Transcriptional regulation mechanisms involved in azole resistance in Candida species: focusing on the transcription factors Rpn4 and Mrr1 Raquel da Silva Califórnia Thesis to obtain the Master of Science Degree in Biotechnology Supervisor: Prof. Dr. Miguel Nobre Parreira Cacho Teixeira Examination Committee Chairperson: Prof. Dr. Ana Cristina Anjinho Madeira Viegas Supervisor: Prof. Dr. Miguel Nobre Parreira Cacho Teixeira Member of the Committee: Dr. Catarina Isabel Ribeiro Pimentel October 2018 ii Acknowledgements For me the development of this thesis was very challenging and involved a very extensive work, whose purpose would not have been reached without the help of some people I will mention below. First of all, I would like to thank my supervisor Professor Miguel Teixeira for the opportunity given by accepting me in his team and in this project. His tremendous support, guidance and motivation, always available to help, were crucial for the success of this work. I would like to thank Professor Isabel Sá-Correia for giving me the chance to join the Biological Sciences Research Group to develop my master thesis work. The achievement of this thesis required an indispensable help from several parts, which deserve my recognition. For the collaboration in the transcriptomic analysis herein accomplished, I thank Professor Geraldine Butler and her team, from University College of Dublin. For the supply of Candida glabrata mutants used in this work, I have to thank Professor Hiroji Chibana, from University of Chiba, Japan. For the study developed in HPLC analysis of ergosterol levels, I thank also Professor Nuno Mira for his availability and assistance. My gratitude should also be expressed towards my colleague, Pedro Pais, for the great help he has given me throughout this period, always available to help and explain anything. Many of the knowledge I would take with me were given by him, so thank you. Also, I a big thank to Mafalda Cavalheiro, who helped in several steps of my work mainly with HPLC, for her inexhaustible support in the lab as well as to all my other lab partners. And last but not least, I thank to Mónica Galocha for her help and friendship unconditionally, and because without her work, this thesis would never have been possible. For all the friendship and laughs, to my fellow of this journey, I leave a big thanks to all my friends, in special to Cristiana Ulpiano, Susana Vagueiro, Rita Simões, Pedro Monteiro, Mariana São Pedro, João Lampreia, Alexandra Balola, João Carvalho e Inês Sá, that were always present during this important stage of my life, even because half developed his thesis in the same building. To my teammates, for making me leave my house with the desire to train, for the moments of distraction and for helping me unwind my head for a few hours. Without enough words, I thank my boyfriend, Alexandre, for the indefatigable and unequaled support and patience, for always listen me carefully even though he does not realize anything I was saying. Thank you for always stay by my side and believe in me, love you more than everything. Most of all, I would like to thank my family, specially to my parents, for all the love and support throughout the course of my life, thanks for believing in me more than myself. My little sister, Rita, I hope to be always an example for her and a sister he will always be proud of. I have no words to say how much I love you all. This work was financially supported by Fundação para a Ciência e Tecnologia (FCT), contracts PTDC/BBB-BIO/4004/2014, PTDC/BII-BIO/28216/2017 and UID/BIO/04565/2013, and Programa Operacional Regional de Lisboa 2020, contract LISBOA-01-0145-FEDER-022231. iii iv Abstract Candida glabrata has emerged as the second most common cause of invasive candidiasis mainly due to the ability of this pathogenic yeast to resist to azole antifungal drugs. The transcriptional control of fluconazole drug response in C. glabrata was analyzed due to preliminary data suggesting that two transcription factors are determinants of azole drug resistance in C. glabrata, CgRpn4 (ORF CAGL0K01727g) and CgMrr1 (ORF CAGL0B03421g). Using RNA- sequencing, the regulon of CgRpn4 and CgMrr1 in control conditions and fluconazole exposure was defined. As predicted based on the role of the Saccharomyces cerevisiae homolog, CgRpn4 was found to be a major regulator of proteasome genes. In the context of fluconazole resistance mechanisms, CgRpn4 was found to be required for the up-regulation of ERG11, encoding the molecular target of azole drugs, as well as many other genes required for ergosterol biosynthesis. Consistently, Δrpn4 deletion mutant display a lower ergosterol concentration under fluconazole exposure. CgMrr1 was found to control the expression of a wide variety of genes, making