Bcr1 plays a central role in the regulation of opaque cell filamentation in Candida albicans.
ABSTRACT: The human fungal pathogen Candida albicans has at least two types of morphological transitions: white to opaque cell transitions and yeast to hyphal transitions. Opaque cells have historically not been known to undergo filamentation under standard filament-inducing conditions. Here we find that Bcr1 and its downstream regulators Cup9, Nrg1 and Czf1 and the cAMP-signalling pathway control opaque cell filamentation in C.?albicans. We have shown that deletion of BCR1, CUP9, NRG1 and CZF1 results in opaque cell filamentation under standard culture conditions. Disruption of BCR1 in white cells has no obvious effect on hyphal growth, suggesting that Bcr1 is an opaque-specific regulator of filamentation under the conditions tested. Moreover, inactivation of the cAMP-signalling pathway or disruption of its downstream transcriptional regulators, FLO8 and EFG1, strikingly attenuates filamentation in opaque cells of the bcr1/bcr1 mutant. Deletion of HGC1, a downstream gene of the cAMP-signalling pathway encoding G1 cyclin-related protein, completely blocks opaque cell filamentation induced by inactivation of BCR1. These results demonstrate that Bcr1 regulated opaque cell filamentation is dependent on the cAMP-signalling pathway. This study establishes a link between the white-opaque switch and the yeast-filamentous growth transition in C.?albicans.
Project description:Candida albicans is the most common cause of invasive fungal infections in humans. Its ability to undergo the morphological transition from yeast to hyphal growth forms is critical for its pathogenesis. Hyphal initiation requires the activation of the cAMP-PKA pathway, which down-regulates the expression of NRG1, the major repressor of hyphal development. Hyphal initiation also requires inoculation of a small amount of C. albicans cells from overnight culture to fresh medium. This inoculation releases the inhibition from farnesol, a quorum-sensing molecule of C. albicans, that accumulated in the spent medium. Here, we show that farnesol inhibits hyphal initiation mainly through blocking the protein degradation of Nrg1. Through screening a kinase mutant library, we identified Sok1 as the kinase required for Nrg1 degradation during inoculation. SOK1 expression is transiently activated on inoculation during hyphal initiation, and overexpression of SOK1 overcomes the farnesol-mediated inhibition of hyphal initiation. Screening a collection of transcription factor mutants, the homeodomain-containing transcription repressor Cup9 is found to be responsible for the repression of SOK1 expression in response to farnesol inhibition. Interestingly, farnesol inhibits Cup9 degradation mediated by the N-end rule E3 ubiquitin ligase, Ubr1. Therefore, hyphal initiation requires both the cAMP-PKA pathway-dependent transcriptional down-regulation of NRG1 and Sok1-mediated degradation of Nrg1 protein. The latter is triggered by the release from farnesol inhibition of Cup9 degradation and consequently, derepression of SOK1 transcription. Neither pathway alone is sufficient for hyphal initiation.
Project description:Microorganisms rarely exist as single species in natural environments. The opportunistic fungal pathogen Candida albicans and lactic acid bacteria (LAB) are common members of the microbiota of several human niches such as the mouth, gut and vagina. Lactic acid bacteria are known to suppress filamentation, a key virulence feature of C. albicans, through the production of lactic acid and other metabolites. Here we report that C. albicans cells switch between two heritable cell types, white and opaque, to undergo filamentation to adapt to diversified environments. We show that acidic pH conditions caused by LAB and low temperatures support opaque cell filamentation, while neutral pH conditions and high temperatures promote white cell filamentation. The cAMP signalling pathway and the Rfg1 transcription factor play major roles in regulating the responses to these conditions. This cell type-specific response of C. albicans to different environmental conditions reflects its elaborate regulatory control of phenotypic plasticity.
Project description:The yeast-filament transition is essential for the virulence of a variety of fungi that are pathogenic to humans. N-acetylglucosamine (GlcNAc), a ubiquitous molecule in both the environment and host, is one of the most potent inducers of filamentation in Candida albicans and thermally dimorphic fungi such as Histoplasma capsulatum and Blastomyces dermatitidis. However, GlcNAc suppresses rather than promotes filamentation in Candida tropicalis, a fungal species that is closely related to C. albicans. Furthermore, we discover that glucose induces filamentous growth in C. tropicalis. Mutation and overexpression assays demonstrate that the conserved cAMP signaling pathway plays a central role in the regulation of filamentation in C. tropicalis. Activation of this pathway promotes filamentation in C. tropicalis, while inactivation of this pathway results in a serious growth defect in filamentation. By screening an overexpression library of 154 transcription factors, we have identified approximately 40 regulators of filamentous growth in C. tropicalis. Although most of the regulators (e.g., Tec1, Gat2, Nrg1, Sfl1, Sfl2, and Ash1) demonstrate a conserved role in the regulation of filamentation, similar to their homologs in C. albicans or S. cerevisiae, some of them are specific to C. tropicalis. For example, Czf1 and Efh1 repress filamentation, while Wor1, Zcf3, and Hcm1 promote filamentation in C. tropicalis. Bcr1, Aaf1, and Csr1 play a specific role in the process of GlcNAc-regulated filamentation. Our findings indicate that multiple interconnected signaling pathways are involved in the regulation of filamentation in C. tropicalis. These mechanisms have conserved and divergent features among different Candida species. Total RNA profiles of cells grown in Lee's glucose or Lee's GlcNAc medium.
