CHARACTERIZATION OF CANDIDA ALBICANS CYCLASE-ASSOCIATED PROTEIN CAP1 AND ITS ROLES IN MORPHOGENESIS — G-actin Assoicates with the Adenylyl Cyclase Cyr1 through Cap1 and Regulates cAMP Synthesis in Candida albicans Hyphal Morphogenesis ZOU HAO NATIONAL UNIVERSITY OF SINGAPORE 2008 CHARACTERIZATION OF CANDIDA ALBICANS CYCLASE-ASSOCIATED PROTEIN CAP1 AND ITS ROLES IN MORPHOGENESIS — G-actin Assoicates with the Adenylyl Cyclase Cyr1 through Cap1 and Regulates cAMP Synthesis in Candida albicans Hyphal Morphogenesis ZOU HAO A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2008 -i- ACKNOWLEDGEMENTS First and foremost, my deepest gratitude goes to my supervisor, Dr Yue Wang (Associate Professor) His constant encouragement, insightful guidance and stimulating discussions help me to walk all the way through the stages of research and thesis-writing Second, I would like to express my sincere gratitude to members of my Ph.D supervisor committee, Associate Professor Mingjie Cai and Associate Professor Edward Manser, for their constructive instructions and suggestions in improving my research work through all these years I also owe my heartfelt gratitude to all the past and present members in YW lab, in particular, Dr Haoming Fang for his teaching me all the microbiological techniques when I joined the lab, Dr Chen Bai for his valuable suggestions on my thesis-writing, and Ms Yanming Wang for her friendship and high-standard technical support Last, I am glad to extend my gratitude to my family including my parents, my parents in law, my dear husband and my beloved son It was really a laborious task to accomplish a Ph.D research, but your extensive supports made the whole process full of pleasure Zou Hao August 2008 - ii - TABLE OF CONTENTS ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF FIGURES vii LIST OF TABLES ix ABBREVIATIONS x SUMMARY xi CHAPTER Introduction 1.1 Pathogenesis of C albicans 1.1.1 Candidiasis 1.1.2 Behaviors of C albicans during infection 1.1.3 Host defense systems 1.2 Polymorphism of C albicans 1.3 The Regulation of morphological transitions 1.3.1 The cAMP-dependent protein kinase A pathway 10 1.3.2 Other pathways involved in hyphal growth 12 1.3.3 Hypha-specific genes 15 1.3.4 Cell cycle inhibition and pseudohyphal growth 18 1.4 Polarity establishment 19 1.4.1 Actin 19 1.4.2 The Rho-GTPase Cdc42 and its regulation 20 1.4.3 Polarisome and Spitzenkörper 22 1.4.4 Early phosphorylation of the septin Cdc11 24 1.5 Cyclase-associated protein (Cap1) 25 1.5.1 Evolutionary conservation of CAP proteins 25 1.5.2 The actin-regulatory function of Cap1 27 1.5.3 Regulation of adenylyl cyclase by Cap1 in fungi 29 1.6 Research objectives 30 CHAPTER Materials and Methods 32 2.1 Reagents 32 - iii - 2.2 Strains and culture conditions 32 2.3 C albicans and S cerevisiae manipulations 34 2.3.1 Transformation 34 2.3.2 Preparation of C albicans genomic DNA 34 2.3.3 Preparation of C albicans RNA 35 2.3.4 Preparation of C albicans total cell lysates 36 2.3.5 Yeast two-hybrid assay 37 2.4 Gene disruption and expression 37 2.4.1 C albicans gene deletion 37 2.4.2 Construction of Cap1 domain-deletion mutants and epitope-tagging of proteins 40 2.5 DNA work 41 2.5.1 Oligonucleotide primers and PCR 41 2.5.2 Recombinant DNA methods 41 2.5.3 Transformation of E coli 42 2.5.4 Plasmid preparation and analysis 43 2.5.5 Preparation of DNA probes 44 2.6 RNA work 45 2.6.1 RT-PCR 45 2.6.2 Real time (RT) PCR 45 2.7 Protein work 46 2.7.1 Western blot analysis 46 2.7.2 Immunoprecipitation (IP) 47 2.7.3 Expression and purification of GST-fusion proteins 47 2.7.4 Actin depletion pull-down assays 48 2.8 Microscopy and fluorescence studies 49 2.8.1 Microscopy 49 2.8.2 Endocytosis assays 49 2.8.3 Actin staining by rhodamine-phalloidin 50 2.9 Adenylyl cyclase activity and cAMP assays 50 2.9.1 cAMP assay in vivo 50 2.9.2 Adenylyl cyclase activity assay in vitro 54 - iv - CHAPTER Characterization of C albicans Cap1 and its Function 56 in Morphogenesis 3.1 Introduction 56 3.2 Expressing Cap1 mutants in C albicans 56 3.3 Interaction of Cap1 with actin and Cyr1 61 3.3.1 The C-terminal part of Cap1 binds ADP-G-actin in vitro 3.3.2 61 The N-terminal part of Cap1 binds the adenylyl cyclase Cyr1 in vitro 3.3.3 62 Cyr1, Cap1 and actin form a complex in vivo 63 3.4 Influence of Cap1 on the actin cytoskeleton 67 3.4.1 Subcellular locations of Cap1 67 3.4.2 Other effects of Cap1 on actin cytoskeleton 70 3.5 Role of Cap1 in Cyr1 activation 3.5.1 71 Cap1 N-terminal part is not sufficient for optimal control of cellular cAMP levels during hyphal induction 3.5.2 71 Cap1 C-terminal part is required for optimal cAMP production during hyphal