Forward genetics in Candida albicans reveals the Arp2/3 complex is required for hyphal formation, but not endocytosis
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ABSTRACT:
ORGANISM(S): Candida albicans
SUBMITTER: Zully Leon
PROVIDER: E-GEOD-19583 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Candida albicans is a diploid fungal pathogen lacking a defined complete sexual cycle, and thus has been refractory to standard forward genetic analysis. Instead, transcription profiling and reverse genetic strategies based on Saccharomyces cerevisiae have typically been used to link genes to functions. To overcome restrictions inherent in such indirect approaches, we have investigated a forward genetic mutagenesis strategy based on the UAU1 technology. We screened 4700 random insertion mutants for defects in hyphal development, and linked two new genes (ARP2 and VPS52) to hyphal growth. Deleting ARP2 abolished hyphal formation, generated round and swollen yeast phase cells, disrupted cortical actin patches and blocked virulence in mice. The mutants also showed a global lack of induction of hyphae-specific genes upon the yeast-to-hyphae switch. Surprisingly, both arp2Î/Î and arp2Î/Îarp3Î/Î mutants were still able to endocytose FM4-64 and Lucifer Yellow, although as shown by time-lapse movies internalization of FM4-64 was somewhat delayed in mutant cells. Thus the non-essential role of the Arp2/3 complex discovered by forward genetic screening in C. albicans showed that uptake of membrane components from the plasma membrane to vacuolar structures is not dependent on this actin nucleating machinery. By forward genetic screening, we have identified genes that are essential for hyphal formation. One of the hits is the Arp2/3 complex, which is essential for hyphal formation, but not for viability in Candida albicans. To gain insights into cellular processes affected by disrupting Arp2/3 complex functions, we performed transcriptional profiling under yeast growth conditions (YPD at 30°C for three hours) or hyphal induction (YPD + 10% FBS at 37°C for three hours) and compared transcriptional consequences of deleting ARP2 to MYO5 and SLA2 microarray data sets (Oberholzer et al., 2006).
2010-10-08 | E-GEOD-19565 | biostudies-arrayexpress
Project description:Deleting components of the Arp2/3 complex in Candida albicans resulted in a global lack of hyphal specific gene induction. This observation suggests that the failure in hyphal growth of Arp2/3 complex mutants could be a result of failure to activate hyphal specific genes. If the hyphal defect was primarily due to failure to activate gene expression, de-repressing hyphal-specific gene expression by deleting the NRG1 repressor could potentially suppress the defect, as deletion of NRG1 leads to constitutive filamentous growth even in the absence of any hyphal induction signals (Garcia-Sanchez et al., 2005, Kadosh & Johnson, 2005). We therefore created an nrg1Î/Îarp2Î/Î mutant. When grown under non-inducing conditions, nrg1Î/Îarp2Î/Î cells showed the arp2Î/Î mutant morphology of round and swollen cells. When induced for hyphal growth, nrg1Î/Îarp2Î/Î cells also exhibited the arp2Î/Î cell morphology and did not form hyphae even after extended overnight incubation times. To determine if the hyphal-specific genes are de-repressed in the nrg1Î/Îarp2Î/Î mutant, we performed transcript profiling. We compared the nrg1Î/Îarp2Î/Î mutant grown under hyphal conditions to the arp2Î/Î mutant grown under the same conditions (10% serum, 37ï°C, three hours), and found that a significant number of hyphal-specific genes that are normally induced when WT cells are undergoing the yeast to hyphae switch (WT-HY) showed greater expression in the nrg1Î/Îarp2Î/Î mutant compared to arp2Î/Î cells (p-value 4.9x10-9). When we examined the set of NRG1-dependent hyphal-specific genes previously identified (Kadosh & Johnson, 2005), we found that seven of 28 genes (HYR1, SAP5, SAP4, KIP4, ORF19.6079, ALS3 and UME6) showed significantly increased expression (⥠2 fold) in nrg1Î/Îarp2Î/Î cells compared to arp2Î/Î cells, while a further four genes (IHD1, CBP1, ORF19.6705 and ALS10) showed moderately increased expression between 1.5 and 2-fold. Thus, while deleting a transcriptional repressor of the filamentation program leads to de-repression of many hyphal genes, the entire regulated gene set is not de-repressed; this presumably reflects the complex interplay that different transcriptional (co-) repressors exert on the yeast-to-hyphae transition (Garcia-Sanchez et al., 2005, Kadosh & Johnson, 2005). We further found that despite the increased induction of some hyphal genes in the nrg1Î/Îarp2Î/Î mutant, a few of those genes are not as highly induced as in WT cells. One gene that was induced in both the ânrg1Î/Îarp2Î/Î vs arp2Î/Îâ and the ânrg1Î/Îarp2Î/Î vs WTâ-comparisons is UME6, a recently identified key regulator of the hyphal program (Banerjee et al., 2008, Zeidler et al., 2009). Interestingly, although constitutive over-expression of UME6 in WT cells resulted in constitutive filamentous growth even in the absence of hyphae signals (Carlisle et al., 2009), the increased expression level of UME6 in the nrg1Î/Îarp2Î/Î mutant is not sufficient to restore filamentation in the absence of a functional Arp2/3 complex. Thus despite partial de-repression of the hyphal program, hyphae do not form, making it likely other roles of the Arp2/3 complex, such as its function in actin patch formation and actin branching, are required for hyphal development.
