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Transcriptional control of dimorphism in Candida albicans
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Transcriptional control of dimorphism in Candida albicans Haoping Liu Candida albicans uses a network of multiple signaling pathways to control the yeast→hypha transition. These include a mitogen-activated protein kinase pathway through Cph1, the cAMP-dependent protein kinase pathway via Efg1, a pH-responsive pathway through Rim101, the Tup1-mediated repression through Rfg1 and Nrg1, and pathways represented by transcription factors Cph2, Tec1 and Czf1. These pathways control the transcription of a common set of hypha-specific genes, many of which encode known virulence factors. The link between the signaling pathways and hyphal elongation is currently unknown, but there is evidence to suggest that Cdc42 likely plays a key role in hyphal morphogenesis. Unlike pseudohyphal growth in Saccharomyces cerevisiae, hyphal elongation is regulated independently of the cell cycle. Cellular differences between pseudohyphae and hyphae are further revealed by septin localization. Addresses Department of Biological Chemistry, 19172 Jamboree Road, University of California at Irvine, Irvine, California 92697-1700, USA; e-mail: [email protected] Current Opinion in Microbiology 2001, 4:728–735 1369-5274/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations bHLH basic helix-loop-helix MAPK mitogen-activated protein kinase PKA cAMP-dependent protein kinase A
Introduction Candida albicans is one of the most frequently isolated fungal pathogens of humans. It is capable of causing superficial mucosal infections as well as systemic infections in immunocompromised individuals. It can grow in a variety of morphological forms, ranging from budding yeast to pseudohyphae (chains of elongated cells with visible constrictions at the sites of septa) and hyphae (linear filaments without visible constrictions at the septa). Its ability to switch from yeast to hyphal growth in response to various environmental signals is not only inherent to its pathogenicity, but also provides an excellent paradigm to understand how signaling pathways coordinate growth and development. Many signaling pathways and regulators involved in hyphal development have been identified as a result of the strong molecular conservation between C. albicans and Saccharomyces cerevisiae in many cellular processes, a C. albicans genome sequencing program (http://www-sequence.stanford.edu/group/candida/) and efficient homologous recombination that allows a variety of molecular manipulations to be possible. The functions of these pathways and regulators in morphogenesis, have been well-reviewed recently [1,2•,3•]. In this review, I concentrate on recent developments, with emphasis on the
relationships between different signaling pathways, and between regulators and downstream effectors.
Transcriptional regulation of hyphal development in C. albicans Hyphal development in C. albicans is determined by a wide range of signals or culture conditions in vitro [1,2•]. Many signaling pathways or regulators have been found to regulate filamentation in one or many of the in vitro hypha-inducing conditions (Figure 1). The mitogen-activated protein kinase pathway for mating and filamentation
In S. cerevisiae, elements of the pheromone-responsive mitogen-activated protein kinase (MAPK) pathway are involved in pseudohyphal and invasive growth. Like S. cerevisiae, Cph1, a homolog of S. cerevisiae Ste12, and a MAPK cascade that includes Cst20 (p21-activated kinase; PAK), Hst7 (MAP kinase kinase; MEK) and Cek1 (MAPK) are also involved in filamentation. Mutants of these genes all display a defect in hyphal development on solid Spider medium. In certain liquid media, the cph1/cph1 mutant is defective in filamentation and in the induction of hypha-specific genes (genes whose expression is associated with cell elongation, rather than with a specific hyphainducing condition) (S Lane, C Birse, S Zhou, R Matson, H Liu, unpublished data). As in S. cerevisiae, Cph1 and members of the MAPK pathway (Cst20, Hst7, Cek1 and a Fus3 MAPK homolog Cek2) are required for mating between MTLa or MTLα strains (J Chen, J Chen, S Lane, H Liu, unpublished data), consistent with the prediction obtained from a comparative genomic analysis between the two yeasts [4]. Overexpression of CPH1 in MTLa and MTLα cells induces the expression of CEK1, CEK2 and genes similar to S. cerevisiae pheromone-induced genes. The cAMP-dependent protein kinase A pathway
The cAMP-dependent protein kinase A (PKA) pathway plays a crucial role in filamentation in S. cerevisiae, C. albicans and other fungi [5]. C. albicans has a single gene homologous to the S. cerevisiae adenylate cyclase gene (CDC35/CYR1). The cyclase is not essential for growth in C. albicans, but is completely required for hyphal development, even when induced by serum (M Whiteway, personal communication). Recently, the adenylate-cyclase-associated protein (CAP1) has been identified and disrupted in C. albicans [6•]. An increase in cytoplasmic cAMP is seen to precede germ tube emergence in wild-type strains, but not in cap1/cap1 mutants. The cap1/cap1 mutant is defective in germ tube formation and hyphal development in all conditions examined, including serum-containing media. The defects are suppressed by exogenous cAMP or dibutyryl cAMP. cap1/cap1 strains are avirulent in a mouse model for systemic candidiasis.
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Figure 1 Regulation of dimorphism in C. albicans by multiple signaling pathways. The Cph1mediated MAPK pathway and the Efg1mediated cAMP pathway are two wellcharacterized signaling pathways in dimorphic regulation. Ras1 may function upstream of both pathways. TEC1 transcription is regulated by Efg1 and Cph2. Rim101 or Czf1 may function through Efg1 or act in parallel with Efg1. Negative regulators Tup1, recruited by Rfg1 or Nrg1, and Rbt1 are also implicated in dimorphic regulation. Not all pathways are shown. Arrows indicate activation and bars indicate inhibition. They do not necessarily indicate a direct interaction. Transcription factors are shown in boxes. These regulators control the transcription of a common set of hypha-specific genes. Pathway-specific outputs are also indicated with dashed lines.
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Both Ras and a receptor-coupled G protein (that is, G protein Gpa2 coupled to receptor Gpr1) act upstream of the adenylate cyclase in S. cerevisiae. They stimulate cAMP synthesis in response to intracellular acidification and extracellular glucose, respectively [7]. C. albicans Ras1 is an important regulator of hyphal development, and is likely to function upstream of the cAMP pathway [8]. Genes similar to GRP1 and GPA2 have been identified by the C. albicans genome-sequencing program. Further studies will be necessary to determine their potential roles in hyphal development. There are only two PKA catalytic subunits, Tpk1 and Tpk2, in C. albicans ([9]; JF Ernst, personal communication). Unlike S. cerevisiae, in which only one of the three catalytic subunits is an activator and the other two are inhibitors of pseudohyphal growth [10], both PKA isoforms in C. albicans are positive regulators of hyphal morphogenesis. Interestingly, Tpk1 and Tpk2 seem to have differential effects on hyphal morphogenesis under different hypha-inducing conditions. Unlike mutants in CYR1 and CAP1, tpk2/tpk2 does not exhibit a strong defect in hyphal morphogenesis in liquid serum-containing media [9], suggesting that the existence of additional cAMP-dependent functions in regulating
hyphal development. Tpk1 is a likely candidate. Phenotypes of a tpk1/tpk1 tpk2/tpk2 double mutant will be able to address this possibility. Efg1, a basic helix-loop-helix (bHLH) protein similar to Phd1 and Sok2 of S. cerevisiae and StuA of Aspergillus nidulans, plays a major role in hyphal morphogenesis [11,12]. efg1/efg1 null mutant strains do not form hyphae under most hypha-inducing conditions, including serum, and are defective in the induction of hypha-specific genes. Efg1 is likely to function downstream of the PKAs, similar to Sok2 in S. cerevisiae [13,14]. TPK2 overexpression cannot suppress the efg1/efg1 defect in hyphal development, whereas overexpression of EFG1 can suppress the filamentation defect in tpk2/tpk2 [9]. The suppression activity of EFG1 depends on threonine-206, a potential phosphorylation site for a PKA [15•]. Substitution of T206 with alanine impairs hyphal formation, whereas glutamate substitution causes hyperfilamentation. Intriguingly, the effects of T206A-Efg1 and T206E-Efg1 variants on hyphal formation are mediumdependent. Unlike the efg1/efg1 null mutant, the T206A Efg1 variant exhibited only a minor defect when induced by serum, indicating the existence of additional modifications or regulations on Efg1 during hyphal development. Efg1
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has recently been shown to bind two palindromic E-box sequences (5′CANNTG3′) upstream of ALS8 [16]. Although the E-box sequence is present at multiple sites upstream of hypha-specific genes, it also appears at approximately the same frequency upstream of randomly chosen genes. Future experiments should address how Efg1 achieves its transcriptional specificity. Efg1 may not be the only target of the cAMP pathway in hyphal morphogenesis. RAS1V13, a dominant active allele of RAS1, can stimulate hyphal development in cph1/cph1 efg1/efg1 strains [17•]. Overproducing a Cdc2-related protein kinase (Crk1) in the cph1/cph1 efg1/efg1 mutant also exhibited a similar phenotype. The crk1/crk1 mutant is defective in hyphal formation in serum and many other hyphainducing media, a phenotype typical of mutants in the cAMP pathway. In S. cerevisiae, additional transcription factors Msn2/Msn6 [18,19] and Sfl1 [10,20] are regulated directly by PKAs and are important for cell morphogenesis and pseudohyphal growth. It will be interesting to determine whether or not their homologs are important for hyphal development in C. albicans.
