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ABSTRACT: Candida albicans forms two types of biofilm depending upon the configuration of the mating type locus. Although architecturally similar, a/α biofilms are impermeable, impenetrable and drug resistant, whereas a/a and α/α biofilms lack these traits. The difference appears to be the result of alternative matrix. Overexpression of BCR1 in a/a cells, a master regulator of the a/α matrix, conferred impermeability, impenetrability and drug resistance to a/a biofilms. Deletion of BCR1 in a/α cells resulted in the loss of these a/α-specific biofilm traits. Using the BCR1 overexpression in a/a cells, we screened 107 genes of interest and identified eight that were Bcr1-upregulated. When each was overexpressed in a/a biofilms, the three a/α traits were partially conferred, and when deleted in a/α cells, the traits were partially lost. Five of the eight genes have been implicated in iron homeostasis, and six encode proteins that are either in the wall or plasma membrane or secreted. All six possess sites for O-linked and N-linked glycosylation that, like GPI anchors, can cross-link to the wall and matrix, suggesting that they may exert a structural role in conferring impermeability, impenetrability and drug resistance, in addition to their physiological functions. The fact that in a screen of 107 genes, all eight of the Bcr1-upregulated genes identified play a role in impermeability, impenetrability and drug resistance suggests that the formation of the a/α matrix is highly complex and involves a larger number of genes than the initial ones identified here.
Eukaryotic Cell 04/2013; · 3.60 Impact Factor
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ABSTRACT: Most experiments exploring the basic biology of pathogenic microbes are performed in vitro under conditions that do not usually mimic those of their host niche. Hence, developmental programs initiated by specific host cues may be missed in vitro. We have tested the effects of growing low-density agar cultures of the yeast pathogen Candida albicans in concentrations of CO(2) found in the gastrointestinal tract. It is demonstrated that in physiological concentrations of CO(2) at 37°C, yeast cells form a heretofore undescribed multicellular "finger" morphology distinct from a previously described stalk-like structure induced by high doses of UV irradiation that kills more than 99.99% of cells. The finger extends aerially, is uniform in diameter, and is visible to the naked eye, attaining lengths of 3 mm. It is composed of a basal yeast cell monolayer adhering to a semispherical crater formed in the agar and connected to a basal bulb of yeast cells at a fragile interface. The bulb extends into the long shaft. We propose that a single, centrally located hypha extending the length of the shaft forms buds at compartment junctions that serve as the source of the yeast cells in the shaft. A mutational analysis reveals finger formation is dependent upon the pathway Ras1→Cdc35→cyclic AMP (cAMP) (PDE2-|)→Tpk2→Tec1. Because of the mechanically fragile interface and the compactness of bulb and shaft, we suggest that the finger may function as a multicellular dispersal mechanism produced in host niches containing high levels of CO(2).
Eukaryotic Cell 08/2012; 11(10):1257-67. · 3.60 Impact Factor
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ABSTRACT: The mating type locus (MTL) of Candida albicans contains the mating type genes and has, therefore, been assumed to play an exclusive role in the mating process. In mating-incompetent a/α cells, two of the mating type genes, MTLa1 and MTLα2, encode components of the a1-α2 corepressor that suppresses mating and switching. But the MTL locus of C. albicans also contains three apparently unrelated "nonsex" genes (NSGs), PIK, PAP and OBP, the first two essential for growth. Since it had been previously demonstrated that deleting either the a/α copy of the entire MTL locus, or either MTLa1 or MTLα2, affected virulence, we hypothesized that the NSGs in the MTL locus may also play a role in pathogenesis. Here by mutational analysis, it is demonstrated that both the mating type and nonsex genes in the MTL locus play roles in a/α biofilm formation, and that OBP is essential for impermeability and fluconazole resistance.