growth 73 3.6 Effects of Cap1 mutants on cell morphology 75 3.6.1 Effects on yeast cells 75 3.6.2 Effects on Cap1 mutants on HU-induced filamentous growth 77 3.6.3 Effects on hyphal growth 78 3.7 Summary 82 CHAPTER The Cellular Status of the Actin Cytoskeleton Affects the 84 cAMP Signaling Pathway through Cap1 4.1 Introduction 84 4.2 External cAMP alleviates the inhibitory effect of LatA on the expression of HSGs 85 4.3 The effect of LatA on HSG expression partly depends on the C-terminal part of Cap1 87 4.4 LatA and other actin-binding drugs affect cAMP production in cell lysates 89 4.5 LatA and Cyto-A affect cAMP production in purified Cyr1/Cap1/actin complex 92 -v- 4.6 Investigations of how actin toxins affect Cyr1 activity through Cap1 94 4.7 Summary 97 CHAPTER Discussions 99 5.1 G-actin plays an important role in regulating Cyr1 activity through Cap1 99 5.2 Repression of the cAMP pathway by actin depolymerizing toxins 101 5.3 The capacity of Cyr1/Cap1/actin ternary complex 104 5.4 The role of the cAMP spike during hyphal induction 105 5.5 Conclusion 106 REFERENCES 109 PUBLICATIONS 119 - vi - List of Figures Figure 1.1.3 A simplified view of T helper responses to C albicans Figure 1.2.1 Different growth forms of C albicans Figure 1.3.1 Regulation of polymorphism in C albicans by multiple signaling pathways 13 Schematic description of Cap1 (C functional domains and conserved regions 26 Figure 1.5.1 albicans) Figure 3.2.1 Chromosomal deletion of CAP1 58-59 Figure 3.3.1 Cap1 binds ADP-actin monomers 62 Figure 3.3.2 Association of Cyr1 and Cap1 63 Figure 3.3.3 Cyr1, Cap1 and actin interactions in vivo 66 Figure 3.4.1 Cellular localizations of Cap1 69 Figure 3.4.2 Cap1 regulation of endocytosis 71 Figure 3.5.1 Increase of cellular cAMP levels during seruminduced hyphal growth 72 Figure 3.5.2 Influence of Cap1 C-terminal part in Cyr1 activation 74-75 Figure 3.6.1 Effects of Cap1 mutants on yeast cells 76 Figure 3.6.2 Effects of CYR1 and CAP1 mutants on HU-induced filamentous growth 78 Hyphal growth of Cap1 mutants under serum induction 80 Figure 3.6.3 Figure 3.6.3.2 Figure 4.2 Hyphal growth of CAP1 mutants in RPMI medium, Lee’s medium, Spider medium and embedding conditions Effect of LatA on the expression of HSGs and the role of Cap1 in this process 81 86-87 Continue on next page - vii - Continue from previous page Figure 4.3 Effect of LatA on the expression of HSGs in strains expressing truncated Cap1 88-89 Figure 4.4 cAMP production in cell lysates 91 Figure 4.5 cAMP production by affinity-purified containing complex in vitro Figure 4.6 Cyr1- Investigation of how the actin toxins affect Cyr1 activity through Cap1 93 95-96 - viii - LIST OF TABLES Table 1.3.3 HSGs are involved in diverse cellular functions 16 Table 2.1 C albicans strains used in this study 33 Table 2.2 Drugs and treatment conditions 33 Table 2.3 Oligonucleotide primers used in this study 38-39 - ix - Chapter5 Discussion Using purified Cyr1/Cap1/actin complexes to study the roles of each component in signal sensing and activation of the Cyr1 activity 108 References REFERENCES Amberg DC, Basart E, Botstein D (1995) Defining protein interactions with yeast actin in vivo Nat Struct Biol 2: 28-35 Ayscough KR (2000) Endocytosis and the development of cell polarity in yeast require a dynamic F-actin cytoskeleton Curr Biol 10: 1587-90 Ayscough KR (2005a) Coupling actin dynamics to the endocytic process in Saccharomyces cerevisiae Protoplasma 226: 81-8 Ayscough KR (2005b) Defining protein modules for endocytosis Cell 123: 188-90 Bahn YS, Sundstrom P (2001) CAP1, an adenylate cyclase-associated protein gene, regulates bud-hypha transitions, filamentous growth, and cyclic AMP levels and is required for virulence of Candida albicans J Bacteriol 183: 3211-23 Balcer HI, Goodman AL, Rodal AA, Smith E, Kugler J, Heuser JE et al (2003) Coordinated regulation of actin filament turnover by a high-molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1 Curr Biol 13: 2159-69 Bassilana M, Hopkins J, Arkowitz RA (2005) Regulation of the Cdc42/Cdc24 GTPase module during Candida albicans hyphal growth Eukaryot Cell 4: 588-603 Berman J, Sudbery PE (2002) Candida Albicans: a molecular revolution built on lessons from budding yeast Nat Rev Genet 3: 918-30 Bertling E, Hotulainen P, Mattila PK, Matilainen T, Salminen M, Lappalainen P (2004) Cyclase-associated protein (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells Mol Biol Cell 15: 2324-34 Bertling E, Quintero-Monzon O, Mattila PK, Goode BL, Lappalainen P (2007) Mechanism and biological role of profilin-Srv2/CAP interaction J Cell Sci 120: 1225-34 Biswas S, Van Dijck P, Datta A (2007) Environmental