TEC1 transcription is regulated by Efg1 and Cph2, but not by Cph1 C. albicans Tec1, a new member of the TEA/ATTS family of transcription factors, has recently been shown to regulate hyphal development and virulence in C. albicans [21•]. AbaA and Tec1 of the same family are involved in the regulation of conidiophore formation in A. nidulans and filamentous growth in S. cerevisiae. In S. cerevisiae, TEC1 transcription is regulated by Ste12, and cooperation between Tec1 and Ste12 is important for pseudohyphal growth [22]. In C. albicans, on the other hand, TEC1 transcription is not regulated by Cph1 (S Lane, C Birse, S Zhou, R Matson, H Liu, unpublished data). As in A. nidulans, where AbaA expression is regulated by StuA, TEC1 transcription requires Efg1 in all media examined (S Lane, C Birse, S Zhou, R Matson, H Liu, unpublished data), consistent with the observation that tec1/tec1 mutants exhibit suppressed filamentation in liquid serum-containing media [21•]. EFG1 overexpression does not suppress the morphological defect of the tec1/tec1 mutant [21•], whereas TEC1 overexpression has a partial phenotype in the efg1/efg1 mutant. These results, coupled with the fact that efg1/efg1 strains have a more severe defect in hyphal development than do tec1/tec1 strains [21•], suggest that TEC1 is one of the downstream effectors of Efg1. Cph2, a myc family bHLH protein, has been found to regulate hyphal development in C. albicans [23•]. cph2/cph2 mutant strains are impaired in hyphal development and in the induction of hypha-specific genes in liquid Lee’s media. TEC1 transcription is highest in Lee’s media, and Cph2 is necessary for the transcriptional induction of TEC1. Cph2 binds directly to two sterolregulatory-element-1-like elements upstream of TEC1. Furthermore, the ectopic expression of TEC1 suppresses
the defect of cph2/cph2 in hyphal development. The function of Cph2 in hyphal transcription is therefore mediated, in part, through Tec1.
Tup1-mediated repression of hyphal development through Rfg1 and Nrg1 Tup1 encodes a transcriptional repressor that negatively controls filamentous growth in C. albicans [24]. Like the wellcharacterized repressor complex Tup1–Ssn6 of S. cerevisiae, CaTup1 is likely to be part of a transcriptional repressor complex that is brought to promoters by sequence-specific DNA-binding proteins. Several genes repressed by Tup1 (RBTs) have been identified in C. albicans [25•]. Many of them are induced during the yeast→hypha transition. But, some RBTs are not regulated during filamentation, indicating that the regulation of filamentation is not the sole function of Tup1. This also suggests that Tup1 itself is not regulated during filamentous growth; rather, its associated DNA-binding proteins are likely to be regulated [25•]. Transcript profiling of RBTs in single, double and triple mutants of TUP1, EFG1 and CPH1 suggests that each gene represents a separate pathway in the transcriptional regulation of filamentation [26]. Recently, Rfg1, a protein related to the S. cerevisiae hypoxic regulator Rox1, has been identified independently by two groups as a negative regulator of filamentous growth [27•,28•]. Rox1 is a high mobility group (HMG) domain DNA-binding protein with a repression domain capable of recruiting the Tup1–Ssn6 repressor complex to repress the expression of hypoxic genes. RFG1 can complement a S. cerevisiae rox1 mutant in the repression of hypoxic genes in a Tup1-dependent manner [28•]. In C. albicans, rfg1/rfg1 mutants cause derepression of filamentation and hyphaspecific RBTs. tup1 is epistatic to rfg1 in terms of colony morphology and the extent of transcriptional derepression of the hypha-specific RBTs [27•]. All these data suggest that Rfg1 directs transcriptional repression at hyphaspecific promoters by recruiting the Tup1–Ssn6 complex. Unlike ROX1, RFG1 transcription is not regulated by oxygen, and is not required for the repression of hypoxic genes in C. albicans. Further studies are required to address how Rfg1 activity is regulated during filamentation. Two groups have independently identified another DNAbinding protein, Nrg1, that might direct the Tup1 repressor complex to the promoters of hypha-specific genes [29•,30•]. S. cerevisiae Nrg1 is a sequence-specific zinc-finger DNAbinding protein that directs the Tup1–Ssn6 complex to repress STA1 transcription in a glucose-dependent manner [31]. C. albicans Nrg1 represses filamentous growth and negatively regulates hypha-specific RBTs in C. albicans. The NRG1 transcript is downregulated during filamentous growth in serum at 37°C [30•]. Other pathways
C. albicans has a conserved pH-response pathway. As in S. cerevisiae and A. nidulans, the transcription factor Rim101
Transcriptional control of dimorphism in Candida albicans Liu
(Prr2) mediates pH response through its proteolysis, which is regulated by Rim8 (Prr1) and Rim20 [32–35]. Rim101 is required for alkaline-induced filamentation, the expression of alkaline-responsive genes such as PHR1, and the repression of the alkaline-repressed gene PHR2. A truncated Rim101 causes filamentation and the expression of alkalineinduced genes in acidic media [35]. Efg1 is required for the Rim101-induced filamentation, but not for the expression of alkaline-induced genes. Therefore, Efg1 either acts downstream of Rim101 or the two factors act in parallel in regulating filamentation. Czf1, a zinc-finger-containing protein, is important for hyphal development when cells are grown in the presence of surrounding matrix [36]. Interestingly, an efg1/efg1 mutant is hyperfilamentous when grown in matrix, indicating that Efg1 represses filamentation under this condition (CA Kumamoto, personal communication). The deletion or ectopic expression of CZF1 does not affect filamentous growth of the efg1/efg1 mutant in the matrix, indicating that Czf1 may act to relieve the Efg1-mediated repression of filamentation. Other known signaling pathways involved in dimorphic regulation include a two-component signaling pathway (for a review, see [3•]), the Hog1 MAPK pathway [37], Rbf1-mediated repression [38] and Mcm1 (S Rupp, personal communication). Transcriptional induction of a common set of hypha-specific genes
The hypha-specific genes identified so far include ECE1, HWP1, HYR1, ALS3, ALS8, RBT1 and RBT4. They encode either cell-wall or secreted proteins. Many have been shown to be important virulence factors for systemic infection. HWP1 encodes a cell-wall protein that can serve as a target for mammalian transglutaminases to form covalent attachments between C. albicans and host epithelial cells [39]. RBT1 encodes a cell-wall protein, and RBT4 encodes a secreted protein similar to a set of pathogenesis-related proteins from plants [25•]. Both genes are necessary for the full virulence of C. albicans in a systemic mouse model. In addition to the hypha-specific genes, three members of the secreted aspartyl proteinase genes, SAPs4–6, are expressed when hyphal development is induced with serum or media containing polypeptides as the sole nitrogen source [40]. SAP genes have been shown to contribute to virulence in both systemic and mucosal candidal infections [41]. The expression of all the aforementioned genes requires Efg1 [16,26,38,42] and, under certain media, also requires Cph1 (S Lane, C Birse, S Zhou, R Matson, H Liu, unpublished data), Cph2 and, potentially, Tec1 [21•,23•]. The hypha-specific genes are also repressed by Tup1–Rfg1 [27•,28•] and Tup1–Nrg1. Czf1-mediated and Rim101-mediated filamentation require Efg1, suggesting that Czf1 and Rim101 may also regulate the expression of the aforementioned hyphaspecific genes. Therefore, the expression of hypha-specific
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genes seems to be regulated by multiple pathways (Figure 1). The regulation of TEC1 expression by Cph2 and Efg1 provides one example of how different pathways converge. Cooperative interaction between hyphal regulators of different pathways at the promoters of hypha-specific genes and pathway crosstalk are other potential mechanisms for the observed convergence in the regulation of hypha-specific gene expression. In addition to hypha-specific genes, many dimorphic regulators or signaling pathways also have pathway-specific genes (Figure 1). For example, PHR1 and PHR2 expression is regulated by Rim101 [34,35], mating genes are activated by Cph1 (J Chen, J Chen, S Lane, H Liu, unpublished data) and Efg1 regulates the expression of phase-specific genes such as WH11 [43]. The expression of pathway-specific genes and hypha-specific genes are not exclusive to each other. It is likely that, under any given growth conditions, a network of signaling pathways are employed to simultaneously assess the availability of multiple nutrients, cell density and other growth conditions. The integrated output of these pathways determines the gene expression and dimorphic transition.
