DNA stress induced filamentous growth in candida albicans

DNA STRESS INDUCED FILAMENTOUS GROWTH IN CANDIDA ALBICANS SHI QINGMEI NATIONAL UNIVERSITY OF SINGAPORE 2006 DNA STRESS INDUCED FILAMENTOUS GROWTH IN CANDIDA ALBICANS SHI QINGMEI A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS First of all, I would like to express my sincere gratitude to my supervisor, associate professor Dr Yue Wang, for his continuous guidance, constant encouragement, stimulating discussion, and patience through the entire course of my Ph.D study I am also thankful to the members of my Ph.D supervisory committee, associate professor Mingjie Cai and associate professor Uttam Surana, for their advises and invaluable discussions I would also like to thank Dr Xinde Zheng, for his sharing ideas and scientific discussions, Yanming Wang for her high standard technical support and kindness, and all the other members of WY labs for the help, support, and friendship Finally, I would like to express my thanks to my husband, for his love, encouragement, and support Shi qingmei June 2006 i TABLE OF CONTENTS ACKONWLEDGEMENTS i TABLE OF CONTENTS ii LIST OF FIGURES vi LIST OF TABLES viii ABBREVIATION ix SUMMARY xi CHAPTER INTRODUCTION 1.1 Polymorphism and virulence of C albicans 1.1.1 Different morphogenic states of C albicans 1.1.2 Morphogenesis and virulence of C albicans 1.2 Transcription pathways that regulate hyphal formation in C albicans 1.2.1 The MAP kinase pathway 1.2.2 The cAMP-dependent protein kinase A pathway 1.2.3 Tup1-mediated repressive pathway 1.2.4 Other pathways 11 1.2.5 Hyphal specific genes (HSGs) 12 1.3 Relationship between filamentous growth and cell cycle progression 13 1.3.1 Relationship between cell cycle progression and filamentous growth of S cerevisiae 13 1.3.2 Relationship between cell cycle progression and hyphal growth of C Albicans 15 1.4 Pseudohyphal growth of C albicans 17 1.5 Relationship between pseudohyphal growth and checkpoint function 18 1.5.1 DNA damage and DNA replication checkpoints 19 1.5.1.1 Sensor proteins of DNA damage and replication checkpoints 20 1.5.1.2 Adaptor proteins of DNA damage and replication checkpoints 24 1.5.1.3 Effector proteins of DNA damage and replication checkpoints 30 ii 1.6 Present study 41 CHAPTER MATERIALS AND METHODS 2.1 Reagents 42 2.2 Strains and Culture conditions 42 2.3 Oligonucleotide primers 42 2.3.1 Primers for gene deletion 44 2.3.2 Primers for cloning work 44 2.3.3 Probes for Northern Blot 47 2.3.4 Primers for site-directed mutagenesis 47 2.4 Recombinant DNA methods 49 2.4.1 Preparation of electro-competent E coli cells 49 2.4.2 Plasmid preparation and analysis 50 2.4.3 Agarose gel electrophoresis 52 2.4.4 DNA probes labeling 52 2.4.5 Southern blot 53 2.4.6 Northern blot 54 2.5 C albicans manipulations 54 2.5.1 Preparation of C albicans competent cells and electroporation 54 2.5.2 Preparation of C albicans genomic DNA 55 2.5.3 Preparation of C albicans RNA 56 2.5.4 Cell synchronization (Centrifugal elutriation) 57 2.6 Gene disruption and integration 58 2.6.1 CaRAD53, CaRAD9, CaMRC1 genes deletion 58 2.6.2 CaMET3 promoter controlled CaRNR2 construct 58 2.6.3 Carad53∆, Carad9∆, Camrc1∆ rescue constructs 59 2.6.4 Site-directed mutagenesis of CaRad53 FHA domain and BRCT2 domain of CaRad9 60 2.6.5 GFP-tagging of CaTUB2 60 2.6.6 Myc-tagging of cyclins 61 iii 2.7 HU and MMS Sensitivity Tests 61 2.8 Flow cytometry analysis 62 2.9 Microscopy and fluorescence studies 62 2.10 Cell viability test (Methylene blue staining) 63 2.11 Protein work 63 2.11.1 C albicans protein purification 63 2.11.2 Western blot 64 2.11.3 Protein dephosphorylation 65 CHAPTER RESULTS 3.1 Low concentrations of HU induce constitutive filamentous growth of C albicans 66 3.2 Inhibition of DNA polymerase α or deletion of CaRNR2 also induces filamentous growth 69 3.3 HU-induced filamentous growth is independent of a number of genes known to be important for hyphal growth 73 3.4 Expression of representative hypha-specific genes is not increased during HUinduced filamentous growth 75 3.5 Constitutive polarization of actin structures and polarisome proteins at the filament tips and formation of cytoplasmic microtubules 77 3.6 DNA damaging agents also induce filamentous growth of C albicans 81 3.7 Identification of CaMRC1, CaRAD53 and CaRAD9 genes of C albicans DNA replication and damage checkpoint pathways 83 3.8 Functional characterization of CaRAD53 in cellular response to DNA replication inhibition and DNA damage 87 3.9 Functional characterization of CaMRC1 in cellular response to DNA replication inhibition and DNA damage 97 3.10 Functional characterization of CaRAD9 in cellular response to DNA replication inhibition and DNA damage 3.11 Role of the FHA Domains in CaRad53-mediated Cellular Functions 100 104 iv 3.12 Swe1 is not required for the genotoxic-stress-induced filamentous growth 111 3.13 Cellular levels of G1 and B-type cyclins during HU-induced filamentous growth 113 CHAPTER DISCUSSION 4.1 Difference between genotoxic-stress-induced filamentous growth and serum induced true hyphal growth in C albicans 115 4.2 The key components of DNA replication and damage checkpoint pathways are conserved 116 4.3 DNA checkpoints are required for the genotoxic-stress-induced filamentous growth 117 4.4 Role of CaRad53 in true hyphal growth 119 4.5 Genetic separation of CaRad53-mediated cell responses to genotoxic stress 120 4.6 Function of G1 and B-type cyclins in HU-induced filamentous growth of C albicans 121 4.7 Growth and checkpoint functions of CaMrc1 123 4.8 Relationship between filamentous growth and virulence 124 REFERENCE 127 PUBLICATIONS 149 v LIST OF FIGURES FIGURE 1.1 Signal transduction pathways regulating hyphal growth in C albicans FIGURE 3.1 HU and aphidicolin induce constitutive filamentous growth of C albicans 71 FIGURE 3.2 Switching off CaRNR2 expression leads to cell elongation 72 FIGURE 3.3 HU-induced filamentous growths of Cacph1∆, Caefg1∆, Cacph1∆/Caefg1∆, Cahgc1∆, and Cacdc35∆ 74 FIGURE 3.4 Northern blot analyses of hypha-specific gene 76 FIGURE 3.5 Actin, CaSpa2-GFP, and microtubule localization in HU-induced filamentous growth of C albicans 80 FIGURE 3.6 DNA damage by MMS or UV causes filamentous growth 82 FIGURE 3.7 Domain organization of C albicans CaRad53, Mrc1 and Rad9 in comparison with S cerevisiae orthologues 85 FIGURE 3.8 Chromosome deletion of RAD53, MRC1, or RAD9 in C albicans strain BWP17 86 FIGURE 3.9 Carad53 morphology and sensitivity to different DNA replication block and DNA damaging agents 90 FIGURE 3.10 characterization of HU- and MMS-induced cell cycle arrest of the Carad53 mutant 91 FIGURE 3.11 Phosphorylation of CaRad53 in response to different treatment 93 FIGURE 3.12 Filamentous growth defects of rad53∆ cells 95 FIGURE 3.13 Characterization of the Camrc1 cells 99 FIGURE 3.14 HU and MMS sensitivity of Carad9∆ 102 FIGURE 3.15 HU, MMS, and UV induced cell cycle arrest and 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