sensing and signal transduction pathways regulating morphopathogenic determinants of Candida albicans Microbiol Mol Biol Rev 71: 348-76 Blanchin-Roland S, Da Costa G, Gaillardin C (2008) Ambient pH signalling in the yeast Yarrowia lipolytica involves YlRim23p/PalC, which interacts with Snf7p/Vps32p, but does not require the long C terminus of YlRim9p/PalI Microbiology 154: 1668-76 Bockmuhl DP, Ernst JF (2001) A potential phosphorylation site for an A-type kinase in the Efg1 regulator protein contributes to hyphal morphogenesis of Candida albicans Genetics 157: 1523-30 109 References Bockmuhl DP, Krishnamurthy S, Gerads M, Sonneborn A, Ernst JF (2001) Distinct and redundant roles of the two protein kinase A isoforms Tpk1p and Tpk2p in morphogenesis and growth of Candida albicans Mol Microbiol 42: 1243-57 Boken DJ, Swindells S, Rinaldi MG (1993) Fluconazole-resistant Candida albicans Clin Infect Dis 17: 1018-21 Borneman AR, Hynes MJ, Andrianopoulos A (2002) A basic helix-loop-helix protein with similarity to the fungal morphological regulators, Phd1p, Efg1p and StuA, controls conidiation but not dimorphic growth in Penicillium marneffei Mol Microbiol 44: 621-31 Brandtzaeg P, Gabrielsen TO, Dale I, Muller F, Steinbakk M, Fagerhol MK (1995) The leucocyte protein L1 (calprotectin): a putative nonspecific defence factor at epithelial surfaces Adv Exp Med Biol 371A: 201-6 Braun BR, Johnson AD (1997) Control of filament formation in Candida albicans by the transcriptional repressor TUP1 Science 277: 105-9 Braun BR, Kadosh D, Johnson AD (2001) NRG1, a repressor of filamentous growth in C.albicans, is down-regulated during filament induction EMBO J 20: 4753-61 Brennwald P, Rossi G (2007) Spatial regulation of exocytosis and cell polarity: yeast as a model for animal cells FEBS Lett 581: 2119-24 Brown AJ, Gow NA (1999) Regulatory networks controlling Candida albicans morphogenesis Trends Microbiol 7: 333-8 Calderone R (2001) Candida and Candidiasis vol one American Society Microbiolgy Cantore ML, Galvagno MA, Passeron S (1983) cAMP levels and in situ measurement of adenylate cyclase and cAMP phosphodiesterase activities during yeast-to-hyphae transition in the dimorphic fungus Mucor rouxii Cell Biol Int Rep 7: 947-54 Chaudhry F, Guerin C, von Witsch M, Blanchoin L, Staiger CJ (2007) Identification of Arabidopsis cyclase-associated protein as the first nucleotide exchange factor for plant actin Mol Biol Cell 18: 3002-14 Chen J, Lane S, Liu H (2002) A conserved mitogen-activated protein kinase pathway is required for mating in Candida albicans Mol Microbiol 46: 1335-44 Clark KL, Feldmann PJ, Dignard D, Larocque R, Brown AJ, Lee MG et al (1995) Constitutive activation of the Saccharomyces cerevisiae mating response pathway by a MAP kinase kinase from Candida albicans Mol Gen Genet 249: 609-21 Cloutier M, Castilla R, Bolduc N, Zelada A, Martineau P, Bouillon M et al (2003) The two isoforms of the cAMP-dependent protein kinase catalytic subunit are 110 References involved in the control of dimorphism in the human fungal pathogen Candida albicans Fungal Genet Biol 38: 133-41 Cooper JA (1987) Effects of cytochalasin and phalloidin on actin J Cell Biol 105: 1473-8 Court H, Sudbery P (2007) Regulation of Cdc42 GTPase activity in the formation of hyphae in Candida albicans Mol Biol Cell 18: 265-81 Crampin H, Finley K, Gerami-Nejad M, Court H, Gale C, Berman J et al (2005) Candida albicans hyphae have a Spitzenkorper that is distinct from the polarisome found in yeast and pseudohyphae J Cell Sci 118: 2935-47 Csank C, Schroppel K, Leberer E, Harcus D, Mohamed O, Meloche S et al (1998) Roles of the Candida albicans mitogen-activated protein kinase homolog, Cek1p, in hyphal development and systemic candidiasis Infect Immun 66: 2713-21 d'Enfert C (2006) Biofilms and their role in the resistance of pathogenic Candida to antifungal agents Curr Drug Targets 7: 465-70 d'Ostiani CF, Del Sero G, Bacci A, Montagnoli C, Spreca A, Mencacci A et al (2000) Dendritic cells discriminate between yeasts and hyphae of the fungus Candida albicans Implications for initiation of T helper cell immunity in vitro and in vivo J Exp Med 191: 1661-74 Davis D (2003) Adaptation to environmental pH in Candida albicans and its relation to pathogenesis Curr Genet 44: 1-7 Davis D, Edwards JE, Jr., Mitchell AP, Ibrahim AS (2000a) Candida albicans RIM101 pH response pathway is required for host-pathogen interactions Infect Immun 68: 5953-9 Davis D, Wilson RB, Mitchell AP (2000b) RIM101-dependent and-independent pathways govern pH responses in Candida albicans Mol Cell Biol 20: 971-8 De Pauw BE (2001) Treatment of documented and suspected