Control of cell polarity during hyphal development During hyphal development, cell-surface expansion is highly restricted to the apical tip of hyphal filaments. This polarized apical growth requires the actin cytoskeleton. C. albicans yeast cells display a temporal change in the organization of the actin cytoskeleton during cell cycle progression like that in S. cerevisiae cells, whereas the actin cytoskeleton is polarized at the tip of all hyphal cells during filamentation [44]. In S. cerevisiae and other organisms, the small GTPase of the Rho subfamily Cdc42 is known to be critical for establishing a polarized actin cytoskeleton in response to extracellular stimuli. In the filamentous ascomycete Ashbya gossypii, three Rho-GTPases control distinct steps during polarized hyphal growth with Cdc42 being required for the establishment of actin polarization [45]. C. albicans Cdc42 is also essential for cell morphogenesis (M Whiteway, personal communication), and is probably responsible for establishing the polarized actin cytoskeleton during hyphal induction, considering that one of its downstream effectors, Cla4, is required for hyphal growth under all conditions examined [46]. Interestingly, Cla4 is also necessary for the maturation of hyphae in A. gossypii [47]. In S. cerevisiae, the activation of Cdc42 involves the temporal and spatial regulation of its sole guanine-nucleotide exchange factor Cdc24 [48]. Whether and how the Cdc42 modular activity is regulated during the yeast→hypha transition has not been elucidated in C. albicans. Studies from S. cerevisiae indicate that pseudohyphal growth is regulated by a change in cell cycle (see the review by Rua, Tobe and Kron in this issue, pp 720–727). However, several lines of evidence show that hyphal
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Alignment of cell cycle events in yeast and hyphae. G1 cells are transferred into yeast growth or hypha-inducing conditions. (a) Germ-tube and hypha-associated actin polarization occur before other cell cycle events, (a–j) and the polarized actin structure persists throughout the cell cycle. (b) A transient septin ring forms at the base of the germ tube. (d,e) Polarization of actin cortical patches, budding/or chitin-ring formation, spindle pole body (SPB) duplication,
nuclear migration and DNA synthesis all occur at the same period of time in yeast and hyphae. Chitin rings probably form at the time of the G1/S transition. (g,h) Mitotic spindle (MS) and (g) DNA separation, the mitotic actin ring and (i) the repolarization of the actin cortical patches to the site of septa all appear with similar dynamics in yeast and hyphae. (j) Unlike yeast cells, septin rings remain after mitosis at septa in a growing hypha. DSPB, duplicated spindle pole bodies.
elongation in C. albicans is controlled independently of the cell cycle. The deletion of a G1 cyclin gene affects only sustained hyphal elongation, but not initial germ tube formation [49]. The phosphorylation state of the conserved Tyr19 of the CDK Cdc28 is cell-cycle-regulated and no difference was observed in the phosphorylation and dephosphorylation kinetics between synchronous yeast and hyphal cells [50•]. The timing of several other cell-cycle-regulated events, including microtubule spindle pole body duplication and spindle elongation, rearrangement of the actin cytoskeleton, actin structures, chitin ring formation and nuclear division are also identical between synchronous yeast and hyphal apical cells (Figure 2) [50•]. Hypha-associated polarization of the actin cytoskeleton occurs before spindle duplication, DNA synthesis and chitin ring formation, and persists throughout the cell cycle during hyphal morphogenesis (Figure 2). In addition, cells from many stages of the cell cycle seem competent to develop hyphae. Hyphae induced from unbudded G1 cells have no restrictions and the first site of septation is positioned in the germ tube, whereas hyphae induced from budded cells retain the constriction and chitin ring from the budded cell during the initial cell cycle.
Cellular differences between pseudohyphae and hyphae have been revealed recently by studying septin localization in C. albicans. Septins, encoded by CDC3, CDC10, CDC11 and CDC12 in S. cerevisiae, are protein components of 10 nm neck filaments that are essential for cytokinesis. In C. albicans pseudohyphae, the Cdc11 ring forms at the neck between the mother and the germ tube, which is followed by nuclear migration, division and cytokinesis across the septin ring [51•]. However, in hyphae cells, a transient Cdc11 ring is observed at the site of evagination, which is not associated with nuclear migration and division (Figure 2). A second Cdc11 ring forms in the germ tube and the septin ring is followed by nuclear migration and the first mitosis happens across the ring. The formation of the transient septin rings in hyphae is probably not regulated by the cell cycle and may be unique to hyphal cells. Cell-cycle-independent septin rings also form at the neck of the mating projection of S. cerevisiae [52], where they are important for the pheromone-induced shape change [53]. Cdc3 also localizes to a ring at the mother–daughter neck in yeast and pseudohyphal cells, and to a ring in the germ tube [54•]. Surprisingly, multiple Cdc3 rings at the septa are observed in a growing hypha, indicating that the Cdc3 ring may not disappear after each cytokinesis
Transcriptional control of dimorphism in Candida albicans Liu
[54•]. The hypha-specific regulation of Cdc3 and Cdc11 localization may also apply to other septins. C. albicans Int1 co-localizes with Cdc3 [54•]. The carboxy terminus of Int1 is similar to that of S. cerevisiae Bud4. Like Bud4, Int1 is important for axial budding in C. albicans. Ectopic INT1 expression in S. cerevisiae causes highly polarized apical growth via Swe1 and Sla2 [55•]. In C. albicans, SLA2 is essential for hyphal cell morphogenesis in either starvation media or serum-containing media. Further experimentation is needed to elucidate whether or not Sla2 is regulated by Int1 and other regulators during hyphal development.
Conclusions and future directions A sketch of the complex regulatory network that controls dimorphism in C. albicans is emerging. Different signaling pathways and transcriptional factors seem to converge to regulate the transcription of a common set of hyphaspecific genes. Molecular mechanisms for the integrative regulation and pathway crosstalk require further experiments. Many of the hypha-specific genes encode known virulence factors, which explains why dimorphic signaling pathways are important for C. albicans pathogenesis. However, none of the hypha-specific genes identified so far is directly linked to cell polarity. Thus, the molecular link between the signaling pathways and hyphal cell polarity is still missing. Development of a C. albicans DNA array for genome-wide gene expression studies is likely to identify the missing connection. It is crucial that we address the function and regulation of a C. albicans protein in C. albicans cells. We have seen several cases in which the C. albicans protein contains a conserved and functional domain, but functions in a pathway different from that of its ortholog in S. cerevisiae, as in the cases of Rfg1 [27•,28•] and Mig1 [56]. Most of the experiments done so far have used molecular manipulations to alter the activity of a gene product in C. albicans in order to determine its function. These experiments do not distinguish a requirement for signal transmission from being a part of the pathway that mediates the signal. Future experiments should try to address whether a regulator is altered in terms of its activity, localization, protein stability or post-translational modifications in response to specific stimuli. A major challenge in the future will be to determine the upstream regulatory components and signals for the orphan transcription factors involved in dimorphic transition.
Acknowledgements I would like to thank: J Chen, S Lane, I Hazan and S Zhou for unpublished results; CA Kumanoto, S Rupp, JF Ernst, AJ Brown, M Whiteway, J Berman, PE Sudbery, P Sundstrom, GR Fink, P Philippsen, J Heitman and S Filler for comments, unpublished results and preprints; and I Hazan for her assistance in producing the graphs. I apologize for not including other data because of space restrictions. The work was supported by funds from the National Institutes of Health (GM55155), Burroughs Wellcome, and University of California Universitywide AIDS Program.
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References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Brown AJ, Gow NA: Regulatory networks controlling Candida albicans morphogenesis. Trends Microbiol 1999, 7:333-338.
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Ernst JF: Transcription factors in Candida albicans — environmental control of morphogenesis. Microbiology 2000, 146:1763-1774. This is an informative and stimulating review that presents the transcriptional regulation of morphogenesis in C. albicans with an emphasis on environmental effects on morphogenesis. 3. •
Whiteway M: Transcriptional control of cell type and morphogenesis in Candida albicans. Curr Opin Microbiol 2000, 3:582-588. This is an up-to-date and insightful review of signalling in C. albicans that details how it is important in the regulation of the yeast→hypha transition and other developmental programs in C. albicans. 4.
Tzung KW, Williams RM, Scherer S, Federspiel N, Jones T, Hansen N, Bivolarevic V, Huizar L, Komp C, Surzycki R et al.: Genomic evidence for a complete sexual cycle in Candida albicans. Proc Natl Acad Sci USA 2001, 98:3249-3253.