Project description:In Candida albicans, a fungal pathogen, the small G-protein Ras1 regulates many important behaviors including white-opaque switching, biofilm formation, and the induction and maintenance of hyphal growth. Like other Ras proteins, Ras1 is activated upon guanine triphosphate binding, and its activity is further modulated by post-translational lipid modifications. Here, we report that the levels of membrane-associated, full-length Ras1 were higher in hyphae than in yeast, and that yeast contained a shorter, soluble Ras1 species that resulted from cleavage. Deletion of the putative cleavage site led to more rapid induction of hyphal growth and delayed hypha-to-yeast transitions. The cleaved Ras1 species was less able to activate its effector, adenylate cyclase (Cyr1), unless tethered to the membrane by a heterologous membrane-targeting domain. Ras1 cleavage was repressed by cAMP-signalling, indicating the presence of a positive feedback loop in which Cyr1 and cAMP influence Ras1. The C.?albicans quorum sensing molecule farnesol, which inhibits Cyr1 and represses filamentation, caused an increase in the fraction of Ras1 in the cleaved form, particularly in nascent yeast formed from hyphae. This newly recognized mode of Ras regulation may control C.?albicans?Ras1 activity in important ways.
Project description:The ability to switch between yeast and filamentous forms is central to Candida albicans biology. The yeast-hyphal transition is implicated in adherence, tissue invasion, biofilm formation, phagocyte escape, and pathogenesis. A second form of morphological plasticity in C. albicans involves epigenetic switching between white and opaque forms, and these two states exhibit marked differences in their ability to undergo filamentation. In particular, filamentous growth in white cells occurs in response to a number of environmental conditions, including serum, high temperature, neutral pH, and nutrient starvation, whereas none of these stimuli induce opaque filamentation. Significantly, however, we demonstrate that opaque cells can undergo efficient filamentation but do so in response to distinct environmental cues from those that elicit filamentous growth in white cells. Growth of opaque cells in several environments, including low phosphate medium and sorbitol medium, induced extensive filamentous growth, while white cells did not form filaments under these conditions. Furthermore, while white cell filamentation is often enhanced at elevated temperatures such as 37°C, opaque cell filamentation was optimal at 25°C and was inhibited by higher temperatures. Genetic dissection of the opaque filamentation pathway revealed overlapping regulation with the filamentous program in white cells, including key roles for the transcription factors EFG1, UME6, NRG1 and RFG1. Gene expression profiles of filamentous white and opaque cells were also compared and revealed only limited overlap between these programs, although UME6 was induced in both white and opaque cells consistent with its role as master regulator of filamentation. Taken together, these studies establish that a program of filamentation exists in opaque cells. Furthermore, this program regulates a distinct set of genes and is under different environmental controls from those operating in white cells.
Project description:The white-opaque switch is a bistable, epigenetic transition affecting multiple traits in Candida albicans including mating, immunogenicity, and niche specificity. To compare how the two cell states respond to external cues, we examined the fitness, phenotypic switching, and filamentation properties of white cells and opaque cells under 1,440 different conditions at 25°C and 37°C. We demonstrate that white and opaque cells display striking differences in their integration of metabolic and thermal cues, so that the two states exhibit optimal fitness under distinct conditions. White cells were fitter than opaque cells under a wide range of environmental conditions, including growth at various pHs and in the presence of chemical stresses or antifungal drugs. This difference was exacerbated at 37°C, consistent with white cells being the default state of C. albicans in the mammalian host. In contrast, opaque cells showed greater fitness than white cells under select nutritional conditions, including growth on diverse peptides at 25°C. We further demonstrate that filamentation is significantly rewired between the two states, with white and opaque cells undergoing filamentous growth in response to distinct external cues. Genetic analysis was used to identify signaling pathways impacting the white-opaque transition both in vitro and in a murine model of commensal colonization, and three sugar sensing pathways are revealed as regulators of the switch. Together, these findings establish that white and opaque cells are programmed for differential integration of metabolic and thermal cues and that opaque cells represent a more metabolically specialized cell state than the default white state.Epigenetic transitions are an important mechanism by which microbes adapt to external stimuli. For Candida albicans, such transitions are crucial for adaptation to complex, fluctuating environments, and therefore contribute to its success as a human pathogen. The white-opaque switch modulates multiple C. albicans attributes, from sexual competency to niche specificity. Here, we demonstrate that metabolic circuits are extensively rewired between white and opaque states, so that the two cell types exhibit optimal fitness under different nutritional conditions and at different temperatures. We thereby establish that epigenetic events can profoundly alter the metabolism of fungal cells. We also demonstrate that epigenetic switching regulates filamentation and biofilm formation, two phenotypes closely associated with pathogenesis. These experiments reveal that white cells, considered the most clinically relevant form of C. albicans, are a "general-purpose" state suited to many environments, whereas opaque cells appear to represent a more metabolically specialized form of the species.