it difficult to ascertain its exact role. In the context of fluconazole resistance, Mrr1 was found to up-regulate genes involved in sphingo/glycerophospholipid biosynthesis. Consistent with a role in the control of plasma membrane lipid composition, Rpn4 and Mrr1 were found to contribute to lower the plasma membrane permeability and intracellular fluconazole accumulation. The obtained results establish two new effectors of azole drug resistance and suggest that their activity in this context is the control of the plasma membrane composition, leading to decreased intracellular accumulation of fluconazole. Keywords: Candida glabrata, fluconazole resistance, CgRpn4, CgMrr1, ergosterol and sphingolipid biosynthesis. v vi Resumo Candida glabrata emergiu como a segunda causa mais comum de candidíase invasiva, principalmente devido à sua reduzida suscetibilidade a antifúngicos. O controlo transcricional em resposta ao fluconazole em C. glabrata foi analisado, porque dados preliminares sugeriram dois fatores de transcrição determinantes na resistência aos azóis, CgRpn4 (ORF CAGL0K01727g) e CgMrr1 (ORF CAGL0B03421g). Através dos dados de RNA-seq, o regulão de CgRpn4 e CgMrr1 em condições de controlo e fluconazole foi definido. Como previsto com base no papel do homólogo de Saccharomyces cerevisiae, o CgRpn4 foi considerado um importante regulador dos genes do proteassoma. No contexto dos mecanismos de resistência ao fluconazole, o CgRpn4 mostrou ser necessário para a ativação do ERG11, codifica o alvo molecular dos azóis, assim como muitos outros genes necessários para a biossíntese do ergosterol. Consistentemente, o mutante de deleção Δrpn4 apresenta uma menor concentração de ergosterol sob exposição ao fluconazole. Quanto ao CgMrr1, constatou-se que controla a expressão de uma ampla variedade de genes, tornando difícil determinar o seu papel exato. No contexto da resistência ao fluconazole, verificou-se que o Mrr1 regula positivamente os genes envolvidos na biossíntese de esfingo/glicerofosfolípidos. Consistente com o papel no controlo da composição lipídica da membrana plasmática, Rpn4 e Mrr1 contribuíram para diminuir a permeabilidade da membrana plasmática e a acumulação de fluconazole intracelular. Os resultados obtidos estabelecem dois novos efetores da resistência aos azóis e sugerem que a sua atividade neste contexto é efetuada através do controlo da composição da membrana plasmática, levando à diminuição da acumulação intracelular de fluconazole. Palavras-chave: Candida glabrata, resistência ao fluconazole, CgRpn4, CgMrr1, vias biossintéticas do ergosterol e dos esfingolipídios. vii viii Table of contents Acknowledgements ................................................................................................................................. iii Abstract.....................................................................................................................................................v Resumo .................................................................................................................................................. vii 1. Introduction ...................................................................................................................................... 1 1.1. Candida infections ................................................................................................................... 1 1.2. Candida glabrata ..................................................................................................................... 1 1.3. Antifungal agents used in the treatment of Candida infections ............................................... 3 1.4. Molecular mechanisms of resistance to azole drugs: focus on fluconazole ........................... 5 1.4.1. Efflux pump overexpression ............................................................................................ 6 1.4.2. Drug target overexpression ............................................................................................. 7 1.4.3. Drug target alteration ....................................................................................................... 7 1.4.4. Bypass pathways ............................................................................................................. 8 1.4.5. Sterol biosynthesis pathway alterations .......................................................................... 8 1.4.6. Mitochondrial DNA deficiency .......................................................................................... 9 1.5. The network of transcription regulators that control azole drug resistance, including an inter- species comparison ..........................................................................................................................