PLoS Pathogens 01/2012; 8(1):e1002476. · 9.13 Impact Factor
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ABSTRACT: Efg1 is a central transcriptional regulator of morphogenesis and metabolism in Candida albicans. In vivo genome-wide ChIP chip and in vitro footprint analyses revealed the Efg1 recognition sequence (EGR-box) TATGCATA in the yeast growth form of this human fungal pathogen. Upstream regions of EFG1 and genes encoding transcriptional regulators of hyphal growth including TCC1, CZF1, TEC1, DEF1 and NRG1 contained EGR- and/or EGR-like boxes. Unexpectedly, after brief hyphal induction the genome-wide Efg1 binding pattern was completely altered and new binding sites of yet unknown specificity had appeared. Hyphal induction abolished Efg1 accumulation on EFG1 and TCC1 promoters and led to rapid decline of both transcripts, although the Efg1 protein persisted in cells. While EFG1 promoter activity in the yeast growth form did not depend on bound Efg1, its downregulation under hyphal induction depended on the presence of Efg1 and the protein kinase A isoform Tpk2. Deletion analyses of the EFG1 upstream region revealed that none of its resident EGR-boxes is uniquely responsible for EFG1 promoter downregulation. These results suggest different binding specificities of Efg1 in yeast growth and in hyphal induction and suggest a brief time window following hyphal induction, in which Efg1 exerts its repressive effect on target promoters.
Molecular Microbiology 09/2011; 82(3):602-18. · 5.01 Impact Factor
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ABSTRACT: Similar multicellular structures can evolve within the same organism that may have different evolutionary histories, be controlled by different regulatory pathways, and play similar but nonidentical roles. In the human fungal pathogen Candida albicans, a quite extraordinary example of this has occurred. Depending upon the configuration of the mating type locus (a/α versus a/a or α/α), C. albicans forms alternative biofilms that appear similar morphologically, but exhibit dramatically different characteristics and are regulated by distinctly different signal transduction pathways. Biofilms formed by a/α cells are impermeable to molecules in the size range of 300 Da to 140 kDa, are poorly penetrated by human polymorphonuclear leukocytes (PMNs), and are resistant to antifungals. In contrast, a/a or α/α biofilms are permeable to molecules in this size range, are readily penetrated by PMNs, and are susceptible to antifungals. By mutational analyses, a/α biofilms are demonstrated to be regulated by the Ras1/cAMP pathway that includes Ras1→Cdc35→cAMP(Pde2-|)→Tpk2(Tpk1)→Efg1→Tec1→Bcr1, and a/a biofilms by the MAP kinase pathway that includes Mfα→Ste2→ (Ste4, Ste18, Cag1)→Ste11→Hst7→Cek2(Cek1)→Tec1. These observations suggest the hypothesis that while the upstream portion of the newly evolved pathway regulating a/a and α/α cell biofilms was derived intact from the upstream portion of the conserved pheromone-regulated pathway for mating, the downstream portion was derived through modification of the downstream portion of the conserved pathway for a/α biofilm formation. C. albicans therefore forms two alternative biofilms depending upon mating configuration.
PLoS Biology 08/2011; 9(8):e1001117. · 11.45 Impact Factor
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ABSTRACT: Like MTL-heterozygous (a/α) cells, white MTL-homozygous (a/a or α/α) cells of Candida albicans, to which a minority of opaque cells of opposite mating type have been added, form thick, robust biofilms. The latter biofilms are uniquely stimulated by the pheromone released by opaque cells and are regulated by the mitogen-activated protein kinase signal transduction pathway. However, white MTL-homozygous cells, to which opaque cells of opposite mating type have not been added, form thinner biofilms. Mutant analyses reveal that these latter biofilms are self-induced. Self-induction of a/a biofilms requires expression of the α-receptor gene STE2 and the α-pheromone gene MFα, and self-induction of α/α biofilms requires expression of the a-receptor gene STE3 and the a-pheromone gene MFa. In both cases, deletion of WOR1, the master switch gene, blocks cells in the white phenotype and biofilm formation, indicating that self-induction depends upon low frequency switching from the white to opaque phenotype. These results suggest a self-induction scenario in which minority opaque a/a cells formed by switching secrete, in a mating-type-nonspecific fashion, α-pheromone, which stimulates biofilm formation through activation of the α-pheromone receptor of majority white a/a cells. A similar scenario is suggested for a white α/α cell population, in which minority opaque α/α cells secrete a-pheromone. This represents a paracrine system in which one cell type (opaque) signals a second highly related cell type (white) to undergo a complex response, in this case the formation of a unisexual white cell biofilm.