neutropenia-associated invasive fungal infections J Chemother 13 Spec No 1: 181-92 Deeks MJ, Rodrigues C, Dimmock S, Ketelaar T, Maciver SK, Malho R et al (2007) Arabidopsis CAP1 - a key regulator of actin organisation and development J Cell Sci 120: 2609-18 DeYoung BJ, Innes RW (2006) Plant NBS-LRR proteins in pathogen sensing and host defense Nat Immunol 7: 1243-9 Dodatko T, Fedorov AA, Grynberg M, Patskovsky Y, Rozwarski DA, Jaroszewski L et al (2004) Crystal structure of the actin binding domain of the cyclase-associated protein Biochemistry 43: 10628-41 Edwards S (1996) Balanitis and balanoposthitis: a review Genitourin Med 72: 155-9 111 References Enloe B, Diamond A, Mitchell AP (2000) A single-transformation gene function test in diploid Candida albicans J Bacteriol 182: 5730-6 Erbagci Z (2004) Topical therapy for dermatophytoses: should corticosteroids be included? Am J Clin Dermatol 5: 375-84 Fang HM, Wang Y (2006) RA domain-mediated interaction of Cdc35 with Ras1 is essential for increasing cellular cAMP level for Candida albicans hyphal development Mol Microbiol 61: 484-96 Feng Q, Summers E, Guo B, Fink G (1999) Ras signaling is required for seruminduced hyphal differentiation in Candida albicans J Bacteriol 181: 6339-46 Fidel PL, Jr., Lynch ME, Sobel JD (1993) Candida-specific cell-mediated immunity is demonstrable in mice with experimental vaginal candidiasis Infect Immun 61: 1990-5 Field J, Vojtek A, Ballester R, Bolger G, Colicelli J, Ferguson K et al (1990) Cloning and characterization of CAP, the S cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein Cell 61: 319-27 Fonzi WA (1999) PHR1 and PHR2 of Candida albicans encode putative glycosidases required for proper cross-linking of beta-1,3- and beta-1,6-glucans J Bacteriol 181: 7070-9 Fonzi WA, Irwin MY (1993) Isogenic strain construction and gene mapping in Candida albicans Genetics 134: 717-28 Frangioni JV, Neel BG (1993) Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins Anal Biochem 210: 179-87 Freeman NL, Lila T, Mintzer KA, Chen Z, Pahk AJ, Ren R et al (1996) A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization Mol Cell Biol 16: 548-56 Futcher AB (1991) Saccharomyces cerevisiae cell cycle: cdc28 and the G1 cyclins Semin Cell Biol 2: 205-12 Gieselmann R, Mann K (1992) ASP-56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase-associated protein from yeast FEBS Lett 298: 149-53 Gottwald U, Brokamp R, Karakesisoglou I, Schleicher M, Noegel AA (1996) Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin Mol Biol Cell 7: 261-72 Gourlay CW, Ayscough KR (2006) Actin-induced hyperactivation of the Ras signaling pathway leads to apoptosis in Saccharomyces cerevisiae Mol Cell Biol 26: 6487-501 112 References Gow NA, Knox Y, Munro CA, Thompson WD (2003) Infection of chick chorioallantoic membrane (CAM) as a model for invasive hyphal growth and pathogenesis of Candida albicans Med Mycol 41: 331-8 Green AR, Begley CG (1992) SCL and related hemopoietic helix-loop-helix transcription factors Int J Cell Cloning 10: 269-76 Grove SN, Bracker CE (1970) Protoplasmic organization of hyphal tips among fungi: vesicles and Spitzenkorper J Bacteriol 104: 989-1009 Gulli MP, Jaquenoud M, Shimada Y, Niederhauser G, Wiget P, Peter M (2000) Phosphorylation of the Cdc42 exchange factor Cdc24 by the PAK-like kinase Cla4 may regulate polarized growth in yeast Mol Cell 6: 1155-67 Harcus D, Nantel A, Marcil A, Rigby T, Whiteway M (2004) Transcription profiling of cyclic AMP signaling in Candida albicans Mol Biol Cell 15: 4490-9 Harris SD, Read ND, Roberson RW, Shaw B, Seiler S, Plamann M et al (2005) Polarisome meets spitzenkorper: microscopy, genetics, and genomics converge Eukaryot Cell 4: 225-9 Hawser SP, Douglas LJ (1994) Biofilm formation by Candida species on the surface of catheter materials in vitro Infect Immun 62: 915-21 Hazan I, Liu H (2002) Hyphal tip-associated localization of Cdc42 is F-actin dependent in Candida albicans Eukaryot Cell 1: 856-64 Head BP, Patel HH, Roth DM, Murray F, Swaney JS, Niesman IR et al (2006) Microtubules and actin microfilaments regulate lipid raft/caveolae localization of adenylyl cyclase signaling components J Biol Chem 281: 26391-9 Henriques M, Azeredo J, Oliveira R (2007) The involvement of physico-chemical interactions in the adhesion of Candida albicans and Candida dubliniensis to epithelial cells Mycoses 50: 391-6 Hubberstey AV, Mottillo EP (2002) Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization FASEB J 16: 487-99 Hull CM, Raisner RM, Johnson AD (2000) Evidence for mating of the "asexual" yeast Candida albicans in a mammalian host Science 289: 307-10 Jain P, Akula I, Edlind