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Bahn YS, Sundstrom P: 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 2001, 183:3211-3223. CAP1, a gene for an adenylate-cyclase-associated protein, was cloned and disrupted in C. albicans. C. albicans strains with inactivated CAP1 were deficient in the ability to form germ tubes, develop hyphae and produce the pathogenesis of candidiasis. An increase in cytoplasmic cAMP was not observed in the cap1/cap1 mutant, and the addition of cAMP could suppress the defect of the cap1/cap1 mutant in germ tube formation and hyphal development. 7.
Colombo S, Ma P, Cauwenberg L, Winderickx J, Crauwels M, Teunissen A, Nauwelaers D, de Winde JH, Gorwa MF, Colavizza D et al.: Involvement of distinct G-proteins, Gpa2 and Ras, in glucose- and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae. EMBO J 1998, 17:3326-3341.
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Sonneborn A, Bockmuhl DP, Gerads M, Kurpanek K, Sanglard D, Ernst JF: Protein kinase A encoded by TPK2 regulates dimorphism of Candida albicans. Mol Microbiol 2000, 35:386-396.
10. Robertson LS, Fink GR: The three yeast A kinases have specific signaling functions in pseudohyphal growth. Proc Natl Acad Sci USA 1998, 95:13783-13787. 11. Lo HJ, Kohler JR, DiDomenico B, Loebenberg D, Cacciapuoti A, Fink GR: Nonfilamentous C. albicans mutants are avirulent. Cell 1997, 90:939-949. 12. Stoldt VR, Sonneborn A, Leuker CE, Ernst JF: 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 1997, 16:1982-1991. 13. Pan X, Heitman J: Sok2 regulates yeast pseudohyphal differentiation via a transcription factor cascade that regulates cell–cell adhesion. Mol Cell Biol 2000, 20:8364-8372. 14. Shenhar G, Kassir Y: A positive regulator of mitosis, Sok2, functions as a negative regulator of meiosis in Saccharomyces cerevisiae. Mol Cell Biol 2001, 21:1603-1612. 15. Bockmuhl DP, Ernst JF: A potential phosphorylation site for an • A type kinase in the Efg1 regulator protein contributes to hyphal morphogenesis of Candida albicans. Genetics 2001, 157:1523-1530. In Efg1, T206 (a potential phosphorylation site for PKA) was found to play an important role in hyphal morphogenesis. T206A Efg1 was defective in hyphal formation under certain hypha-inducing conditions, whereas overexpression of the T206E variant did not suppress the tpk2/tpk2 mutant.
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16. Leng P, Lee PR, Wu H, Brown AJ: Efg1, a morphogenetic regulator in Candida albicans, is a sequence-specific DNA binding protein. J Bacteriol 2001, 183:4090-4093. 17. •
Chen J, Zhou S, Wang Q, Chen X, Pan T, Liu H: Crk1, a novel Cdc2 related protein kinase, is required for hyphal development and virulence in Candida albicans. Mol Cell Biol 2000, 20:8696-8708. A gene encoding a Cdc2-related kinase (Crk1) has been cloned and deleted in C. albicans. crk1/crk1 mutants were impaired in hyphal development and in the induction of hypha-specific genes in all conditions examined, and were avirulent in mice. RAS1V13 or CRK1N generated similar hyphal colonies in cph1/cph1 efg1/efg1 double mutants. Epistasis studies suggested that Crk1 may function at a step downstream of Ras1, which is consistent with the observation that ectopic CRK1N expression in S. cerevisiae promoted pseudohyphal/invasive growth in a flo8-dependent, but Ste12and Phd1-independent, manner.
29. Murad AM, Leng P, Straffon M, Wishart J, Macaskill S, MacCallum D, • Schnell N, Talibi D, Marechal D, Tekaia F et al.: NRG1 represses yeast-hypha morphogenesis and hypha-specific gene expression in Candida albicans. EMBO J 2001, 20:4742-4752. This and [30•] report the identification of a DNA-binding protein, Nrg1, that represses filamentous growth in C. albicans probably by acting through Tup1. Like tup1 mutants, deletion of NRG1 in C. albicans enhances filamentous and invasive growth and derepresses hypha-specific genes. Paper [29•] shows that Nrg1 response elements are responsible for the Nrg1-mediated repression. Paper [30•] suggests that downregulation of the NRG1 transcript is involved in the induction of filamentous growth in serum at 37°C. 30. Braun BR, Kadosh D, Johnson AD: NRG1, a repressor of • filamentous growth in C. albicans, is down-regulated during filament induction. EMBO J 2001, 20:4753-4761. See annotation to [29•].
18. Gorner W, Durchschlag E, Martinez-Pastor MT, Estruch F, Ammerer G, Hamilton B, Ruis H, Schuller C: Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. Genes Dev 1998, 12:586-597.
31. Park SH, Koh SS, Chun JH, Hwang HJ, Kang HS: Nrg1 is a transcriptional repressor for glucose repression of STA1 gene expression in Saccharomyces cerevisiae. Mol Cell Biol 1999, 19:2044-2050.
19. Ho J, Bretscher A: Ras regulates the polarity of the yeast actin cytoskeleton through the stress response pathway. Mol Biol Cell 2001, 12:1541-1555.
32. Porta A, Ramon AM, Fonzi WA: PRR1, a homolog of Aspergillus nidulans palF, controls pH-dependent gene expression and filamentation in Candida albicans. J Bacteriol 1999, 181:7516-7523.
20. Conlan RS, Tzamarias D: Sfl1 functions via the co-repressor Ssn6–Tup1 and the cAMP-dependent protein kinase Tpk2. J Mol Biol 2001, 309:1007-1015.
33. Ramon A, Porta A, Fonzi WA: Effects of environmental pH on morphological development of Candida albicans is mediated via the PacC-related transcription factor encoded by PRR2. J Bacteriol 1999, 181:7524-7530.
21. Schweizer A, Rupp S, Taylor BN, Rollinghoff M, Schroppel K: The • TEA/ATTS transcription factor CaTec1p regulates hyphal development and virulence in Candida albicans. Mol Microbiol 2000, 38:435-445. A functional homolog of S. cerevisiae TEC1 has been cloned and deleted in C. albicans. CaTEC1 is predominantly expressed in the hyphal form. tec1/tec1 strains are defective in hyphal development and induction of SAP4-6 transcription, and show reduced virulence in a systemic candidiasis model. 22. Madhani HD, Fink GR: Combinatorial control required for the specificity of yeast MAPK signaling. Science 1997, 275:1314-1317. 23. Lane S, Zhou S, Pan T, Dai Q, Liu H: The basic helix-loop-helix • transcription factor cph2 regulates hyphal development in Candida albicans partly via Tec1. Mol Cell Biol 2001, 21:6418-6428. A new C. albicans bHLH transcription factor of the Myc subfamily (Cph2) has been cloned by its ability to promote pseudohyphal growth in S. cerevisiae. C. albicans cph2/cph2 strains show impairment in hyphal development and in the induction of hypha-specific genes specifically in Lee’s medium. The function of Cph2 in hyphal transcription is mediated, in part, through Tec1. Cph2 directly binds to two sterol regulatory element-1-like sequences upstream of TEC1, and is necessary for the transcriptional induction of TEC1 in Lee’s medium. The ectopic expression of TEC1 suppresses the defect of cph2/cph2 in hyphal development. 24. Braun BR, Johnson AD: Control of filament formation in Candida albicans by the transcriptional repressor TUP1. Science 1997, 277:105-109. 25. Braun BR, Head WS, Wang MX, Johnson AD: Identification and • characterization of TUP1-regulated genes in Candida albicans. Genetics 2000, 156:31-44. This data-packed paper describes the identification of seven genes (RBTs) repressed by TUP1. Gene products of five out of the seven RBTs are induced during the yeast-to-hyphal switch. Among them, Rbt1 and Rbt4 are found to be important for C. albicans virulence in two mouse models. 26. Braun BR, Johnson AD: TUP1, CPH1 and EFG1 make independent contributions to filamentation in Candida albicans. Genetics 2000, 155:57-67. 27. •
Kadosh D, Johnson AD: Rfg1, a protein related to the Saccharomyces cerevisiae hypoxic regulator Rox1, controls filamentous growth and virulence in Candida albicans. Mol Cell Biol 2001, 21:2496-2505. This paper, together with [28•], reports the identification of Rfg1, a transcription factor related to Rox1 of S. cerevisiae, as a regulator of filamentation and virulence. RFG1 can repress a hypoxic gene in an S. cerevisiae rox1 mutant in a Tup1-dependent manner, and Rfg1 binds specifically to an S. cerevisiae hypoxic operator DNA sequence. Hyperfilamentation and derepression of hypha-specific genes are observed in C. albicans rfg1/rfg1 mutants. tup1 mutation is epistatic to rfg1 mutation in filamentation or the expression of hypha-specific genes. However, Rfg1 does not regulate hypoxic transcripts in response to oxygen starvation in C. albicans. 28. Khalaf RA, Zitomer RS: The DNA binding protein Rfg1 is a repressor • of filamentation in Candida albicans. Genetics 2001, 157:1503-1512. See annotation to [27•].
