White-Opaque Switching in Natural MTLa/alpha Isolates of Candida albicans: Evolutionary Implications for Roles in Host Adaptation, Pathogenesis and Sex
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ABSTRACT: The discovery of white-opaque switching in natural MTLa/alpha isolates of Candida albicans sheds new light on the evolution of phenotypic plasticity and host adaptation. Comparing gene expression of white and opaque cells of a MTL a/alpha strain
Project description:Modes of sexual reproduction in eukaryotic organisms are highly diversified. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally and morphologically differentiated white and opaque cells show a coordinating behavior in the process of mating. Although white cells are mating-incompetent, they are induced to produce sexual pheromones when treated with opposite pheromones or interacted with opaque cells of an opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections and thus facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTLa) in sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling and thus create an environment conducive to sexual mating. This coordination behavior of the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans. total RNA profiles of white cell treated with pheromone
Project description:The capacity of the commensal yeast Candida albicans to grow in several forms, referred to as phenotypic plasticity, is critical for its survival, and abilities to thrive and cause infection in the human host. In this study, we report a novel phenotype of C. albicans, referred as the “gray” phenotype. The gray cell type, together with the previously discovered “white” and “opaque” cell types, forms a tristable phenotypic switching system. The three phenotypes differ in cellular and colony appearance, global transcriptional profiles, secreted aspartyl proteinase (Sap) activities and virulence in different infection models. The addition of the gray cell type to the phenotypic transition systems may enhance fitness and confers an adaptive advantage for C. albicans in the host. We further demonstrate that two key regulators of white-opaque switching, Wor1 and Efg1, are not required for maintenance of the gray phenotype, suggesting that a different regulatory circuitry may be involved in the regulation of gray cell formation. Our study provides an example of multiple stable and heritable switching systems, indicating that the regulation of morphological forms to adapt to environmental changes could be much more elaborate than previously thought. total RNA profiles of 24h old white, gray and opaque cell types
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:Single-celled organisms have different strategies to sense and utilize nutrients in their ever-changing environments. The opportunistic fungal pathogen Candida albicans is a common member of the human microbiota, especially that of the gastrointestinal (GI) tract. An important question concerns how C. albicans gained a competitive advantage over other microbes to become a successful commensal and opportunistic pathogen. Here, we report that C. albicans uses N-acetylglucosamine (GlcNAc), an abundant carbon source present in the GI tract, as a signal for nutrient availability. When placed in water, C. albicans cells normally enter the G0 phase and remain viable for weeks. However, they quickly lose viability when cultured in water containing only GlcNAc. We term this phenomenon GlcNAc-induced cell death (GICD). GlcNAc triggers the upregulation of ribosomalbiogenesis genes, alterations of mitochondrial metabolism, and the accumulation of reactive oxygen species (ROS), followed by rapid cell death via both apoptotic and necrotic mechanisms. Multiple pathways, including the conserved cyclic AMP (cAMP) signaling and GlcNAc catabolic pathways, are involved in GICD. GlcNAc acts as a signaling molecule to regulate multiple cellular programs in a coordinated manner and therefore maximizes the efficiency of nutrient use. This adaptive behavior allows C. albicans’ more efficient colonization of the gut. Expression profiles of Candida alibcans in three different 5 hours) were determined by high throughput sequencing technology.
Project description:Proteomics on B. thuringiensis CT_43 cells in GYS medium. Two biological replicate cell samples were collected at time points of 7 h, 9 h, 13 h and 22 h, respectively. The crude proteins were purified using the ReadyPrep 2-D Cleanup Kit, underwent the reductive alkylation, tryptically digested, and were labeled with 8-plex iTRAQ reagents as follows: 7 h-1, 113; 7 h-2, 114; 9 h-1, 115; 9 h-2, 116; 13 h-1, 117; 13 h-2, 118; 22 h-1, 119; and 22 h-2, 121. The labeled samples were pooled and resolved into 12 fractions, which were loaded onto LC-MSMS.
