The Ndr/LATS kinase Cbk1 regulates a specific subset of Ace2 functions and suppresses the hyphae-to-yeast transition in Candida albicans
Ontology highlight
ABSTRACT: The Regulation of Ace2 and Morphogenesis (RAM) pathway is an important regulatory network in the human fungal pathogen Candida albicans. The RAM pathway’s two most well-studied components, the NDR/Lats kinase Cbk1 and its putative substrate, the transcription factor Ace2, have a wide range of phenotypes and functions. It is not clear, however, which of these functions are specifically due to the phosphorylation of Ace2 by Cbk1. To address this question, we first compared the transcriptional profiles of CBK1 and ACE2 deletion mutants. This analysis indicates that, of the large number of genes whose expression is affected by deletion of CBK1 and ACE2, only 5.5% of those genes are concordantly regulated. Our data also suggest that Ace2 directly or indirectly represses a large set of genes during hyphal morphogenesis. Second, we generated strains containing ACE2 alleles with alanine mutations at the Cbk1 phosphorylation sites. Phenotypic and transcriptional analysis of these ace2 mutants indicates that, as in Saccharomyces cerevisiae, Cbk1 regulation is important for daughter cell localization of Ace2 and cell separation during yeast phase growth. In contrast, Cbk1 phosphorylation of Ace2 plays a minor role in C. albicans yeast-to-hyphae transition. We have, however, discovered a new function for the Cbk1-Ace2 axis. Specifically, Cbk1 phosphorylation of Ace2 prevents the hyphae-to-yeast transition. To our knowledge, this is one of the first regulators of the C. albicans hyphae-to-yeast transition to be described. Finally, we present an integrated model for the role of Cbk1 in the regulation of hyphal morphogenesis in C. albicans.
Project description:Candida albicans, the most common cause of human fungal infections, undergoes a reversible morphological transition from yeast to pseudohyphal and hyphal filaments, which is required for virulence. For many years, the relationship between global gene expression patterns associated with determination of specific C. albicans morphologies has remained obscure. Using a strain that can be genetically manipulated to sequentially transition from yeast to pseudohyphae to hyphae in the absence of complex environmental cues and upstream signaling pathways, we demonstrate by whole-genome transcriptional profiling that genes associated with pseudohyphae represent a subset of those associated hyphae and are generally expressed at lower levels; interestingly, no genes appeared to be expressed exclusively in pseudohyphae. Our results also strongly suggest that in addition to dosage, extended duration of filament-specific gene expression is sufficient to drive the C. albicans yeast-pseudohyphal-hyphal transition. Finally, we describe the first transcriptional profile of the C. albicans reverse hyphal-pseudohyphal-yeast transition and demonstrate that this transition not only involves down-regulation of known hyphal-specific genes but also differential expression of additional genes which have not previously been associated with the forward transition, including many involved in protein synthesis. These findings provide new insight into genome-wide mechanisms important for determining fungal morphology and suggest that in addition to similarities, there are also fundamental differences in global gene expression as pathogenic filamentous fungi undergo forward and reverse morphological transitions.
Project description:We investigated the roles of mitochondrial dynamics in hyphal growth of C. albicans using the small molecule inhibitor MDIVI-1. Strikingly, the small molecule inhibitor represses both the yeast-to hyphae transition and ongoing filamentation, and its effects on morphogenesis can be uncoupled from general growth inhibition. RNAseq experiments of inhibitor-treated cells coupled with Candida mutant analyses suggest the existence of a novel mechanism of action to represses hyphal growth. The inhibitor was protective to host cells, increasing the survival of bone-marrow derived macrophages in ex vivo macrophage-Candida infection assays, suggesting it has potential as a therapeutic.
Project description:Candida albicans is an important fungal pathogen in humans. Several virulence factors of C. albicans have been reported, including a morphological transition from yeast to filamentous forms (hyphae and pseudohyphae). Mss11 is a transcriptional activator required for hyphal formation. To reveal the potential target genes of Mss11, DNA microarray analysis was performed to compare wild type and mss11-deleted mutant.