34. Davis D, Wilson RB, Mitchell AP: RIM101-dependent and -independent pathways govern pH responses in Candida albicans. Mol Cell Biol 2000, 20:971-978. 35. El Barkani A, Kurzai O, Fonzi WA, Ramon A, Porta A, Frosch M, Muhlschlegel FA: Dominant active alleles of RIM101 (PRR2) bypass the pH restriction on filamentation of Candida albicans. Mol Cell Biol 2000, 20:4635-4647. 36. Brown DH Jr, Giusani AD, Chen X, Kumamoto CA: Filamentous growth of Candida albicans in response to physical environmental cues and its regulation by the unique CZF1 gene. Mol Microbiol 1999, 34:651-662. 37.
Monge RA, Navarro-Garcia F, Molero G, Diez-Orejas R, Gustin M, Pla J, Sanchez M, Nombela C: Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans. J Bacteriol 1999, 181:3058-3068.
38. Sharkey LL, McNemar MD, Saporito-Irwin SM, Sypherd PS, Fonzi WA: HWP1 functions in the morphological development of Candida albicans downstream of EFG1, TUP1, and RBF1. J Bacteriol 1999, 181:5273-5279. 39. Staab JF, Bradway SD, Fidel PL, Sundstrom P: Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1. Science 1999, 283:1535-1538. 40. Hube B, Monod M, Schofield DA, Brown AJ, Gow NA: Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol Microbiol 1994, 14:87-99. 41. Schaller M, Korting HC, Schafer W, Bastert J, Chen W, Hube B: Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis. Mol Microbiol 1999, 34:169-180. 42. Schroppel K, Sprosser K, Whiteway M, Thomas DY, Rollinghoff M, Csank C: Repression of hyphal proteinase expression by the mitogen-activated protein (MAP) kinase phosphatase Cpp1p of Candida albicans is independent of the MAP kinase Cek1p. Infect Immun 2000, 68:7159-7161. 43. Srikantha T, Tsai LK, Daniels K, Soll DR: EFG1 null mutants of Candida albicans switch but cannot express the complete phenotype of white-phase budding cells. J Bacteriol 2000, 182:1580-1591. 44. Anderson JM, Soll DR: Differences in actin localization during bud and hypha formation in the yeast Candida albicans. J Gen Microbiol 1986, 132:2035-2047. 45. Wendland J, Philippsen P: Cell polarity and hyphal morphogenesis are controlled by multiple rho- protein modules in the filamentous ascomycete Ashbya gossypii. Genetics 2001, 157:601-610. 46. Leberer E, Ziegelbauer K, Schmidt A, Harcus D, Dignard D, Ash J, Johnson L, Thomas DY: Virulence and hyphal formation of Candida albicans require the Ste20p-like protein kinase CaCla4p. Curr Biol 1997, 7:539-546.
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47.
Ayad-Durieux Y, Knechtle P, Goff S, Dietrich F, Philippsen P: A PAKlike protein kinase is required for maturation of young hyphae and septation in the filamentous ascomycete Ashbya gossypii. J Cell Sci 2000, 113:4563-4575.
48. Gulli MP, Peter M: Temporal and spatial regulation of Rho-type guanine-nucleotide exchange factors: the yeast perspective. Genes Dev 2001, 15:365-379. 49. Loeb JD, Sepulveda-Becerra M, Hazan I, Liu H: A G1 cyclin is necessary for maintenance of filamentous growth in Candida albicans. Mol Cell Biol 1999, 19:4019-4027. 50. Hazan I, Sepulveda-Becerra M, Liu H: Hyphal elongation is • independent of cell cycle in Candida albicans. Mol Biol Cell, in press. This paper carefully compared several cell cycle events between yeast and hyphae and concluded that progression of the cell cycle is similar in yeast and hyphal tip cells. Furthermore, it showed that germ tube emergence and hypha-associated actin polarization can occur prior to the G1/S transition, or in later stages of the cell cycle, including M phase. It also showed that Tyr19 phosphorylation of Cdc28 is not employed to regulate hyphal elongation. 51. Sudbery PE: The germ tube of Candida albicans hyphae and • Pseudohyphae show different patterns of septin ring localization. Mol Microbiol 2001, 41:19-31. Using an antibody to S. cerevisiae Cdc11, Cdc11 was localized in pseudohyphal and hyphal cells of C. albicans. A Cdc11 ring forms at the neck between the mother and the germ tube in pseudohyphae. The septin ring forms prior to nuclear migration and disappears after cytokinesis. In hyphae, a transient Cdc11 ring also forms at the site of evagination. However, the ring is not associated with nuclear division. Later, a second Cdc11 ring forms in the germ tube, which is followed by nuclear migration and nuclear division. 52. Longtine MS, Fares H, Pringle JR: Role of the yeast Gin4p protein kinase in septin assembly and the relationship between septin assembly and septin function. J Cell Biol 1998, 143:719-736. 53. Giot L, Konopka JB: Functional analysis of the interaction between Afr1p and the Cdc12p septin, two proteins involved in pheromoneinduced morphogenesis. Mol Biol Cell 1997, 8:987-998.
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54. Gale C, Gerami-Nejad M, McClellan M, Vandoninck S, Longtine MS, • Berman J: Candida albicans Int1p interacts with the septin ring in yeast and hyphal cells. Mol Biol Cell 2001, 12:3538-3549. INT1 expression in S. cerevisiae generates spiral-like septin and Int1 structures at the periphery of polarized cells, and results in phenotypes similar to those of septin mutants. Septins interact with Int1 and are required for spiral structure formation. In C. albicans, Int1 and Cdc3 co-localize to a ring at septa at the neck in pseudohyphae or in the germ tube of hyphae. Unlike pseudohyphae, Int1/septin rings are seen at multiple septa in a growing hypha. 55. Asleson CM, Bensen ES, Gale CA, Melms AS, Kurischko C, • Berman J: Candida albicans INT1-induced filamentation in Saccharomyces cerevisiae depends on Sla2p. Mol Cell Biol 2001, 21:1272-1284. INT1-induced filamentation in S. cerevisiae depends on a small subset of actin mutations and a limited number of actin-interacting proteins including Sla2. Interestingly, C. albicans sla2/sla2 mutants do not form germ tubes or hyphae under all conditions. 56. Zaragoza O, Rodriguez C, Gancedo C: Isolation of the MIG1 gene from Candida albicans and effects of its disruption on catabolite repression. J Bacteriol 2000, 182:320-326.
Now in press The work referred to in the text as (S Lane, C Birse, S Zhou, R Matson, H Liu, unpublished data) and (M Whiteway, personal communication) are now in press: 57. •
Lane S, Birse C, Zhou S, Matson R, Liu H: DNA array studies demonstrate convergent regulation of virulence factors by Cph1, Cph2, and Efg1 in Candida albicans. J Biol Chem 2002, in press. This study suggests that distinct filamentation signaling pathways converge to regulate a common set of differentially expressed genes. The regulation of TEC1 expression by Efg1 and Cph2 is suggested to be one of the mechanisms for the observed convergence. 58. Rocha CRC, Schroppel K, Harcus D, Marcil A, Dignard D, Taylor BN, Thomas DT, Whiteway M, Leberer E: Signalling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Mol Biol Cell 2001, in press.
Transcriptional control of dimorphism in Candida albicans Haoping Liu Candida albicans uses a network of multiple signaling pathways to control the yeast→hypha transition. These include a mitogen-activated protein kinase pathway through Cph1, the cAMP-dependent protein kinase pathway via Efg1, a pH-responsive pathway through Rim101, the Tup1-mediated repression through Rfg1 and Nrg1, and pathways represented by transcription factors Cph2, Tec1 and Czf1. These pathways control the transcription of a common set of hypha-specific genes, many of which encode known virulence factors. The link between the signaling pathways and hyphal elongation is currently unknown, but there is evidence to suggest that Cdc42 likely plays a key role in hyphal morphogenesis. Unlike pseudohyphal growth in Saccharomyces cerevisiae, hyphal elongation is regulated independently of the cell cycle. Cellular differences between pseudohyphae and hyphae are further revealed by septin localization. Addresses Department of Biological Chemistry, 19172 Jamboree Road, University of California at Irvine, Irvine, California 92697-1700, USA; e-mail: [email protected] Current Opinion in Microbiology 2001, 4:728–735 1369-5274/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations bHLH basic helix-loop-helix MAPK mitogen-activated protein kinase PKA cAMP-dependent protein kinase A
Introduction Candida albicans is one of the most frequently isolated fungal pathogens of humans. It is capable of causing superficial mucosal infections as well as systemic infections in immunocompromised individuals. It can grow in a variety of morphological forms, ranging from budding yeast to pseudohyphae (chains of elongated cells with visible constrictions at the sites of septa) and hyphae (linear filaments without visible constrictions at the septa). Its ability to switch from yeast to hyphal growth in response to various environmental signals is not only inherent to its pathogenicity, but also provides an excellent paradigm to understand how signaling pathways coordinate growth and development. Many signaling pathways and regulators involved in hyphal development have been identified as a result of the strong molecular conservation between C. albicans and Saccharomyces cerevisiae in many cellular processes, a C. albicans genome sequencing program (http://www-sequence.stanford.edu/group/candida/) and efficient homologous recombination that allows a variety of molecular manipulations to be possible. The functions of these pathways and regulators in morphogenesis, have been well-reviewed recently [1,2•,3•]. In this review, I concentrate on recent developments, with emphasis on the
relationships between different signaling pathways, and between regulators and downstream effectors.