T (2003) Cyclic AMP signaling pathway modulates susceptibility of candida species and Saccharomyces cerevisiae to antifungal azoles and other sterol biosynthesis inhibitors Antimicrob Agents Chemother 47: 3195-201 Johnson DI (1999) Cdc42: An essential Rho-type GTPase controlling eukaryotic cell polarity Microbiol Mol Biol Rev 63: 54-105 113 References Jong AY, Chen SH, Stins MF, Kim KS, Tuan TL, Huang SH (2003) Binding of Candida albicans enolase to plasmin(ogen) results in enhanced invasion of human brain microvascular endothelial cells J Med Microbiol 52: 615-22 Jung WH, Stateva LI (2003) The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans Microbiology 149: 2961-76 Kadosh D, Johnson AD (2001) Rfg1, a protein related to the Saccharomyces cerevisiae hypoxic regulator Rox1, controls filamentous growth and virulence in Candida albicans Mol Cell Biol 21: 2496-505 Kaksonen M, Sun Y, Drubin DG (2003) A pathway for association of receptors, adaptors, and actin during endocytic internalization Cell 115: 475-87 Kasper LH, Buzoni-Gatel D (1998) Some Opportunistic Parasitic Infections in AIDS: Candidiasis, Pneumocystosis, Cryptosporidiosis, Toxoplasmosis Parasitol Today 14: 150-6 Kawamukai M, Ferguson K, Wigler M, Young D (1991) Genetic and biochemical analysis of the adenylyl cyclase of Schizosaccharomyces pombe Cell Regul 2: 15564 Kim K, Galletta BJ, Schmidt KO, Chang FS, Blumer KJ, Cooper JA (2006) Actinbased motility during endocytosis in budding yeast Mol Biol Cell 17: 1354-63 Kim K, Yamashita A, Wear MA, Maeda Y, Cooper JA (2004) Capping protein binding to actin in yeast: biochemical mechanism and physiological relevance J Cell Biol 164: 567-80 Kirkpatrick CH, Rich RR, Graw RG, Jr., Smith TK, Mickenberg I, Rogentine GN (1971) Treatment of chronic mucocutaneous moniliasis by immunologic reconstitution Clin Exp Immunol 9: 733-48 Klengel T, Liang WJ, Chaloupka J, Ruoff C, Schroppel K, Naglik JR et al (2005) Fungal adenylyl cyclase integrates CO2 sensing with cAMP signaling and virulence Curr Biol 15: 2021-6 Kobe B, Deisenhofer J (1994) The leucine-rich repeat: a versatile binding motif Trends Biochem Sci 19: 415-21 Kohler JR, Fink GR (1996) Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signaling components have defects in hyphal development Proc Natl Acad Sci U S A 93: 13223-8 Kufer TA (2008) Signal transduction pathways used by NLR-type innate immune receptors Mol Biosyst 4: 380-6 Kuo SC, Lampen JO (1975) Action of cytochalasin A, a sulfhydryl-reactive agent, on sugar metabolism and membrane-bound adenosine triphosphatase of yeast Biochim Biophys Acta 389: 145-53 114 References Lamarche-Vane N, Hall A (1998) CdGAP, a novel proline-rich GTPase-activating protein for Cdc42 and Rac J Biol Chem 273: 29172-7 Lamb TM, Mitchell AP (2003) The transcription factor Rim101p governs ion tolerance and cell differentiation by direct repression of the regulatory genes NRG1 and SMP1 in Saccharomyces cerevisiae Mol Cell Biol 23: 677-86 Lane S, Birse C, Zhou S, Matson R, Liu H (2001a) DNA array studies demonstrate convergent regulation of virulence factors by Cph1, Cph2, and Efg1 in Candida albicans J Biol Chem 276: 48988-96 Lane S, Zhou S, Pan T, Dai Q, Liu H (2001b) The basic helix-loop-helix transcription factor Cph2 regulates hyphal development in Candida albicans partly via TEC1 Mol Cell Biol 21: 6418-28 Lassing I, Schmitzberger F, Bjornstedt M, Holmgren A, Nordlund P, Schutt CE et al (2007) Molecular and structural basis for redox regulation of beta-actin J Mol Biol 370: 331-48 Leberer E, Harcus D, Broadbent ID, Clark KL, Dignard D, Ziegelbauer K et al (1996) Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans Proc Natl Acad Sci U S A 93: 13217-22 Leberer E, Ziegelbauer K, Schmidt A, Harcus D, Dignard D, Ash J et al (1997) Virulence and hyphal formation of Candida albicans require the Ste20p-like protein kinase CaCla4p Curr Biol 7: 539-46 Levin DE, Errede B (1995) The proliferation of MAP kinase signaling pathways in yeast Curr Opin Cell Biol 7: 197-202 Li CR, Wang YM, De Zheng X, Liang HY, Tang JC, Wang Y (2005) The formin family protein CaBni1p has a role in cell polarity control during both yeast and hyphal growth in Candida albicans J Cell Sci 118: 2637-48 Liu H (2001) Transcriptional control of dimorphism in Candida albicans Curr Opin Microbiol 4: 728-35 Liu H (2002) Co-regulation of pathogenesis with dimorphism and phenotypic switching in Candida albicans, a commensal and a pathogen Int J Med Microbiol 292: 299-311 Lopez-Ribot JL (2005) Candida albicans biofilms: more than filamentation Curr Biol 15: R453-5 Magee BB, Magee PT (2000) Induction of mating in Candida albicans by construction