Eukaryotic Cell 06/2011; 10(6):753-60. · 3.60 Impact Factor
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ABSTRACT: Few mating-regulated genes have been characterized in Candida albicans. C. albicans FIG1 (CaFIG1) is a fungus-specific and mating-induced gene encoding a putative 4-transmembrane domain protein that shares sequence similarities with members of the claudin superfamily. In Saccharomyces cerevisiae, Fig1 is required for shmoo fusion and is upregulated in response to mating pheromones. Expression of CaFIG1 was also strongly activated in the presence of cells of the opposite mating type. CaFig1-green fluorescent protein (GFP) was visible only during the mating response, when it localized predominantly to the plasma membrane and perinuclear zone in mating projections and daughter cells. At the plasma membrane, CaFig1-GFP was visualized as discontinuous zones, but the distribution of perinuclear CaFig1-GFP was homogeneous. Exposure to pheromone induced a 5-fold increase in Ca(2+) uptake in mating-competent opaque cells. Uptake was reduced substantially in the fig1Δ null mutant. CaFig1 is therefore involved in Ca(2+) influx and localizes to membranes that are destined to undergo fusion during mating.
Eukaryotic Cell 01/2011; 10(3):435-44. · 3.60 Impact Factor
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ABSTRACT: Among the hemiascomycetes, only Candida albicans must switch from the white phenotype to the opaque phenotype to mate. In the recent evolution of this transition, mating-incompetent white cells acquired a unique response to mating pheromone, resulting in the formation of a white cell biofilm that facilitates mating. All of the upstream components of the white cell response pathway so far analyzed have been shown to be derived from the ancestral pathway involved in mating, except for the mitogen-activated protein (MAP) kinase scaffold protein, which had not been identified. Here, through binding and mutational studies, it is demonstrated that in both the opaque and the white cell pheromone responses, Cst5 is the scaffold protein, supporting the evolutionary scenario proposed. Although Cst5 plays the same role in tethering the MAP kinases as Ste5 does in Saccharomyces cerevisiae, Cst5 is approximately one-third the size and has only one rather than four phosphorylation sites involved in activation and cytoplasmic relocalization.
mBio 01/2011; 2(1):e00237-10. · 5.31 Impact Factor
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PLoS Pathogens 01/2010; 6(3). · 9.13 Impact Factor
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ABSTRACT: To mate, the fungal pathogen Candida albicans must undergo homozygosis at the mating-type locus and then switch from the white to opaque phenotype. Paradoxically, opaque cells were found to be unstable at physiological temperature, suggesting that mating had little chance of occurring in the host, the main niche of C. albicans. Recently, however, it was demonstrated that high levels of CO(2), equivalent to those found in the host gastrointestinal tract and select tissues, induced the white to opaque switch at physiological temperature, providing a possible resolution to the paradox. Here, we demonstrate that a second signal, N-acetylglucosamine (GlcNAc), a monosaccharide produced primarily by gastrointestinal tract bacteria, also serves as a potent inducer of white to opaque switching and functions primarily through the Ras1/cAMP pathway and phosphorylated Wor1, the gene product of the master switch locus. Our results therefore suggest that signals produced by bacterial co-members of the gastrointestinal tract microbiota regulate switching and therefore mating of C. albicans.
PLoS Pathogens 01/2010; 6(3):e1000806. · 9.13 Impact Factor
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ABSTRACT: The way in which signal transduction pathways evolve remains a mystery, primarily because we have few examples of ones that have newly evolved. There are numerous examples of how signal transduction pathways in the same organism selectively share components, most notably between the signal transduction pathways in Saccharomyces cerevisiae for the mating process, the filamentation process, cell wall integrity, ascospore formation, and osmoregulation. These examples, however, have not provided insights into how such pathways evolve. Here, through construction of an overexpression library for 107 transcription factors, and through mutational analyses, we have identified the transcription factor Tec1 as the last component of the newly evolved signal transduction pathway that regulates the pheromone response of the white cell phenotype in Candida albicans. The elucidation of this last component, Tec1, establishes a comprehensive description of the pheromone response pathway in the white cell phenotype of C. albicans, providing a unique perspective on how new signal transduction pathways may evolve. The three portions of this new regulatory pathway appear to have been derived from three different ancestral programs still functional in C. albicans. The upstream portion, including signals, receptors, the trimeric G protein complex, and the MAP kinase cascade, was derived intact from the upstream portion of the opaque pheromone response pathway of the mating process; Tec1, the transcription factor targeted by the MAP kinase pathway, was derived from a filamentation pathway; and the white-specific downstream target genes were derived from an ancestral biofilm process. The evolution of this pheromone response pathway provides a possible paradigm for how such signal transduction pathways evolve.