Transcriptional regulation of hyphal development in C. albicans Hyphal development in C. albicans is determined by a wide range of signals or culture conditions in vitro [1,2•]. Many signaling pathways or regulators have been found to regulate filamentation in one or many of the in vitro hypha-inducing conditions (Figure 1). The mitogen-activated protein kinase pathway for mating and filamentation
In S. cerevisiae, elements of the pheromone-responsive mitogen-activated protein kinase (MAPK) pathway are involved in pseudohyphal and invasive growth. Like S. cerevisiae, Cph1, a homolog of S. cerevisiae Ste12, and a MAPK cascade that includes Cst20 (p21-activated kinase; PAK), Hst7 (MAP kinase kinase; MEK) and Cek1 (MAPK) are also involved in filamentation. Mutants of these genes all display a defect in hyphal development on solid Spider medium. In certain liquid media, the cph1/cph1 mutant is defective in filamentation and in the induction of hypha-specific genes (genes whose expression is associated with cell elongation, rather than with a specific hyphainducing condition) (S Lane, C Birse, S Zhou, R Matson, H Liu, unpublished data). As in S. cerevisiae, Cph1 and members of the MAPK pathway (Cst20, Hst7, Cek1 and a Fus3 MAPK homolog Cek2) are required for mating between MTLa or MTLα strains (J Chen, J Chen, S Lane, H Liu, unpublished data), consistent with the prediction obtained from a comparative genomic analysis between the two yeasts [4]. Overexpression of CPH1 in MTLa and MTLα cells induces the expression of CEK1, CEK2 and genes similar to S. cerevisiae pheromone-induced genes. The cAMP-dependent protein kinase A pathway
The cAMP-dependent protein kinase A (PKA) pathway plays a crucial role in filamentation in S. cerevisiae, C. albicans and other fungi [5]. C. albicans has a single gene homologous to the S. cerevisiae adenylate cyclase gene (CDC35/CYR1). The cyclase is not essential for growth in C. albicans, but is completely required for hyphal development, even when induced by serum (M Whiteway, personal communication). Recently, the adenylate-cyclase-associated protein (CAP1) has been identified and disrupted in C. albicans [6•]. An increase in cytoplasmic cAMP is seen to precede germ tube emergence in wild-type strains, but not in cap1/cap1 mutants. The cap1/cap1 mutant is defective in germ tube formation and hyphal development in all conditions examined, including serum-containing media. The defects are suppressed by exogenous cAMP or dibutyryl cAMP. cap1/cap1 strains are avirulent in a mouse model for systemic candidiasis.
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Figure 1 Regulation of dimorphism in C. albicans by multiple signaling pathways. The Cph1mediated MAPK pathway and the Efg1mediated cAMP pathway are two wellcharacterized signaling pathways in dimorphic regulation. Ras1 may function upstream of both pathways. TEC1 transcription is regulated by Efg1 and Cph2. Rim101 or Czf1 may function through Efg1 or act in parallel with Efg1. Negative regulators Tup1, recruited by Rfg1 or Nrg1, and Rbt1 are also implicated in dimorphic regulation. Not all pathways are shown. Arrows indicate activation and bars indicate inhibition. They do not necessarily indicate a direct interaction. Transcription factors are shown in boxes. These regulators control the transcription of a common set of hypha-specific genes. Pathway-specific outputs are also indicated with dashed lines.
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Both Ras and a receptor-coupled G protein (that is, G protein Gpa2 coupled to receptor Gpr1) act upstream of the adenylate cyclase in S. cerevisiae. They stimulate cAMP synthesis in response to intracellular acidification and extracellular glucose, respectively [7]. C. albicans Ras1 is an important regulator of hyphal development, and is likely to function upstream of the cAMP pathway [8]. Genes similar to GRP1 and GPA2 have been identified by the C. albicans genome-sequencing program. Further studies will be necessary to determine their potential roles in hyphal development. There are only two PKA catalytic subunits, Tpk1 and Tpk2, in C. albicans ([9]; JF Ernst, personal communication). Unlike S. cerevisiae, in which only one of the three catalytic subunits is an activator and the other two are inhibitors of pseudohyphal growth [10], both PKA isoforms in C. albicans are positive regulators of hyphal morphogenesis. Interestingly, Tpk1 and Tpk2 seem to have differential effects on hyphal morphogenesis under different hypha-inducing conditions. Unlike mutants in CYR1 and CAP1, tpk2/tpk2 does not exhibit a strong defect in hyphal morphogenesis in liquid serum-containing media [9], suggesting that the existence of additional cAMP-dependent functions in regulating
hyphal development. Tpk1 is a likely candidate. Phenotypes of a tpk1/tpk1 tpk2/tpk2 double mutant will be able to address this possibility. Efg1, a basic helix-loop-helix (bHLH) protein similar to Phd1 and Sok2 of S. cerevisiae and StuA of Aspergillus nidulans, plays a major role in hyphal morphogenesis [11,12]. efg1/efg1 null mutant strains do not form hyphae under most hypha-inducing conditions, including serum, and are defective in the induction of hypha-specific genes. Efg1 is likely to function downstream of the PKAs, similar to Sok2 in S. cerevisiae [13,14]. TPK2 overexpression cannot suppress the efg1/efg1 defect in hyphal development, whereas overexpression of EFG1 can suppress the filamentation defect in tpk2/tpk2 [9]. The suppression activity of EFG1 depends on threonine-206, a potential phosphorylation site for a PKA [15•]. Substitution of T206 with alanine impairs hyphal formation, whereas glutamate substitution causes hyperfilamentation. Intriguingly, the effects of T206A-Efg1 and T206E-Efg1 variants on hyphal formation are mediumdependent. Unlike the efg1/efg1 null mutant, the T206A Efg1 variant exhibited only a minor defect when induced by serum, indicating the existence of additional modifications or regulations on Efg1 during hyphal development. Efg1
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has recently been shown to bind two palindromic E-box sequences (5′CANNTG3′) upstream of ALS8 [16]. Although the E-box sequence is present at multiple sites upstream of hypha-specific genes, it also appears at approximately the same frequency upstream of randomly chosen genes. Future experiments should address how Efg1 achieves its transcriptional specificity. Efg1 may not be the only target of the cAMP pathway in hyphal morphogenesis. RAS1V13, a dominant active allele of RAS1, can stimulate hyphal development in cph1/cph1 efg1/efg1 strains [17•]. Overproducing a Cdc2-related protein kinase (Crk1) in the cph1/cph1 efg1/efg1 mutant also exhibited a similar phenotype. The crk1/crk1 mutant is defective in hyphal formation in serum and many other hyphainducing media, a phenotype typical of mutants in the cAMP pathway. In S. cerevisiae, additional transcription factors Msn2/Msn6 [18,19] and Sfl1 [10,20] are regulated directly by PKAs and are important for cell morphogenesis and pseudohyphal growth. It will be interesting to determine whether or not their homologs are important for hyphal development in C. albicans.