of MTLa and MTLalpha strains Science 289: 310-3 115 References Maidan MM, De Rop L, Serneels J, Exler S, Rupp S, Tournu H et al (2005) The G protein-coupled receptor Gpr1 and the Galpha protein Gpa2 act through the cAMPprotein kinase A pathway to induce morphogenesis in Candida albicans Mol Biol Cell 16: 1971-86 Malic S, Hill KE, Ralphs JR, Hayes A, Thomas DW, Potts AJ et al (2007) Characterization of Candida albicans infection of an in vitro oral epithelial model using confocal laser scanning microscopy Oral Microbiol Immunol 22: 188-94 Mallet L, Renault G, Jacquet M (2000) Functional cloning of the adenylate cyclase gene of Candida albicans in Saccharomyces cerevisiae within a genomic fragment containing five other genes, including homologues of CHS6 and SAP185 Yeast 16: 959-66 Martin GA, Bollag G, McCormick F, Abo A (1995) A novel serine kinase activated by rac1/CDC42Hs-dependent autophosphorylation is related to PAK65 and STE20 EMBO J 14: 1970-8 Martin R, Walther A, Wendland J (2005) Ras1-induced hyphal development in Candida albicans requires the formin Bni1 Eukaryot Cell 4: 1712-24 Martin SG, Rincon SA, Basu R, Perez P, Chang F (2007) Regulation of the formin for3p by cdc42p and bud6p Mol Biol Cell 18: 4155-67 Mavor AL, Thewes S, Hube B (2005) Systemic fungal infections caused by Candida species: epidemiology, infection process and virulence attributes Curr Drug Targets 6: 863-74 Mencacci A, Cenci E, Del Sero G, d'Ostiani CF, Montagnoli C, Bacci A et al (1999) Innate and adaptive immunity to Candida albicans: A new view of an old paradigm Rev Iberoam Micol 16: 4-7 Merson-Davies LA, Odds FC (1989) A morphology index for characterization of cell shape in Candida albicans J Gen Microbiol 135: 3143-52 Mitchell AP (1998) Dimorphism and virulence in Candida albicans Curr Opin Microbiol 1: 687-92 Moran GP, MacCallum DM, Spiering MJ, Coleman DC, Sullivan DJ (2007) Differential regulation of the transcriptional repressor NRG1 accounts for altered host-cell interactions in Candida albicans and Candida dubliniensis Mol Microbiol 66: 915-29 Moriyama K, Yahara I (2002) Human CAP1 is a key factor in the recycling of cofilin and actin for rapid actin turnover J Cell Sci 115: 1591-601 Morrison CJ, Hurst SF, Reiss E (2003) Competitive binding inhibition enzymelinked immunosorbent assay that uses the secreted aspartyl proteinase of Candida albicans as an antigenic marker for diagnosis of disseminated candidiasis Clin Diagn Lab Immunol 10: 835-48 116 References Morton WM, Ayscough KR, McLaughlin PJ (2000) Latrunculin alters the actinmonomer subunit interface to prevent polymerization Nat Cell Biol 2: 376-8 Naglik J, Albrecht A, Bader O, Hube B (2004) Candida albicans proteinases and host/pathogen interactions Cell Microbiol 6: 915-26 Navarro-Garcia F, Alonso-Monge R, Rico H, Pla J, Sentandreu R, Nombela C (1998) A role for the MAP kinase gene MKC1 in cell wall construction and morphological transitions in Candida albicans Microbiology 144 ( Pt 2): 411-24 Nobile CJ, Nett JE, Andes DR, Mitchell AP (2006) Function of Candida albicans adhesin Hwp1 in biofilm formation Eukaryot Cell 5: 1604-10 Odds F (1988) Candida and Candidiasis Bailliere Tindall, London Odds FC (1993) Resistance of yeasts to azole-derivative antifungals J Antimicrob Chemother 31: 463-71 Olazabal IM, Machesky LM (2001) Abp1p and cortactin, new "hand-holds" for actin J Cell Biol 154: 679-82 Olver WJ, Scott F, Shankland GS (2006) Successful treatment of Candida krusei fungemia with amphotericin B and caspofungin Med Mycol 44: 655-7 Pelkmans L, Puntener D, Helenius A (2002) Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae Science 296: 535-9 Perepnikhatka V, Fischer FJ, Niimi M, Baker RA, Cannon RD, Wang YK et al (1999) Specific chromosome alterations in fluconazole-resistant mutants of Candida albicans J Bacteriol 181: 4041-9 Pruyne D, Legesse-Miller A, Gao L, Dong Y, Bretscher A (2004) Mechanisms of polarized growth and organelle segregation in yeast Annu Rev Cell Dev Biol 20: 55991 Qualmann B, Kessels MM (2002) Endocytosis and the cytoskeleton Int Rev Cytol 220: 93-144 Quinlan JJ, Nickels JT, Jr., Wu WI, Lin YP, Broach JR, Carman GM (1992) The 45and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae are regulated differentially by phosphorylation via cAMP-dependent protein kinase J Biol Chem 267: 18013-20 Rabkin JM, Oroloff SL, Corless CL, Benner KG, Flora KD, Rosen HR et al (2000) Association of fungal infection and increased mortality in liver transplant recipients Am J Surg 179: 426-30 Raghavan R, Date A, Bhaktaviziam A (1987) Fungal and nocardial infections of the kidney Histopathology 11: 9-20 117 References Ramage