Roles of CASPA2 and HGC1 in morphological control and virulence in candida albicans

ROLES OF CASPA2 AND HGC1 IN MORPHOLOGICAL CONTROL AND VIRULENCE IN CANDIDA ALBICANS ZHENG XINDE NATIONAL UNIVERSITY OF SINGAPORE 2005 ROLES OF CASPA2 AND HGC1 IN MORPHOLOGICAL CONTROL AND VIRULENCE IN CANDIDA ALBICANS ZHENG XINDE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 TABLE OF CONTENTS ACKNOWLEDGEMENTS LIST OF CONTENTS LIST OF FIGURES LIST OF TABLES ABBREVIATIONS SUMMARY ii iii vi viii ix xi CHAPTER Introduction 1.1 Candida albicans: a polymorphic fungal pathogen 1.2 Transcriptional regulation of hyphal growth in C albicans 1.2.1 The MAP kinase pathway 1.2.2 The cAMP-dependent protein kinase A pathway 1.2.3 Hyphal specific genes 1.2.4 CaTup1-mediated repression of hyphal development 1.2.5 pH responsive pathway 1.2.6 Other factors involved in hyphal growth 3 10 12 14 1.3 Morphological control in C albicans 1.3.1 Actin and polarized growth 1.3.2 Morphological machinery controlling polarized growth 16 16 17 1.3.3 1.3.4 20 22 Cell cycle and morphological control in C albicans Septin ring and morphological control Chapter Materials and Methods 2.1 2.2 2.3 Reagents Strains and culture conditions Oligonucleotide primers 25 25 27 2.3.1 27 Primers used in the study of CaSPA2 2.5 28 29 2.4.1 2.4.2 2.4.3 2.4.4 2.4.5 2.4 2.3.2 Primers used in the study of HGC1 Recombinant DNA methods 29 30 31 32 32 Preparation of electrocompetent E coli cells Plasmid preparation and analysis Preparation of DNA probes Southern blot Northern blot C albicans manipulations 2.5.1 Transformation 33 33 iii 2.5.2 Preparation of C albicans genomic DNA 33 2.5.3 Preparation of C albicans RNA 34 35 35 2.6.1 2.6.2 CaSPA2, CaTUP1, CaNRG1, HGC1 gene deletion Plasmid constructs for GFP tagging 35 36 2.6.3 2.6 2.5.4 Cell synchronization (Centrifugal elutriation) Gene disruption and integration CaSPA2 domain-deletion constructs 37 2.6.4 Constructs in characterization of HGC1 Microscopy and fluorescence studies 2.7.1 Calcofluor and phalloidin staining Flow cytometric analysis Protein work 37 38 39 39 40 2.9.1 C albicans protein extract preparation 2.9.2 Western blot 2.9.3 Immunoprecipitation and kinase assays 2.10 Virulence test in mice 40 40 41 41 2.7 2.8 2.9 CHAPTER The role of CaSPA2 in polarity establishment and maintenance in C albicans 3.1 3.2 3.3 Introduction Comparison of Spa2 and CaSpa2 amino acid sequence Subcellular localization of CaSpa2 in yeast and hyphal cells 43 44 45 3.4 Construction of Caspa2∆ 49 3.5 Defects of Caspa2∆ cells in polarized growth 50 3.6 Actin localization in Caspa2∆ cells 55 3.7 Multinucleate Caspa2∆ cells 55 3.8 Defects in microtubule structures in Caspa2∆ cells 59 3.9 The role of different domains of CaSpa2 in C albicans growth 61 3.10 3.11 Caspa2∆ exhibited no virulence Discussion 3.11.1 Persistent and cell cycle phase independent tip localization of CaSpa2 3.11.2 Functions domains of CaSpa2 3.11.3 Function of CaSpa2 in nuclear movement 62 62 63 64 65 CHAPTER Functional characterization of HGC1 4.1 Introduction 4.2 67 Identification of a G1 cyclin-related protein in C albicans 68 iv 4.3 The expression pattern of CLN21 71 4.4 hgc1∆ was defective in hyphal growth 73 4.5 HGC1 is not required for the expression of HWP1, HYR1 and ECE1 79 4.6 HGC1 expression is regulated by cAMP/PKA pathway and CaTup1 80 4.7 Constitutive overexpression of HGC1 alone is not sufficient to induce hyphal growth Physical and functional interaction between Hgc1 and CaCdc28 Hgc1 is required to maintain hyphal tip localization of actin and CaSpa2 Hgc1 is required for virulence Discussion 4.11.1 Role of Hgc1 in hyphal morphogenesis 4.11.2 The unknown factors in germ tube formation 4.11.3 Role of Hgc1 in virulence 81 83 86 87 88 88 90 90 4.8 4.9 4.10 4.11 REFERENCE PUBLICATIONS 92 105 v List of Figures Figure 1.1 Multiple signal transduction pathways involved in hyphal program control in C albicans Figure 3.1 Partial amino acid sequence alignment of S cerevisiae Spa2 and CaSpa2 45 Figure 3.2 CaSpa2-GFP localized to sites of cell growth in C albicans 48 Figure 3.3 CaSpa2-GFP persistently localized to the tips of filaments in Catup1∆ and Canrg1∆ mutants 49 Figure 3.4 Caspa2∆ mutant exhibited defects in morphology and budding pattern during yeast growth 51 Figure 3.5 