TEC1 transcription is regulated by Efg1 and Cph2, but not by Cph1 C. albicans Tec1, a new member of the TEA/ATTS family of transcription factors, has recently been shown to regulate hyphal development and virulence in C. albicans [21•]. AbaA and Tec1 of the same family are involved in the regulation of conidiophore formation in A. nidulans and filamentous growth in S. cerevisiae. In S. cerevisiae, TEC1 transcription is regulated by Ste12, and cooperation between Tec1 and Ste12 is important for pseudohyphal growth [22]. In C. albicans, on the other hand, TEC1 transcription is not regulated by Cph1 (S Lane, C Birse, S Zhou, R Matson, H Liu, unpublished data). As in A. nidulans, where AbaA expression is regulated by StuA, TEC1 transcription requires Efg1 in all media examined (S Lane, C Birse, S Zhou, R Matson, H Liu, unpublished data), consistent with the observation that tec1/tec1 mutants exhibit suppressed filamentation in liquid serum-containing media [21•]. EFG1 overexpression does not suppress the morphological defect of the tec1/tec1 mutant [21•], whereas TEC1 overexpression has a partial phenotype in the efg1/efg1 mutant. These results, coupled with the fact that efg1/efg1 strains have a more severe defect in hyphal development than do tec1/tec1 strains [21•], suggest that TEC1 is one of the downstream effectors of Efg1. Cph2, a myc family bHLH protein, has been found to regulate hyphal development in C. albicans [23•]. cph2/cph2 mutant strains are impaired in hyphal development and in the induction of hypha-specific genes in liquid Lee’s media. TEC1 transcription is highest in Lee’s media, and Cph2 is necessary for the transcriptional induction of TEC1. Cph2 binds directly to two sterolregulatory-element-1-like elements upstream of TEC1. Furthermore, the ectopic expression of TEC1 suppresses
the defect of cph2/cph2 in hyphal development. The function of Cph2 in hyphal transcription is therefore mediated, in part, through Tec1.
Tup1-mediated repression of hyphal development through Rfg1 and Nrg1 Tup1 encodes a transcriptional repressor that negatively controls filamentous growth in C. albicans [24]. Like the wellcharacterized repressor complex Tup1–Ssn6 of S. cerevisiae, CaTup1 is likely to be part of a transcriptional repressor complex that is brought to promoters by sequence-specific DNA-binding proteins. Several genes repressed by Tup1 (RBTs) have been identified in C. albicans [25•]. Many of them are induced during the yeast→hypha transition. But, some RBTs are not regulated during filamentation, indicating that the regulation of filamentation is not the sole function of Tup1. This also suggests that Tup1 itself is not regulated during filamentous growth; rather, its associated DNA-binding proteins are likely to be regulated [25•]. Transcript profiling of RBTs in single, double and triple mutants of TUP1, EFG1 and CPH1 suggests that each gene represents a separate pathway in the transcriptional regulation of filamentation [26]. Recently, Rfg1, a protein related to the S. cerevisiae hypoxic regulator Rox1, has been identified independently by two groups as a negative regulator of filamentous growth [27•,28•]. Rox1 is a high mobility group (HMG) domain DNA-binding protein with a repression domain capable of recruiting the Tup1–Ssn6 repressor complex to repress the expression of hypoxic genes. RFG1 can complement a S. cerevisiae rox1 mutant in the repression of hypoxic genes in a Tup1-dependent manner [28•]. In C. albicans, rfg1/rfg1 mutants cause derepression of filamentation and hyphaspecific RBTs. tup1 is epistatic to rfg1 in terms of colony morphology and the extent of transcriptional derepression of the hypha-specific RBTs [27•]. All these data suggest that Rfg1 directs transcriptional repression at hyphaspecific promoters by recruiting the Tup1–Ssn6 complex. Unlike ROX1, RFG1 transcription is not regulated by oxygen, and is not required for the repression of hypoxic genes in C. albicans. Further studies are required to address how Rfg1 activity is regulated during filamentation. Two groups have independently identified another DNAbinding protein, Nrg1, that might direct the Tup1 repressor complex to the promoters of hypha-specific genes [29•,30•]. S. cerevisiae Nrg1 is a sequence-specific zinc-finger DNAbinding protein that directs the Tup1–Ssn6 complex to repress STA1 transcription in a glucose-dependent manner [31]. C. albicans Nrg1 represses filamentous growth and negatively regulates hypha-specific RBTs in C. albicans. The NRG1 transcript is downregulated during filamentous growth in serum at 37°C [30•]. Other pathways
C. albicans has a conserved pH-response pathway. As in S. cerevisiae and A. nidulans, the transcription factor Rim101
Transcriptional control of dimorphism in Candida albicans Liu
(Prr2) mediates pH response through its proteolysis, which is regulated by Rim8 (Prr1) and Rim20 [32–35]. Rim101 is required for alkaline-induced filamentation, the expression of alkaline-responsive genes such as PHR1, and the repression of the alkaline-repressed gene PHR2. A truncated Rim101 causes filamentation and the expression of alkalineinduced genes in acidic media [35]. Efg1 is required for the Rim101-induced filamentation, but not for the expression of alkaline-induced genes. Therefore, Efg1 either acts downstream of Rim101 or the two factors act in parallel in regulating filamentation. Czf1, a zinc-finger-containing protein, is important for hyphal development when cells are grown in the presence of surrounding matrix [36]. Interestingly, an efg1/efg1 mutant is hyperfilamentous when grown in matrix, indicating that Efg1 represses filamentation under this condition (CA Kumamoto, personal communication). The deletion or ectopic expression of CZF1 does not affect filamentous growth of the efg1/efg1 mutant in the matrix, indicating that Czf1 may act to relieve the Efg1-mediated repression of filamentation. Other known signaling pathways involved in dimorphic regulation include a two-component signaling pathway (for a review, see [3•]), the Hog1 MAPK pathway [37], Rbf1-mediated repression [38] and Mcm1 (S Rupp, personal communication). Transcriptional induction of a common set of hypha-specific genes
The hypha-specific genes identified so far include ECE1, HWP1, HYR1, ALS3, ALS8, RBT1 and RBT4. They encode either cell-wall or secreted proteins. Many have been shown to be important virulence factors for systemic infection. HWP1 encodes a cell-wall protein that can serve as a target for mammalian transglutaminases to form covalent attachments between C. albicans and host epithelial cells [39]. RBT1 encodes a cell-wall protein, and RBT4 encodes a secreted protein similar to a set of pathogenesis-related proteins from plants [25•]. Both genes are necessary for the full virulence of C. albicans in a systemic mouse model. In addition to the hypha-specific genes, three members of the secreted aspartyl proteinase genes, SAPs4–6, are expressed when hyphal development is induced with serum or media containing polypeptides as the sole nitrogen source [40]. SAP genes have been shown to contribute to virulence in both systemic and mucosal candidal infections [41]. The expression of all the aforementioned genes requires Efg1 [16,26,38,42] and, under certain media, also requires Cph1 (S Lane, C Birse, S Zhou, R Matson, H Liu, unpublished data), Cph2 and, potentially, Tec1 [21•,23•]. The hypha-specific genes are also repressed by Tup1–Rfg1 [27•,28•] and Tup1–Nrg1. Czf1-mediated and Rim101-mediated filamentation require Efg1, suggesting that Czf1 and Rim101 may also regulate the expression of the aforementioned hyphaspecific genes. Therefore, the expression of hypha-specific
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genes seems to be regulated by multiple pathways (Figure 1). The regulation of TEC1 expression by Cph2 and Efg1 provides one example of how different pathways converge. Cooperative interaction between hyphal regulators of different pathways at the promoters of hypha-specific genes and pathway crosstalk are other potential mechanisms for the observed convergence in the regulation of hypha-specific gene expression. In addition to hypha-specific genes, many dimorphic regulators or signaling pathways also have pathway-specific genes (Figure 1). For example, PHR1 and PHR2 expression is regulated by Rim101 [34,35], mating genes are activated by Cph1 (J Chen, J Chen, S Lane, H Liu, unpublished data) and Efg1 regulates the expression of phase-specific genes such as WH11 [43]. The expression of pathway-specific genes and hypha-specific genes are not exclusive to each other. It is likely that, under any given growth conditions, a network of signaling pathways are employed to simultaneously assess the availability of multiple nutrients, cell density and other growth conditions. The integrated output of these pathways determines the gene expression and dimorphic transition.