G, Saville SP, Wickes BL, Lopez-Ribot JL (2002) Inhibition of Candida albicans biofilm formation by farnesol, a quorum-sensing molecule Appl Environ Microbiol 68: 5459-63 Ramon AM, Fonzi WA (2003) Diverged binding specificity of Rim101p, the Candida albicans ortholog of PacC Eukaryot Cell 2: 718-28 Richards MJ, Edwards JR, Culver DH, Gaynes RP (1999) Nosocomial infections in medical intensive care units in the United States National Nosocomial Infections Surveillance System Crit Care Med 27: 887-92 Richardson MD (1991) Opportunistic and pathogenic fungi J Antimicrob Chemother 28 Suppl A: 1-11 Rittig MG, Schroppel K, Seack KH, Sander U, N'Diaye EN, Maridonneau-Parini I et al (1998) Coiling phagocytosis of trypanosomatids and fungal cells Infect Immun 66: 4331-9 Rolstad BS, Erwin-Toth PL (2004) Peristomal skin complications: prevention and management Ostomy Wound Manage 50: 68-77 Romani L, Bistoni F, Puccetti P (1997) Initiation of T-helper cell immunity to Candida albicans by IL-12: the role of neutrophils Chem Immunol 68: 110-35 Ruhnke M (2004) Mucosal and systemic fungal infections in patients with AIDS: prophylaxis and treatment Drugs 64: 1163-80 Sagot I, Rodal AA, Moseley J, Goode BL, Pellman D (2002) An actin nucleation mechanism mediated by Bni1 and profilin Nat Cell Biol 4: 626-31 Sambrook J, Fritsch, EF, Maniatis, T (1989) Molecular Cloning, vol Cold Spring Harbor Laboratory Press Sanglard D, Kuchler K, Ischer F, Pagani JL, Monod M, Bille J (1995) Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters Antimicrob Agents Chemother 39: 2378-86 Schweizer A, Rupp S, Taylor BN, Rollinghoff M, Schroppel K (2000) The TEA/ATTS transcription factor CaTec1p regulates hyphal development and virulence in Candida albicans Mol Microbiol 38: 435-45 Scott VR, Boehme R, Matthews TR (1988) New class of antifungal agents: jasplakinolide, a cyclodepsipeptide from the marine sponge, Jaspis species Antimicrob Agents Chemother 32: 1154-7 Sertil O, Cohen BD, Davies KJ, Lowry CV (1997) The DAN1 gene of S cerevisiae is regulated in parallel with the hypoxic genes, but by a different mechanism Gene 192: 199-205 118 References Shi QM, Wang YM, Zheng XD, Lee RT, Wang Y (2007) Critical role of DNA checkpoints in mediating genotoxic-stress-induced filamentous growth in Candida albicans Mol Biol Cell 18: 815-26 Shima F, Okada T, Kido M, Sen H, Tanaka Y, Tamada M et al (2000) Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Rasbinding site which mediates its Ras-dependent activation Mol Cell Biol 20: 26-33 Shima F, Yamawaki-Kataoka Y, Yanagihara C, Tamada M, Okada T, Kariya K et al (1997) Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein Mol Cell Biol 17: 1057-64 Shin JH, Og YG, Cho D, Kee SJ, Shin MG, Suh SP et al (2005) Molecular epidemiological analysis of bloodstream isolates of Candida albicans from a university hospital over a five-year period J Microbiol 43: 546-54 Sinha I, Wang YM, Philp R, Li CR, Yap WH, Wang Y (2007) Cyclin-dependent kinases control septin phosphorylation in Candida albicans hyphal development Dev Cell 13: 421-32 Snyder M (1989) The SPA2 protein of yeast localizes to sites of cell growth J Cell Biol 108: 1419-29 Song Y, Kim JY (2006) Role of CaBud6p in the polarized growth of Candida albicans J Microbiol 44: 311-9 Sonneborn A, Bockmuhl DP, Gerads M, Kurpanek K, Sanglard D, Ernst JF (2000) Protein kinase A encoded by TPK2 regulates dimorphism of Candida albicans Mol Microbiol 35: 386-96 Spiliotis ET, Nelson WJ (2006) Here come the septins: novel polymers that coordinate intracellular functions and organization J Cell Sci 119: 4-10 Sprague GF, Cullen PJ, Goehring AS (2004) Yeast signal transduction: regulation and interface with cell biology Adv Exp Med Biol 547: 91-105 St Georgiev V (2000) Membrane transporters and antifungal drug resistance Curr Drug Targets 1: 261-84 Staab JF, Bahn YS, Tai CH, Cook PF, Sundstrom P (2004) Expression of transglutaminase substrate activity on Candida albicans germ tubes through a coiled, disulfide-bonded N-terminal domain of Hwp1 requires C-terminal glycosylphosphatidylinositol modification J Biol Chem 279: 40737-47 Staab JF, Bradway SD, Fidel PL, Sundstrom P (1999) Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1 Science 283: 1535-8 Stahlhut M, van Deurs B (2000) Identification of filamin as a novel ligand for caveolin-1: evidence for the organization of caveolin-1-associated membrane domains by the actin cytoskeleton Mol Biol Cell 11: 325-37 119 References Steiner P, Rudolph B, Muller D, Eilers M (1996) The functions of Myc in cell cycle progression and apoptosis Prog Cell Cycle Res 2: 73-82 Stoldt VR, Sonneborn A, Leuker CE, Ernst JF (1997) Efg1p, an