PLoS Biology 01/2010; 8(5):e1000363. · 11.45 Impact Factor
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ABSTRACT: To mate, MTL-homozygous strains of the yeast pathogen Candida albicans must switch from the white to opaque phase. Mating-competent opaque cells then release pheromone that induces polarization, a G1 block and conjugation tube formation in opaque cells of opposite mating type. Pheromone also induces mating-incompetent white cells to become adhesive and cohesive, and form thicker biofilms that facilitate mating. The pheromone response pathway of white cells shares the upstream components of that of opaque cells, but targets a different transcription factor. Here we demonstrate that the genes up-regulated by the pheromone in white cells are activated through a common cis-acting sequence, WPRE, which is distinct from the cis-acting sequence, OPRE, responsible for up-regulation in opaque cells. Furthermore, we find that these white-specific genes play roles in white cell biofilm formation, and are essential for biofilm formation in the absence of an added source of pheromone, suggesting either an autocrine or pheromone-independent mechanism. These results suggest an intimate, complex and unique relationship between switching, mating and MTL-homozygous white cell biofilm formation, the latter a presumed virulence factor in C. albicans.
PLoS Pathogens 10/2009; 5(10):e1000601. · 9.13 Impact Factor
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ABSTRACT: Recent comparative genomics and mutational studies of the genes regulating mating and meiosis in fungi provide new insights into not only the variability of the key genes, but also the plasticity of the regulatory circuitry in the evolution of mating systems.
Current biology: CB 08/2009; 19(13):R509-11. · 10.99 Impact Factor
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Geraldine Butler,
Matthew D Rasmussen,
Michael F Lin,
Manuel A S Santos,
Sharadha Sakthikumar,
Carol A Munro,
Esther Rheinbay,
Manfred Grabherr,
Anja Forche,
Jennifer L Reedy, [......], Thyagarajan Srikantha,
Qiandong Zeng,
Judith Berman,
Matthew Berriman,
Joseph Heitman,
Neil A R Gow,
Michael C Lorenz,
Bruce W Birren,
Manolis Kellis,
Christina A Cuomo
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ABSTRACT: Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.
Nature 07/2009; 459(7247):657-62. · 36.28 Impact Factor
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ABSTRACT: To mate, Candida albicans must undergo homozygosis at the mating type-like locus MTL[1, 2], then switch from the white to opaque phenotype [3, 4]. Paradoxically, when opaque cells are transferred in vitro to 37 degrees C, the temperature of their animal host, they switch en masse to white [5-7], suggesting that their major niche might not be conducive to mating. It has been suggested that pheromones secreted by opaque cells of opposite mating type [8] or the hypoxic condition of host niches [9, 10] stabilize opaque cells. There is, however, an additional possibility, namely that CO(2), which achieves levels in the host 100 times higher than in air [11-13], stabilizes the opaque phenotype. CO(2) has been demonstrated to regulate the bud-hypha transition in C. albicans[14, 15], expression of virulence genes in bacteria [16], and mating events in Cryptococcus neoformans[14, 17]. We tested the possibility that CO(2) stabilizes the opaque phenotype, and found that physiological levels of CO(2) induce white-to-opaque switching and stabilize the opaque phenotype at 37 degrees C. It exerts this control equally under anaerobic and aerobic conditions. These results suggest that the high levels of CO(2) in the host induce and stabilize the opaque phenotype, thus facilitating mating.
Current biology: CB 03/2009; 19(4):330-4. · 10.99 Impact Factor
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ABSTRACT: Candida albicans strains homozygous at the mating type locus can switch from white to opaque, and must do so to mate. Opaque cells then secrete mating pheromones that stimulate opaque cells of opposite mating type to undergo mating. These same pheromones stimulate mating-incompetent white cells to become cohesive and adhesive, and enhance white cell biofilm development, a pathogenic trait. Stimulation is mediated through the same receptor, G protein complex and mitogen-activated protein kinase pathway. Here we present evidence that a C. albicans-specific 55-amino-acid region of the first intracellular loop, IC1, of the alpha-pheromone receptor Ste2p, is required for the alpha-pheromone response of white cells, but not that of opaque cells. This represents a unique regulatory configuration in which activation of a common pathway by the same ligand, the same receptor and the same signal transduction pathway is dependent on a unique region of an intracellular loop of the common receptor in one of the two responding phenotypes.