2010-01-22 | E-GEOD-19582 | biostudies-arrayexpress
Project description:Background : Candida albicans is a diploid pathogenic fungus not yet amenable to routine genetic investigations. Understanding aspects of the regulation of its biological functions and the assembly of its protein complexes would lead to further insight into the biology of this common disease-causing microbial agent. Results: We have developed a toolbox allowing in vivo protein tagging by PCR-mediated homologous recombination with TAP, HA and MYC tags. The transformation cassettes were designed to accommodate a common set of integration primers. The tagged proteins can be used to perform tandem affinity purification (TAP) or chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP). Tandem affinity purification of C. albicans Nop1 revealed the high conservation of the small processome composition in yeasts. Data obtained with in vivo TAP-tagged Tbf1, Cbf1 and Mcm1 recapitulates previously published genome-wide location profiling by ChIP-CHIP. We also designed a new reporter system for in vivo analysis of transcriptional activity of gene loci in C. albicans. Conclusion: This toolbox provides a basic setup to perform purification of protein complexes and increase the number of annotated transcriptional regulators and genetic circuits in C. albicans. Two independent biological replicates of ChIP-CHIP of Mcm1-TAP in yeast and hyphal states. ChIP-CHIP of Cbf1-TAP and Tbf1-TAP.
2010-05-16 | E-GEOD-13427 | biostudies-arrayexpress
Project description:In this study, we have investigated Mediator function in the human fungal pathogen C. albicans. An initial screening of conditionally regulated Mediator subunits showed that the Med7 of C. albicans was not essential, in contrast to the situation noted for Saccharomyces cerevisiae. While loss of CaMed7 did not lead to loss of viability under normal growth conditions, it dramatically influenced the pathogenM-bM-^@M-^Ys ability to grow in different carbon sources, to form hyphae and biofilms, and to colonize the gastrointestinal tracts of mice. We used location profiling to determine Mediator binding under yeast and hyphal morphologies characterized by different transcription profiles. We observed a core set of specific and common genes bound by Med7 under both conditions; this specific core set is expanded considerably during hyphal growth, supporting the idea that Mediator binding correlates with changes in transcriptional activity and that this binding is condition specific. Med7 bound not only in the promoter regions of active genes but also of inactive genes and within coding regions and at the 3M-bM-^@M-^Y ends of genes. By combining genome-wide location profiling, expression analyses and phenotyping, we have identified different Med7 regulons including genes related to glycolysis and the Filamentous Growth Regulator family. We performed genome-wide occupancy experiments (YPD, 30oC and YPD, 37oC) with Med7p to determine its binding sites.
2014-09-18 | E-GEOD-61518 | biostudies-arrayexpress
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. CLAM-bM-^@M-^Ys 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 CLAM-bM-^@M-^Ys ability to inhibit hyphal growth in C. albicans. Two-color experimental design that consistently used growth in Spider Media at 30M-bM-^DM-^C as the control. We tested the effect of high temperature as well as the effect of adding 100 mM-BM-5 CLA at either low or high temperature. RNA from each replicate came from independent cultures.
2011-03-10 | E-GEOD-25822 | biostudies-arrayexpress
Project description:Biofilm development by Candida albicans requires cell adhesion for the initial establishment of the biofilm and the continued stability after hyphal development occurs; however, the regulation of the process has not been fully established. Using chromatin immunoprecipitation coupled to microarray analysis (ChIP-chip) we have characterized a regulon containing the Mcm1p factor that is required for the initial surface adhesion during biofilm formation. In the yeast Saccharomyces cerevisiae several Mcm1p regulons have been characterized in which regulatory specificity is achieved through co-factors binding a sequence adjacent to the Mcm1p-binding site. This new Mcm1p regulon in C. albicans also requires a co-factor, which we identify as the transcription factor Ahr1p. However, in contrast to the other yeast regulons, Ahr1p alone binds the target promoters, which include several key adhesion genes, and recruits Mcm1p to these sites. Through transcription profiling and qPCR analysis, we demonstrate that this Ahr1p-Mcm1p complex directly activates these adhesion genes. When the regulon was disrupted by deleting AHR1, the strain displayed reduced adherence to a polystyrene surface. We also demonstrate a role for the regulon in hyphal growth and in virulence. Our work thus establishes a new mechanism of Mcm1p-directed regulation distinct from those observed for other Mcm1p co-regulators. We performed genome wide occupancy experiments (YPD, 30oC) with Ahr1p and Mcm1p to determine their binding sites. To confirm an interaction between the two factors we also performed genome wide occupancy with Mcm1p in an ahr1 deletion strain. To complement with genome wide occupancy experiments, we performed a transcription profile with an ahr1 deletion strain under yeast conditions (YPD, 30oC).
