ABSTRACT Title of Dissertation: TRANSLOCATION OF PROTEIN CARGO INTO CANDIDA ALBICANS USING CELL- PENETRATING PEPTIDES Sayanee Adhikari, Doctor of Philosophy, 2020 Dissertation directed by: Professor Amy J. Karlsson, Department of Chemical and Biomolecular Engineering Fungal infections caused by Candida albicans pose a serious threat to public health. The rising drug resistance towards azoles, the current first-line antifungal treatment, warrants novel approaches to designing and delivering new antifungal agents that target C. albicans cells. To increase the intracellular delivery of bioactive molecular cargo, we studied the use of cell-penetrating peptides (CPPs) as delivery vehicles. CPPs have been extensively used to deliver different cargoes into mammalian cells, but limited work has focused on delivery into fungal cells. To improve understanding of CPP-mediated delivery to C. albicans, we studied their ability to deliver green fluorescent protein (GFP) intracellularly. For our work, we chose the CPPs, MPG and Hst5, that have previously shown translocation into fungal cells without cargo and recombinantly produced these CPP fusions to GFP in Escherichia coli. We investigated the CPP-mediated translocation of GFP using flow cytometry. Fusion of GFP to MPG resulted in translocation into 40% of C. albicans cells, which was significantly higher than 13% cells that demonstrate translocation of GFP without a CPP. However, Hst5 did not translocate GFP into cells, with only 5% of cells exhibiting Hst5-GFP translocation. Our results demonstrate that MPG can deliver GFP, while Hst5 is not as promising. These results are consistent with molecular dynamics simulations that show MPG enters a model membrane preferentially with the N-terminal residues, whereas Hst5 fails to enter the membrane. Our results emphasize the potential of CPPs in delivering large cargo to C. albicans cells and the advantage of using both experiments and simulations to study the translocation of CPPs into C. albicans. To explore factors affecting translocation efficacy, we evaluated the aggregation of CPP-GFP fusion constructs. Using dynamic light scattering and interference scattering microscopy, our results identified aggregation of our fusions at high concentration as a possible limitation to translocation, motivating future studies of the causes of aggregation and its relationship to translocation efficiency. Our work has shown that CPPs can deliver large biomolecular cargo into fungal cells and has laid the foundation for furtHer studies to design better CPPs and to explore mechanisms limiting translocation of CPPs into fungal pathogens. TRANSLOCATION OF PROTEIN CARGO INTO CANDIDA ALBICANS USING CELL-PENETRATING PEPTIDES by Sayanee Adhikari Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2020 Advisory Committee: Professor Amy J. Karlsson, Chair Professor Jeffery B. Klauda Professor Ganesh Sriram Professor Taylor J. Woehl Professor Birthe V. Kjellerup © Copyright by Sayanee Adhikari 2020 © Copyright of subsections of Chapter 2 by American Institute of Chemical Engineers Sayanee Adhikari, Jesse A. Leissa, Amy J. Karlsson, AIChE Journal 66 (3), e16776 2019 Dedication I dedicate my PH.D. Dissertation to my parents Mr. Sujit Kumar Adhikari and Mrs. Basabi Adhikari, and my Husband Angshuk Chakraborty. Thank you, God, for helping me become who I am today. ii Acknowledgements The past 5 years in the Karlsson Lab has been an unbelievable experience. This journey would not be possible without my advisor, Dr. Amy Karlsson. I would like to take this opportunity to express my gratitude for your constant mentoring, support and encouragement. Your faith in me has built the basis of my research projects, has helped me through many ups and downs, and helped me to become an independent researcher. I am thankful to my committee members Dr. Jeffery Klauda, Dr. Ganesh Sriram, Dr. Taylor Woehl and Dr. Birthe Kjellerup for your guidance and support. I am extremely grateful to be a part of the Karlsson Lab. These are a few of the nicest people in the lab that I have met. I would like to thank my previous team members and wonderful friends Dr. Zifan Gong, Dr. Svetlana Ikonomova and Dr. Parisa Moghaddam-Taaheri for supporting me in my initial years in the lab. You have always been there for me and I don’t have adequate words to express my appreciation towards you. I would also like to thank Mahdi Ghorbani for being a good friend and for being a source of creative brainstorming. Thank you to Jesse Leissa for the constantly helping me in the lab and keeping the lab organized. Marzyeh Kheradmand-Hajibasi and Jiwon Wu recently joined our lab and has been instrumental in shaping up a lot of the experiments. I have been closely working with them for the past few months and they both have learned things very quickly and efficiently. They have good-naturedly listened to all my experiences in the lab and I trust that they will be very successful with all they do. Wish you both a very good luck! I would also like to take this opportunity to thank all the undergrads and the high-school students in the lab. Turki Alahamadi, Katherine Dura and iii Rosemary Iwuala for all their help in the project. Thank you Dinara Konakbayeva, Wright Makambi, Katherine Sniezek, Andrea Fox, and Funke Okunrinboye for making the lab an enjoyable place. In addition to members of the lab, I need to thank all others who have been instrumental in my PhD journey. I would like to thank Niti Agrawal who has always stood by me all these years in a journey that we started together and are at a stretch where we embark on the next phase. I want to thank Shakiba Nikfarjam who has been a great friend and helped me with my research projects. Dr. Derrick Ko and Dr. Viviana Monje-Galvan have been great friends. Thank you to Dan Lugar for being an amazing friend and listening to my grumbles. Most essentially, I want to thank my friends and family. Thank you to my parents for the overwhelming love, inspiration, faith and pride for the past so many years; without you I would not have been here. Every bit of my success belongs to you. Thank you to my uncle for enabling me to realize my dreams and work towards achieving them. I am thankful to my sister and brother-in-law, for being there always. Thank you to my husband and my best friend, for being supportive and for trusting in my abilities. Finally, thank you to all my friends without whose friendship and constant support I wouldn’t be here today. I would also like to thank the National Science Foundation (CBET Award #1511718) for making this work possible. I would like to thank Dr. Xianbing Zeng and Kenneth Class and for their help with the flow cytometry, and Dr. Dorothy Beckett for help with CD experiments. I would also like to thank Dr. John Fisher’s lab for their help in the fluorescence assays. I would also like to thank Dr. Mikhail Anisimov for his help with Dynamic light Scattering studies. iv Table of contents Dedication ..................................................................................................................... ii Acknowledgements ...................................................................................................... iii Table of contents ........................................................................................................... v List of tables ................................................................................................................. xi List of figures .............................................................................................................. xii List of abbreviations .................................................................................................. xiv Chapter 1: Background and motivation ........................................................................ 1 1.1 Fungal pathogen C. albicans and existing treatments ................................... 2 1.2 Cell-penetrating peptides (CPPs) .................................................................. 5 1.3 CPPs, their classifications and mechanism of entry ..................................... 6 1.4 CPP as delivery vectors ................................................................................ 9 1.4.1 Delivery of proteins as a cargo ........................................................... 10 1.4.2 Delivery of nucleic acids as a cargo ................................................... 11 1.4.3 Delivery of nanoparticles as cargo ...................................................... 12 1.4.4 Delivery of small-molecule drugs as cargo ........................................ 13 1.5 Overview of thesis ...................................................................................... 14 1.6 References ................................................................................................... 16 v Chapter 2: Methods to engineer peptides .................................................................... 27 2.1 Introduction ................................................................................................. 27 2.2 Approaches for introducing and screening diversity .................................. 29 2.2.1 Rational design .................................................................................... 30 2.2.2 Directed evolution ............................................................................... 32 2.2.3 Computational