Molecular Microbiology 01/2009; 71(4):925-47. · 5.01 Impact Factor
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ABSTRACT: Candida glabrata undergoes reversible, high-frequency core switching between phenotypes that include dark brown (DB), light brown (LB) and white (Wh). These phenotypes in turn can switch to the irregular wrinkle (IWr) phenotype. Natural isolates, however, express predominantly the DB phenotype, leading to the hypothesis that it has a colonization advantage over the other switch phenotypes. Using the mouse model of systemic infection, results are presented which support this hypothesis. DB has an advantage over other switch phenotypes in colonizing the two major target organs in the mouse model, the spleen and liver. A time-course study reveals that colonization of the major target organs occurs very rapidly (within 2 h) after host injection, and that the DB advantage for spleen and liver colonization is immediate. The DB advantage is maintained during clearing from spleen, liver and kidneys, and during delayed transient brain colonization. These results demonstrate that DB has a colonization advantage over other switch phenotypes, and that the switch phenotype expressed by a colonizing population therefore plays a fundamental role in virulence. It is therefore essential that switching be considered in both in vivo and in vitro studies of C. glabrata virulence.
Microbiology 12/2008; 154(Pt 11):3309-18. · 3.06 Impact Factor
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ABSTRACT: Candida albicans must undergo a switch from white to opaque to mate. Opaque cells then release mating type-specific pheromones that induce mating responses in opaque cells. Uniquely in C. albicans, the same pheromones induce mating-incompetent white cells to become cohesive, form an adhesive basal layer of cells on a surface, and then generate a thicker biofilm that, in vitro, facilitates mating between minority opaque cells. Through mutant analysis, it is demonstrated that the pathways regulating the white and opaque cell responses to the same pheromone share the same upstream components, including receptors, heterotrimeric G protein, and mitogen-activated protein kinase cascade, but they use different downstream transcription factors that regulate the expression of genes specific to the alternative responses. This configuration, although common in higher, multicellular systems, is not common in fungi, and it has not been reported in Saccharomyces cerevisiae. The implications in the evolution of multicellularity in higher eukaryotes are discussed.
Molecular biology of the cell 04/2008; 19(3):957-70. · 5.98 Impact Factor
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ABSTRACT: In the mouse model for systemic infection, natural a/alpha strains of C. albicans are more virulent and more competitive than their spontaneous MTL-homozygous offspring, which arise primarily by loss of one chromosome 5 homologue followed by duplication of the retained homologue (uniparental disomy). Deletion of either the a or alpha copy of the MTL locus of natural a/alpha strains results in a small decrease in virulence, and a small decrease in competitiveness. Loss of the heterozygosity of non-MTL genes along chromosome 5, however, results in larger decreases in virulence and competitiveness. Natural MTL-homozygous strains are on average less virulent than natural MTL-heterozygous strains and arise by multiple mitotic cross-overs along chromosome 5 outside of the MTL region. These results are consistent with the hypothesis that a competitive advantage of natural a/alpha strains over MTL-homozygous offspring maintains the mating system of C. albicans.
Molecular Microbiology 07/2007; 64(6):1587-604. · 5.01 Impact Factor
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ABSTRACT: In Candida albicans, the a1-alpha2 complex represses white-opaque switching, as well as mating. Based upon the assumption that the a1-alpha2 corepressor complex binds to the gene that regulates white-opaque switching, a chromatinimmunoprecipitation-microarray analysis strategy was used to identify 52 genes that bound to the complex. One of these genes, TOS9, exhibited an expression pattern consistent with a "master switch gene." TOS9 was only expressed in opaque cells, and its gene product, Tos9p, localized to the nucleus. Deletion of the gene blocked cells in the white phase, misexpression in the white phase caused stable mass conversion of cells to the opaque state, and misexpression blocked temperature-induced mass conversion from the opaque state to the white state. A model was developed for the regulation of spontaneous switching between the opaque state and the white state that includes stochastic changes of Tos9p levels above and below a threshold that induce changes in the chromatin state of an as-yet-unidentified switching locus. TOS9 has also been referred to as EAP2 and WOR1.
Eukaryotic Cell 11/2006; 5(10):1674-87. · 3.60 Impact Factor