Control of cell polarity during hyphal development During hyphal development, cell-surface expansion is highly restricted to the apical tip of hyphal filaments. This polarized apical growth requires the actin cytoskeleton. C. albicans yeast cells display a temporal change in the organization of the actin cytoskeleton during cell cycle progression like that in S. cerevisiae cells, whereas the actin cytoskeleton is polarized at the tip of all hyphal cells during filamentation [44]. In S. cerevisiae and other organisms, the small GTPase of the Rho subfamily Cdc42 is known to be critical for establishing a polarized actin cytoskeleton in response to extracellular stimuli. In the filamentous ascomycete Ashbya gossypii, three Rho-GTPases control distinct steps during polarized hyphal growth with Cdc42 being required for the establishment of actin polarization [45]. C. albicans Cdc42 is also essential for cell morphogenesis (M Whiteway, personal communication), and is probably responsible for establishing the polarized actin cytoskeleton during hyphal induction, considering that one of its downstream effectors, Cla4, is required for hyphal growth under all conditions examined [46]. Interestingly, Cla4 is also necessary for the maturation of hyphae in A. gossypii [47]. In S. cerevisiae, the activation of Cdc42 involves the temporal and spatial regulation of its sole guanine-nucleotide exchange factor Cdc24 [48]. Whether and how the Cdc42 modular activity is regulated during the yeast→hypha transition has not been elucidated in C. albicans. Studies from S. cerevisiae indicate that pseudohyphal growth is regulated by a change in cell cycle (see the review by Rua, Tobe and Kron in this issue, pp 720–727). However, several lines of evidence show that hyphal
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Figure 2
Hyphae
Yeast
DNA synthesis
(a)
(b)
(c) Actin
(d)
(e)
DNA
(f) Septin
(g) SPB
(h) DSPB
(i)
(j) MS Current Opinion in Microbiology
Alignment of cell cycle events in yeast and hyphae. G1 cells are transferred into yeast growth or hypha-inducing conditions. (a) Germ-tube and hypha-associated actin polarization occur before other cell cycle events, (a–j) and the polarized actin structure persists throughout the cell cycle. (b) A transient septin ring forms at the base of the germ tube. (d,e) Polarization of actin cortical patches, budding/or chitin-ring formation, spindle pole body (SPB) duplication,
nuclear migration and DNA synthesis all occur at the same period of time in yeast and hyphae. Chitin rings probably form at the time of the G1/S transition. (g,h) Mitotic spindle (MS) and (g) DNA separation, the mitotic actin ring and (i) the repolarization of the actin cortical patches to the site of septa all appear with similar dynamics in yeast and hyphae. (j) Unlike yeast cells, septin rings remain after mitosis at septa in a growing hypha. DSPB, duplicated spindle pole bodies.
elongation in C. albicans is controlled independently of the cell cycle. The deletion of a G1 cyclin gene affects only sustained hyphal elongation, but not initial germ tube formation [49]. The phosphorylation state of the conserved Tyr19 of the CDK Cdc28 is cell-cycle-regulated and no difference was observed in the phosphorylation and dephosphorylation kinetics between synchronous yeast and hyphal cells [50•]. The timing of several other cell-cycle-regulated events, including microtubule spindle pole body duplication and spindle elongation, rearrangement of the actin cytoskeleton, actin structures, chitin ring formation and nuclear division are also identical between synchronous yeast and hyphal apical cells (Figure 2) [50•]. Hypha-associated polarization of the actin cytoskeleton occurs before spindle duplication, DNA synthesis and chitin ring formation, and persists throughout the cell cycle during hyphal morphogenesis (Figure 2). In addition, cells from many stages of the cell cycle seem competent to develop hyphae. Hyphae induced from unbudded G1 cells have no restrictions and the first site of septation is positioned in the germ tube, whereas hyphae induced from budded cells retain the constriction and chitin ring from the budded cell during the initial cell cycle.
Cellular differences between pseudohyphae and hyphae have been revealed recently by studying septin localization in C. albicans. Septins, encoded by CDC3, CDC10, CDC11 and CDC12 in S. cerevisiae, are protein components of 10 nm neck filaments that are essential for cytokinesis. In C. albicans pseudohyphae, the Cdc11 ring forms at the neck between the mother and the germ tube, which is followed by nuclear migration, division and cytokinesis across the septin ring [51•]. However, in hyphae cells, a transient Cdc11 ring is observed at the site of evagination, which is not associated with nuclear migration and division (Figure 2). A second Cdc11 ring forms in the germ tube and the septin ring is followed by nuclear migration and the first mitosis happens across the ring. The formation of the transient septin rings in hyphae is probably not regulated by the cell cycle and may be unique to hyphal cells. Cell-cycle-independent septin rings also form at the neck of the mating projection of S. cerevisiae [52], where they are important for the pheromone-induced shape change [53]. Cdc3 also localizes to a ring at the mother–daughter neck in yeast and pseudohyphal cells, and to a ring in the germ tube [54•]. Surprisingly, multiple Cdc3 rings at the septa are observed in a growing hypha, indicating that the Cdc3 ring may not disappear after each cytokinesis
Transcriptional control of dimorphism in Candida albicans Liu
[54•]. The hypha-specific regulation of Cdc3 and Cdc11 localization may also apply to other septins. C. albicans Int1 co-localizes with Cdc3 [54•]. The carboxy terminus of Int1 is similar to that of S. cerevisiae Bud4. Like Bud4, Int1 is important for axial budding in C. albicans. Ectopic INT1 expression in S. cerevisiae causes highly polarized apical growth via Swe1 and Sla2 [55•]. In C. albicans, SLA2 is essential for hyphal cell morphogenesis in either starvation media or serum-containing media. Further experimentation is needed to elucidate whether or not Sla2 is regulated by Int1 and other regulators during hyphal development.
Conclusions and future directions A sketch of the complex regulatory network that controls dimorphism in C. albicans is emerging. Different signaling pathways and transcriptional factors seem to converge to regulate the transcription of a common set of hyphaspecific genes. Molecular mechanisms for the integrative regulation and pathway crosstalk require further experiments. Many of the hypha-specific genes encode known virulence factors, which explains why dimorphic signaling pathways are important for C. albicans pathogenesis. However, none of the hypha-specific genes identified so far is directly linked to cell polarity. Thus, the molecular link between the signaling pathways and hyphal cell polarity is still missing. Development of a C. albicans DNA array for genome-wide gene expression studies is likely to identify the missing connection. It is crucial that we address the function and regulation of a C. albicans protein in C. albicans cells. We have seen several cases in which the C. albicans protein contains a conserved and functional domain, but functions in a pathway different from that of its ortholog in S. cerevisiae, as in the cases of Rfg1 [27•,28•] and Mig1 [56]. Most of the experiments done so far have used molecular manipulations to alter the activity of a gene product in C. albicans in order to determine its function. These experiments do not distinguish a requirement for signal transmission from being a part of the pathway that mediates the signal. Future experiments should try to address whether a regulator is altered in terms of its activity, localization, protein stability or post-translational modifications in response to specific stimuli. A major challenge in the future will be to determine the upstream regulatory components and signals for the orphan transcription factors involved in dimorphic transition.
Acknowledgements I would like to thank: J Chen, S Lane, I Hazan and S Zhou for unpublished results; CA Kumanoto, S Rupp, JF Ernst, AJ Brown, M Whiteway, J Berman, PE Sudbery, P Sundstrom, GR Fink, P Philippsen, J Heitman and S Filler for comments, unpublished results and preprints; and I Hazan for her assistance in producing the graphs. I apologize for not including other data because of space restrictions. The work was supported by funds from the National Institutes of Health (GM55155), Burroughs Wellcome, and University of California Universitywide AIDS Program.
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54. Gale C, Gerami-Nejad M, McClellan M, Vandoninck S, Longtine MS, • Berman J: Candida albicans Int1p interacts with the septin ring in yeast and hyphal cells. Mol Biol Cell 2001, 12:3538-3549. INT1 expression in S. cerevisiae generates spiral-like septin and Int1 structures at the periphery of polarized cells, and results in phenotypes similar to those of septin mutants. Septins interact with Int1 and are required for spiral structure formation. In C. albicans, Int1 and Cdc3 co-localize to a ring at septa at the neck in pseudohyphae or in the germ tube of hyphae. Unlike pseudohyphae, Int1/septin rings are seen at multiple septa in a growing hypha. 55. Asleson CM, Bensen ES, Gale CA, Melms AS, Kurischko C, • Berman J: Candida albicans INT1-induced filamentation in Saccharomyces cerevisiae depends on Sla2p. Mol Cell Biol 2001, 21:1272-1284. INT1-induced filamentation in S. cerevisiae depends on a small subset of actin mutations and a limited number of actin-interacting proteins including Sla2. Interestingly, C. albicans sla2/sla2 mutants do not form germ tubes or hyphae under all conditions. 56. Zaragoza O, Rodriguez C, Gancedo C: Isolation of the MIG1 gene from Candida albicans and effects of its disruption on catabolite repression. J Bacteriol 2000, 182:320-326.
Now in press The work referred to in the text as (S Lane, C Birse, S Zhou, R Matson, H Liu, unpublished data) and (M Whiteway, personal communication) are now in press: 57. •
Lane S, Birse C, Zhou S, Matson R, Liu H: DNA array studies demonstrate convergent regulation of virulence factors by Cph1, Cph2, and Efg1 in Candida albicans. J Biol Chem 2002, in press. This study suggests that distinct filamentation signaling pathways converge to regulate a common set of differentially expressed genes. The regulation of TEC1 expression by Efg1 and Cph2 is suggested to be one of the mechanisms for the observed convergence. 58. Rocha CRC, Schroppel K, Harcus D, Marcil A, Dignard D, Taylor BN, Thomas DT, Whiteway M, Leberer E: Signalling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Mol Biol Cell 2001, in press.