Project description:Ace2 transcription factor family genes are found in many fungal genomes and are required for regulation of expression of genes involved in cell separation. We used transcriptional profiling to identify the targets of Ace2 in Candida albicans, and we show that these include several cell wall components, such as glucanases and glycosylphosphatidylinositol-anchored proteins. Expression is downregulated in ace2 deletion mutants in both yeast and hyphal cells. In addition, deleting ace2 results in dramatic changes in expression of metabolic pathways. Expression of glycolytic enzymes is reduced, while expression of respiratory genes (including those involved in the tricarboxylic acid cycle, oxidative phosphorylation, and ATP synthesis) is increased. Similar changes occur in both yeast and hyphal cells. In contrast, genes required for acetyl-coenzyme A and lipid metabolism are upregulated in an ace2 deletion mutant grown predominantly as yeast cells but are downregulated in hyphae. These results suggest that in wild-type strains, Ace2 acts to increase glycolysis and reduce respiration. This is supported by the observation that deleting ace2 results in increased resistance to antimycin A, a drug that inhibits respiration. We also show that Ace2 is required for filamentation in response to low oxygen concentrations (hypoxia). We suggest that filamentation is induced in wild-type cells by reducing respiration (using low oxygen or respiratory drugs) and that mutants with increased respiratory activity fail to undergo filamentation under these conditions. This SuperSeries is composed of the SubSeries listed below.
Project description:Candida albicans is a human gut commensal and an opportunistic pathogen. The ability to transition from yeasts to invasive hyphae is a central virulence attribute, but it is unclear how these two cell types function during commensal growth. Using FISH to visualize C. albicans in a murine gut colonization model, we observed the co-occurrence of yeasts and hyphae throughout the gastrointestinal tract. Forward genetic analysis revealed that major transcriptional activators of yeast-to-hypha morphogenesis have unexpected activity as potent inhibitors of commensalism. In vivo FISH, transcriptomic, and genetic analyses indicate that the morphogenesis program inhibits commensal fitness, not through control of cell shape as expected from in vitro studies, but by activating expression of a hypha-specific pro-inflammatory secreted protease and a hyphal cell surface adhesin. These results reveal a tradeoff between fungal programs supporting commensalism and virulence within the commensal niche in which selection against hypha-specific markers limits the disease-causing potential of this ubiquitous commensal-pathogen.
Project description:Candida albicans is a human gut commensal and an opportunistic pathogen. The ability to transition from yeasts to invasive hyphae is a central virulence attribute, but it is unclear how these two cell types function during commensal growth. Using FISH to visualize C. albicans in a murine gut colonization model, we observed the co-occurrence of yeasts and hyphae throughout the gastrointestinal tract. Forward genetic analysis revealed that major transcriptional activators of yeast-to-hypha morphogenesis have unexpected activity as potent inhibitors of commensalism. In vivo FISH, transcriptomic, and genetic analyses indicate that the morphogenesis program inhibits commensal fitness, not through control of cell shape as expected from in vitro studies, but by activating expression of a hypha-specific pro-inflammatory secreted protease and a hyphal cell surface adhesin. These results reveal a tradeoff between fungal programs supporting commensalism and virulence within the commensal niche in which selection against hypha-specific markers limits the disease-causing potential of this ubiquitous commensal-pathogen.
Project description:Goal of this study was to determine metabolic adaptation processes in C. albicans associated to hyphal morphogenesis. Accessory to the metabolic profiling the corresponding transcriptome was investigated. To identify media-specific and general adaptation three different hyphae stimuli were used (M199 pH 7.4, Human serum and N-Aectylglucosamine) were used and compared again two respective yeast conditions (SD and M199 pH 4). Two filamentation-affected mutant strains were included - lacking either two central regulators of filamentation (cph1Δ/efg1Δ) or a downstream effector (hgc1Δ) - for the identification of specfic morphotype-dependent metabolic adaptation in repsonse to hyphae stimuli. Two different time points (90 min and 240 min after transition to test medium) were investigated to define the progression of the examined adaptation processes.