Caspa2∆ mutant showed defects in hyphal growth 53 Figure 3.6 Caspa2∆ was defective in filamentous growth on solid media 54 Figure 3.7 Actin localization in Caspa2∆ 56 Figure 3.8 Caspa2∆ mutant exhibited defects in nuclear localization 58 Figure 3.9 Spindles and cytoplasmic microtubules in Caspa2∆ 60 Figure 4.1 Relationship of Cln21 with other cyclin family proteins in S cerevisiae and C albicans 70 Figure 4.2 The expression pattern of CLN21 (I) 71 Figure 4.3 The expression of CLN21 (II) 74 Figure 4.4 HGC1 gene deletion 75 Figure 4.5 HGC1 is required for hyphal morphogenesis 77 Figure 4.6 HGC1 is required for the filamentous phenotype of Catup1∆ 79 Figure 4.7 Deletion of HGC1 did not affect the expression of HWP1, HYR1 and ECE1 80 Figure 4.8 HGC1 was not expressed in efg1∆ and Cacdc35∆ but induced normally in cph1∆ under inducing conditions 81 vi Figure 4.9 Northern blot confirmation of constitutive HGC1 expression driven by CaACT1 promoter in CAI4 and hgc1∆ 82 Figure 4.10 Interactions of Hgc1 with Cdc28 85 Figure 4.11 Role of Hgc1 in maintaining tip localization of actin and CaSpa2 87 Figure 4.12 hgc1∆ exhibits markedly reduced virulence 88 vii List of TABLES Table 2.1 C albicans and S cerevisiae strains used in this study 26 Table 3.1 Multinucleate Caspa2∆ yeast cells 58 Table 3.2 Investigation of functional domains of CaSpa2 62 viii ABBREVIATIONS a.a amino acid 5-FOA 5-fluoro orotic acid bp base pair CDK cyclin-dependent kinase Cys cysteine DAPI 4',6-diamidino-2-phenylindole DTT dithiothreitol EDTA ethylenediamine tetraacetic acid g gram GFP green fluorescence protein h hour HA haemagglutinin HSG hyphal specific gene HU hydroxyurea Kb kilobase kDa kiloDalton MAPK mitogen activated protein kinase mCi millicurie Met methionine mg milligram minute Noc nocodazole OD optical density ORF open reading frame PAGE polyacrylamide gel eletrophoresis PBS phosphate buffered saline ix PCR polymerase chain reaction UV ultraviolet µl microlitre µM micromolar x Chapter Functional characterization of Hgc1 now, the role of morphological transition in virulence is still not unequivocally established The efg1 cph1 mutant, in which both the cAMP/PKA and MAPK pathways are blocked, shows most severe defects in hyphal growth in vitro, and its virulence is greatly reduced in a mouse model of systemic infection However, the role of morphogenesis transition in virulence cannot be established because of the extensive effects due to the loss of Efg1p and Cph1p Moreover, it was found that the signaling pathways for hyphal growth not only control the morphogenesis, but also activate or derepress the expression of a group of proteins involved in diverse, infection-related functions such as adhesins, secreted aspartyl proteinases, and iron acquisition (Staab et al., 1999; Braun and Johnson, 2000; Kapteyn et al., 2000; Ramanan and Wang, 2000; Naglik et al., 2003) The specific function of HGC1 in promoting C albicans hyphal morphogenesis and its requirement for virulence provides strong evidence that morphological transition is required for the full virulence of C.albicans, though we cannot totally exclude the possibility that some other virulence features may be compromised in the HGC1 deletion mutants The unique function of Hgc1p, plus the previous findings of a variety of virulence-related features controlled by hyphal program, further reveals that C albicans has evolved a ‘pathogenesis program’ which includes a range of distinct cellular functions, each with specific contribution to different aspects of infection These genes are collectively responsible for the full virulence of the pathogen 91 Reference Reference 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albicans J Cell Sci (in press) 105 .. .ROLES OF CASPA2 AND HGC1 IN MORPHOLOGICAL CONTROL AND VIRULENCE IN CANDIDA ALBICANS ZHENG XINDE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL... role of CaSPA2 in polarity establishment and maintenance in C albicans 3.1 3.2 3.3 Introduction Comparison of Spa2 and CaSpa2 amino acid sequence Subcellular localization of CaSpa2 in yeast and. .. of Hgc1 with Cdc28 85 Figure 4.11 Role of Hgc1 in maintaining tip localization of actin and CaSpa2 87 Figure 4.12 hgc1? ?? exhibits markedly reduced virulence 88 vii List of TABLES Table 2.1 C albicans