate uptake in the ato2 single mutant after glucose-grown cells were passaged into minimal media with acetate as sole carbon source. This study also uncovered a role of Ady2 and Ato2 in ammonia export and that they preferentially import acetate versus other carboxylates. Major C. neoformans virulence factors: growth at 37°C and capsule synthesis were impaired by loss of Ady2 and Ato2. Moreover, Ady2 and Ato2 were essential for virulence in mice such that ady2ato2 infected mice survived as long as uninfected controls. Additionally, Ady2 and Ato2 were additively required during coculture with human and mouse-derived phagocytes. Therefore, acetate transporters play a significant role in the survival and virulence of C. neoformans. iii Dedication To the great fountain of knowledge, the Creator; to the ever - nourishing bonds of family and irrevocable friendship, and to my unyielding ‘eternal optimist’ - thou knowest thyself! iv Acknowledgements I am very thankful for my parents: dad- Samson Ole Kisirkoi and mom- Florence Kanorio Kisirkoi who encouraged me into furthering education with a lot of confidence in my abilities. My siblings: sister- Sarah Naserian, brothers- Timothy Lemayian and Caleb Lekishon, niece- Joy Enkirotet, best friends Winnie Selle, and Steven Cogill have been very patient sounding boards. I am grateful to Clemson University for an opportunity to pursue a PhD degree and providing an environment for collaborative research. The guidance of my advisor, Dr. Kerry Smith has been helpful towards gaining valuable skills and his recommendation letters have helped me obtain career- advancement opportunities for which I am grateful. My thanks extend to Dr. Cheryl- Ingram Smith, who has been a source of ideas during lab-meetings and as part of my thesis committee. I am filled with warmth at my numerous interactions with Dr. Julia Frugoli both during our lab TA appointments and as part of my committee. Besides gaining from her wealth of knowledge in science, teaching, and grant writing, I have enjoyed scrumptious meaty meals at her house parties. I am thankful for Drs. Frugoli‘s and Amy Lawton-Rauh’s TA award nominations as well as for writing me recommendation letters to career advancement opportunities. The generosity of another one of my committee members, Dr. Meredith Morris has been helpful towards gaining cell biology skills by allowing our lab to access her lab space for flow cytometry and microscopy, thank you! I am also thankful to Dr. Lukasz Kozubowski, for thoughtful questions which spur curiosity and for generosity to provide vectors containing fluorescent tags. Support from Dr. Kerry Smith’s senior graduate students- Drs. Tonya v Taylor and Katie Glenn, and Dr. Ingram-Smith’s senior graduate student, Thanh Dang were a great help both in shaping my research and in adjusting to a new culture. The EPIC family at large has been instrumental in providing a collaborative environment for equipment and expertise-sharing. Especially, I am grateful to Drs. Kimberley Paul and Jimmy Suryadi for their help with troubleshooting my radiolabeled experiments. Dr. Paul went out of her way to connect me with Dr. Sandra Gray of the endocrine physiology lab (thank you so much!) when our scintillation counter broke down. Peipei Wu and Evan (Yijian) Qiu been a great source of support. Thanks Bridget Luckie for stimulating scientific discussions that made for an amazing final year! I have no words to thank Dr. Gary Powell and his wife, Connie Powell for their friendship, encouragement, generosity, and mentorship to me and my family. Levi Blue, Anna Williams, Ekei Eyo, Nicole Barclay, Kemi Hambolu, Martin Kimani, Wangechi Taiti, Betiglu Eshete and his family and the rest of my Clemson family, thank you for a home away from home! The 2016 Molecular Mycology Course held at MBL was a great platform to learn practical host- microbe interactions skills in a collaborative environment, thanks for the opportunity and scholarship award! I am obliged to the the Wade-Stackhouse fellowship, Professional Enrichment Grants, and the NIH and NSF, whose funding support has supported the work in this dissertation. My appreciation extends to Dr. Stuart Levitz who graciously opened his home and lab for a collaboration that has greatly enriched my PhD research. Dr. Charlie Specht and Chrono Lee of the Levitz lab have also been quite resourceful to my research topic, thanks! There is not enough room to thank all that contributed to my PhD journey, but my heartfelt gratitude goes to everyone for every little and big thing! vi Table of Contents Page Title Page...................................................................................................................... i Abstract .............................................................................................................. ii Dedication ......................................................................................................... iv Acknowledgements ............................................................................................ v Table of Contents .............................................................................................. vii CHAPTER ONE ............................................................................................................ 1 Global disease burden and control of Cryptococcus neoformans ........................ 1 Serotyping and diagnosing Cryptococcus infection ............................................ 2 Anticryptococcal therapy .................................................................................... 4 Cryptococcus neoformans host-pathogen interactions ........................................ 6 Virulence determinants of Cryptococcus neoformans ......................................... 9 Role of fungal acetate metabolism in Cryptococcus neoformans pathogenesis ................................................................................................ 11 Export and uptake of weak carboxylic acids ..................................................... 17 Transport of acetate

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