Project description:The polymorphic yeast Candida albicans exists in blastospore and filamentous forms. The switch from one morphological state to the other coincides with the expression of virulence factors, which makes the yeast-to-hypha transition an attractive target for the development of new antifungal agents. Because an untapped therapeutic potential resides in small molecules that hinder C. albicans filamentation, we characterized the inhibitory effect of conjugated linoleic acid (CLA) on hyphal growth and addressed its mechanism of action. CLA inhibited hyphal growth in a dose-dependent fashion, in both liquid- and solid-inducing media. The fatty acid blocked germ tube formation and impeded hyphal elongation. Global transcriptional profiling revealed that CLA downregulated the expression of hypha-specific genes and abrogated the induction of several morphogenesis regulators, including RAS1, TEC1 and UME6. CLA’s repressive effect on TEC1 expression was Ras1-dependent, but Efg1-independent. CLA treatment resulted in the delocalization of Ras1 and its degradation, resulting in the downregulation of the Ras1-cAMP-PKA signaling pathway. This study provides the biological and molecular explanations that underlie CLA’s ability to inhibit hyphal growth in C. albicans.
Project description:Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to systemic diseases known as candidiasis. C. albicans can grow in various morphological forms, including unicellular budding yeasts, filamentous pseudophyphae and true hyphae, and the ability to switch from yeast to hyphal forms is a key survival mechanism in the adaptation of the pathogen to the microenvironments encountered within the host. The filamentation is regulated by multiple signalling pathways and can be induced, in vitro, in several growth media, often leading to contradictory results in the literature. In this study, we use quantitative proteomic analyses to compare the response of C. albicans yeast cells grown in the minimal medium YNB to four media widely used in the literature to induce the yeast to hyphae transition: YNB-Serum, YNB-N-Acetyl-glucosamine (YNB-NAG), Lee medium and the rich Spider medium. We show here that each growth medium induces a unique pattern of response in C. albicans cells, and that some conditions trigger an original and specific metabolic adaptive response. Moreover, comparison of the proteomic profiles indicates modifications of the thiol-dependent oxidative stress status of the cells, especially in YNB-Serum and Lee medium, and, to a lesser extent, in Spider medium, confirming the role of oxidative stress in the filamentation process. Overall, our data indicate that some hypha-inducing media routinely used in the literature are associated with significant changes in in proteomic signature and that should be more often taken account when exploring the filamentation process of the pathogen.
Project description:Sfl1p and Sfl2p are two homologous heat shock factor-type transcriptional regulators that antagonistically control morphogenesis in Candida albicans, while being required for full pathogenesis and virulence. To understand how Sfl1p and Sfl2p exert their function, we combined genome-wide location and expression analyses to reveal their transcriptional targets in vivo together with the associated changes of the C. albicans transcriptome. We show that Sfl1p and Sfl2p bind to the promoter of at least 113 common targets through divergent binding motifs and modulate directly the expression of key transcriptional regulators of C. albicans morphogenesis and/or virulence. Surprisingly, we found that Sfl2p additionally binds to the promoter of 75 specific targets, including a high proportion of hyphal-specific genes (HSGs; HWP1, HYR1, ECE1, others), revealing a direct link between Sfl2p and hyphal development. Data mining pointed to a regulatory network in which Sfl1p and Sfl2p act as both transcriptional activators and repressors. Sfl1p directly represses the expression of positive regulators of hyphal growth (BRG1, UME6, TEC1, SFL2), while upregulating both yeast form-associated genes (RME1, RHD1,YWP1) and repressors of morphogenesis (SSN6, NRG1). On the other hand, Sfl2p directly upregulates HSGs and activators of hyphal growth (UME6, TEC1), while downregulating yeast form-associated genes and repressors of morphogenesis (NRG1, RFG1, SFL1). Using genetic interaction analyses, we provide further evidences that Sfl1p and Sfl2p antagonistically control C. albicans morphogenesis through direct modulation of the expression of important regulators of hyphal growth. Bioinformatic analyses suggest that binding of Sfl1p and Sfl2p to their targets occurs with the co-binding of Efg1p and/or Ndt80p. Indeed, we show that Sfl1p and Sfl2p targets are bound by Efg1p and that both Sfl1p and Sfl2p associate in vivo with Efg1p. Taken together, our data suggest that Sfl1p and Sfl2p act as central “switch on/off” proteins to coordinate the regulation of C. albicans morphogenesis.