Project description:Pathogenic fungi are capable of switching between different phenotypes, each of which has a different biological advantage. In the most prevalent human fungal pathogen, Candida albicans, phenotypic transitions not only improve its adaptation to a continuously changing host microenvironment but also regulate sexual mating. In this report, we show that Candida tropicalis, another important human opportunistic pathogen, undergoes reversible and heritable phenotypic switching, referred to as the "white-opaque" transition. Here we show that N-acetylglucosamine (GlcNAc), an inducer of white-to-opaque switching in C. albicans, promotes opaque-cell formation and mating and also inhibits filamentation in a number of natural C. tropicalis strains. Our results suggest that host chemical signals may facilitate this phenotypic switching and mating of C. tropicalis, which had been previously thought to reproduce asexually. Overexpression of the C. tropicalis WOR1 gene in C. albicans induces opaque-cell formation. Additionally, an intermediate phase between white and opaque was observed in C. tropicalis, indicating that the switching could be tristable.
Project description:The human pathogen Candida albicans can assume either of two distinct cell types, designated "white" and "opaque." Each cell type is maintained for many generations; switching between them is rare and stochastic, and occurs without any known changes in the nucleotide sequence of the genome. The two cell types differ dramatically in cell shape, colony appearance, mating competence, and virulence properties. In this work, we investigate the transcriptional circuitry that specifies the two cell types and controls the switching between them. First, we identify two new transcriptional regulators of white-opaque switching, Czf1 and white-opaque regulator 2 (Wor2). Analysis of a large set of double mutants and ectopic expression strains revealed genetic relationships between CZF1, WOR2, and two previously identified regulators of white-opaque switching, WOR1 and EFG1. Using chromatin immunoprecipitation, we show that Wor1 binds the intergenic regions upstream of the genes encoding three additional transcriptional regulators of white-opaque switching (CZF1, EFG1, and WOR2), and also occupies the promoters of numerous white- and opaque-enriched genes. Based on these interactions, we have placed these four genes in a circuit controlling white-opaque switching whose topology is a network of positive feedback loops, with the master regulator gene WOR1 occupying a central position. Our observations indicate that a key role of the interlocking feedback loop network is to stably maintain each epigenetic state through many cell divisions.
Project description:Morphological transitions play an important role in virulence and virulence-related processes in a wide variety of pathogenic fungi, including the most commonly isolated human fungal pathogen Candida albicans. While environmental signals, transcriptional regulators, and target genes associated with C. albicans morphogenesis are well-characterized, considerably little is known about morphological regulatory mechanisms and the extent to which they are evolutionarily conserved in less pathogenic and less filamentous non-albicans Candida species (NACS). We have identified specific optimal filament-inducing conditions for three NACS (C. tropicalis, C. parapsilosis, and C. guilliermondii), which are very limited, suggesting that these species may be adapted for niche-specific filamentation in the host. Only a subset of evolutionarily conserved C. albicans filament-specific target genes were induced upon filamentation in C. tropicalis, C. parapsilosis, and C. guilliermondii. One of the genes showing conserved expression was UME6, a key filament-specific regulator of C. albicans hyphal development. Constitutive high-level expression of UME6 was sufficient to drive increased filamentation as well as biofilm formation and partly restore conserved filament-specific gene expression in both C. tropicalis and C. parapsilosis, suggesting that evolutionary differences in filamentation ability among pathogenic Candida species may be partially attributed to alterations in the expression level of a conserved filamentous growth machinery. In contrast to UME6, NRG1, an important repressor of C. albicans filamentation, showed only a partly conserved role in controlling NACS filamentation. Overall, our results suggest that C. albicans morphological regulatory functions are partially conserved in NACS and have evolved to respond to more specific sets of host environmental cues.
Project description:Members of the dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK) family perform a variety of functions in eukaryotes. We used gene disruption, targeted pharmacologic inhibition, and genome-wide transcriptional profiling to dissect the function of the Yak1 DYRK in the human fungal pathogen Candida albicans. C. albicans strains with mutant yak1 alleles showed defects in the yeast-to-hypha transition and in maintaining hyphal growth. They also could not form biofilms. Despite their in vitro filamentation defect, C. albicans yak1Delta/yak1Delta mutants remained virulent in animal models of systemic and oropharyngeal candidiasis. Transcriptional profiling showed that Yak1 was necessary for the up-regulation of only a subset of hypha-induced genes. Although downstream targets of the Tec1 and Bcr1 transcription factors were down-regulated in the yak1Delta/yak1Delta mutant, TEC1 and BCR1 were not. Furthermore, 63% of Yak1-dependent, hypha-specific genes have been reported to be negatively regulated by the transcriptional repressor Tup1 and inactivation of TUP1 in the yak1Delta/yak1Delta mutant restored filamentation, suggesting that Yak1 may function upstream of Tup1 in governing hyphal emergence and maintenance.