Project description:C. albicans white, opaque and white engineered cells were incubated on Spider medium for 8 hours. Crosses were between a and alpha cells of each phenotype, and a white cells alone or a opaque cells alone were also incubated as controls. White cells are no
Project description:In Candida albicans the Efg1 transcription factor (a member of the APSES family) is an important regulator of hyphal growth, and of the white-to-opaque transition. In contrast, we show that the Efg1 ortholog in Candida parapsilosis is a major regulator of a different morphological switch at the colony level, from a concentric to smooth morphology. The rate of switching is at least 100-fold increased in an efg1 knockout relative to wild type. Deleting efg1 also reduces biofilm formation, and results in increased sensitivity to SDS, Congo red, caspofungin and calcofluor white in cells of both morphologies. Biofilm reduction is more dramatic in in vitro than in in vivo models. We use ChIP-seq to show that Efg1 binds to 502 promoter regions, including 70 potential transcription factors or regulatory proteins. Several of the transcription factors belong to networks that regulate biofilm development and white-opaque switching in C. albicans. Efg1 also binds to its own promoter. The binding site for C. parapsilosis Efg1 resembles that of orthologs in other fungi. Many Efg1 targets are probably also regulated by the Ndt80 transcription factor. We show that a paralog of Efg1 (Efh1) is restricted to Candida species. Efh1 does not regulate concentric-smooth phenotype switching, biofilm formation or stress response in C. parapsilosis. Our analysis supports the hypothesis that Efg1 has an ancient role as regulator of development in fungi, but we have identified a new role in C. parapsilosis as a regulator of colony switching that is distinct from the white-opaque switch in C. albicans. RNA was isolated from C. parapsilosis wild type (three biological replicates, concentric phenotype), and from efg1 deletion strains (three biological replicates from both concentric and smooth phenotype). Gene expression was determined using strand-specific RNA-seq.
Project description:Transcriptional profiling of C. tropicalis a/alpha cells (CAY1511) in white state, opaque state, overexpressing Wor1, or wor1 mutants hybridized against a universal mixed reference sample from all 4 states. 4 condition experiment: white, opaque, tdh3-wor1, Δ/Δwor1; 4 biological replicates of each
Project description:In Candida albicans, the a1-alpha2 complex represses white-opaque switching as well as mating. A ChIP-chip strategy was, therefore, used to screen for genes with a1-alpha2 binding sites and expression patterns consistent with a master switch gene (MSG). Of 51 genes identified with an a1-alpha2 binding site, one gene, TOS9, also referred to as EAP2, exhibited an expression pattern consistent with a MSG. TOS9 is expressed in opaque, not white a/a and alpha/alpha cells and Tos9p localizes to the opaque cell nucleus. Deletion of TOS9 blocks cells in the white phase, and misexpression in the white phase of the parent tos9+/tos9+ strain results in mass conversion to opaque. Expression of TOS9 under control of a MET promoter rescues the TOS9 null mutant tos9-/tos9- phenotype. Temperature-induced mass conversion of opaque to white in the parent strain results in the immediate cessation of TOS9 transcription, and loss of Tos9p prior to the point of commitment to white (the switch event), which occurs at the time of the second cell doubling. Misexpression of TOS9, as well as inhibition of the second round of DNA replication inhibits the temperature-induced switch from opaque to white. Based on these observations, a model is developed for the regulation and role of TOS9 in switching. Keywords: ChIP chip
Project description:Transcriptional profiling of a-type wor1 deleted cells and mixed a-type and alpha-type opaque cells under in vitro biofilm-forming conditions. Specifically, they were grown for two days at room temperature in a 12-well poly-styrene plate containing 1 ml of Lee's + Glucose liquid media. Samples were hybridized against a universal mixed reference sample of a-type cells in white and opaque states grown in Spider liquid media. 2 condition experiment: white wor1-deletion mutant a-type cells, opaque mixed a-type and alpha-type cells; two biological replicates each.