essential regulator of morphogenesis of the human pathogen Candida albicans, is a member of a conserved class of bHLH proteins regulating morphogenetic processes in fungi EMBO J 16: 1982-91 Tada T, Simonetta A, Batterton M, Kinoshita M, Edbauer D, Sheng M (2007) Role of Septin cytoskeleton in spine morphogenesis and dendrite development in neurons Curr Biol 17: 1752-8 Ushinsky SC, Harcus D, Ash J, Dignard D, Marcil A, Morchhauser J et al (2002) CDC42 is required for polarized growth in human pathogen Candida albicans Eukaryot Cell 1: 95-104 VandenBerg AL, Ibrahim AS, Edwards JE, Jr., Toenjes KA, Johnson DI (2004) Cdc42p GTPase regulates the budded-to-hyphal-form transition and expression of hypha-specific transcripts in Candida albicans Eukaryot Cell 3: 724-34 Vinces MD, Haas C, Kumamoto CA (2006) Expression of the Candida albicans morphogenesis regulator gene CZF1 and its regulation by Efg1p and Czf1p Eukaryot Cell 5: 825-35 Vojtek A, Haarer B, Field J, Gerst J, Pollard TD, Brown S et al (1991) Evidence for a functional link between profilin and CAP in the yeast S cerevisiae Cell 66: 497-505 Walther A, Wendland J (2004) Polarized hyphal growth in Candida albicans requires the Wiskott-Aldrich Syndrome protein homolog Wal1p Eukaryot Cell 3: 471-82 Warenda AJ, Konopka JB (2002) Septin function in Candida albicans morphogenesis Mol Biol Cell 13: 2732-46 Whiteway M, Bachewich C (2007) Morphogenesis in Candida albicans Annu Rev Microbiol 61: 529-53 Whiteway M, Dignard D, Thomas DY (1992) Dominant negative selection of heterologous genes: isolation of Candida albicans genes that interfere with Saccharomyces cerevisiae mating factor-induced cell cycle arrest Proc Natl Acad Sci U S A 89: 9410-4 Whiteway M, Oberholzer U (2004) Candida morphogenesis and host-pathogen interactions Curr Opin Microbiol 7: 350-7 Wolyniak MJ, Sundstrom P (2007) Role of actin cytoskeletal dynamics in activation of the cyclic AMP pathway and HWP1 gene expression in Candida albicans Eukaryot Cell 6: 1824-40 120 References Wormley FL, Jr., Chaiban J, Fidel PL, Jr (2001) Cell adhesion molecule and lymphocyte activation marker expression during experimental vaginal candidiasis Infect Immun 69: 5072-9 Xu XL, Lee RT, Fang HM, Wang YM, Li R, Zou H et al (2008) Bacterial peptidoglycan triggers Candida albicans hyphal growth by directly activating the adenylyl cyclase Cyr1p Cell Host Microbe 4: 28-39 Yarmola EG, Somasundaram T, Boring TA, Spector I, Bubb MR (2000) Actinlatrunculin A structure and function Differential modulation of actin-binding protein function by latrunculin A J Biol Chem 275: 28120-7 Yokoyama K, Kaji H, Nishimura K, Miyaji M (1990) The role of microfilaments and microtubules in apical growth and dimorphism of Candida albicans J Gen Microbiol 136: 1067-75 Yong PV, Chong PP, Lau LY, Yeoh RS, Jamal F (2008) Molecular identification of Candida orthopsilosis isolated from blood culture Mycopathologia 165: 81-7 Yu G, Swiston J, Young D (1994) Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins J Cell Sci 107 ( Pt 6): 1671-8 Zheng X, Wang Y (2004) Hgc1, a novel hypha-specific G1 cyclin-related protein regulates Candida albicans hyphal morphogenesis EMBO J 23: 1845-56 Zheng XD, Lee RT, Wang YM, Lin QS, Wang Y (2007) Phosphorylation of Rga2, a Cdc42 GAP, by CDK/Hgc1 is crucial for Candida albicans hyphal growth EMBO J 26: 3760-9 Zheng XD, Wang YM, Wang Y (2003) CaSPA2 is important for polarity establishment and maintenance in Candida albicans Mol Microbiol 49: 1391-405 Zink S, Nass T, Rosen P, Ernst JF (1996) Migration of the fungal pathogen Candida albicans across endothelial monolayers Infect Immun 64: 5085-91 121 Publications PUBLICATIONS Xu XL, Lee RT, Fang HM, Wang YM, Li R, Zou H et al (2008) Bacterial peptidoglycan triggers Candida albicans hyphal growth by directly activating the adenylyl cyclase Cyr1p Cell Host Microbe 4: 28-39 Zou H, Fang HM, Wang Y (2008) G-actin assoicates with the Adenylyl Cyclase Cyr1 through Cap1 and regulates cAMP synthesis in Candida albicans hyphal morphogenesis (Submitted) 122 .. .CHARACTERIZATION OF CANDIDA ALBICANS CYCLASE- ASSOCIATED PROTEIN CAP1 AND ITS ROLES IN MORPHOGENESIS — G- actin Assoicates with the Adenylyl Cyclase Cyr1 through Cap1 and Regulates cAMP Synthesis. .. resulting in impaired response to the hyphal- inducing signals Furthermore, the finding that the purified Cyr1 /Cap1/ actin complex can increase cAMP synthesis in response to hyphal- inducing molecules... activate cAMP synthesis by binding to the LRR domain of Cyr1, suggesting that Ras1 may not be essential for hyphal induction In some conditions, the G protein- coupled receptor Gpr1 and the G? ? protein