2010-11-10 | E-GEOD-25174 | biostudies-arrayexpress
Project description:Coordinated ribosomal protein (RP) gene expression is crucial for cellular viability, but the transcriptional network controlling this regulon has only been well characterized in the yeast Saccharomyces cerevisiae. We have used whole-genome transcriptional and location profiling to establish that, in Candida albicans, the RP regulon is controlled by the Myb-domain protein Tbf1 working in conjunction with Cbf1. These two factors bind both the promoters of RP genes and the rDNA locus; Tbf1 activates transcription at these loci and is essential. Orthologs of Tbf1 bind TTAGGG telomeric repeats in most eukaryotes, and TTAGGG cis-elements are present upstream of RP genes in plants and fungi, suggesting that Tbf1 was involved in both functions in ancestral eukaryotes. In all Hemiascomycetes, Rap1 substituted Tbf1 at telomeres and in the S. cerevisiae lineage this substitution also occurred independently at RP genes, illustrating the extreme adaptability and flexibility of transcriptional regulatory networks. This SuperSeries is composed of the following subset Series: GSE10458: Transcription Factor Substitution during the Evolution of Fungal Ribosome Regulation_ChIP-CHIP GSE10499: Transcription Factor Substitution during the Evolution of Fungal Ribosome Regulation_expression profiling Keywords: SuperSeries Refer to individual Series
2010-05-25 | E-GEOD-10622 | biostudies-arrayexpress
Project description:Transcriptional profiling of CDC53 down-regulated Candida albicans cells compared to control cells Keywords: comparative genomic hybridization, genetic modification Candida albicans is an important opportunistic human fungal pathogen, which can cause mucosal as well as systemic infections in immunocompromised patients. Critical for the virulence of C. albicans is its ability to undergo a morphological transition from yeast to hyphal growth mode. Proper induction of filamentation is dependent on the ubiquitination pathway, which targets proteins for proteasome-mediated protein degradation or activates them for signaling events. In the present study, we evaluated the role of ubiquitination in C. albicans by impairing the function of the major ubiquitin-ligase complex SCF. This was done by depleting its backbone, the cullin Cdc53p (orf19.1674), using a tetracycline down-regulatable promoter system. Cdc53p-depleted cells displayed an invasive phenotype and constitutive filamentation under conditions favouring yeast growth mode, both on solid and in liquid media. In addition, these cells exhibited an early onset of cell death, as judged from propidium iodide staining, suggesting that CDC53 is an essential gene in C. albicans. To identify Cdc53p-dependent pathways in C. albicans, a genome-wide expression analysis was carried out that revealed a total of 425 differentially expressed genes (fold change ⥠2, p-value ⤠0.05) with 192 up- and 233 down-regulated genes in the CDC53-repressed mutant as compared to the control strain. GO term analysis identified biological processes significantly affected by Cdc53p depletion, including amino acid starvation response, with 14 genes being targets of the transcriptional regulator Gcn4p, and reductive iron transport. These results indicate that Cdc53p enables C. albicans to adequately respond to environmental signals. Two-strain experiment, CDC53-repressible cells vs. control cells, both grown in the presence of 20 µg/ml Doxycycline. Four independent biological replicates of total RNA of each strain, dye-swap.
2010-05-14 | E-GEOD-13976 | biostudies-arrayexpress
Project description:Coordinated ribosomal protein (RP) gene expression is crucial for cellular viability, but the transcriptional network controlling this regulon has only been well characterized in the yeast Saccharomyces cerevisiae. We have used whole-genome transcriptional and location profiling to establish that, in Candida albicans, the RP regulon is controlled by the Myb-domain protein Tbf1 working in conjunction with Cbf1. These two factors bind both the promoters of RP genes and the rDNA locus; Tbf1 activates transcription at these loci and is essential. Orthologs of Tbf1 bind TTAGGG telomeric repeats in most eukaryotes, and TTAGGG cis-elements are present upstream of RP genes in plants and fungi, suggesting that Tbf1 was involved in both functions in ancestral eukaryotes. In all Hemiascomycetes, Rap1 substituted Tbf1 at telomeres and in the S. cerevisiae lineage this substitution also occurred independently at RP genes, illustrating the extreme adaptability and flexibility of transcriptional regulatory networks. Keywords: Expression profiling Expression profiling of tetracycline addition time-course in the tetO-TBF1/tbf1 conditional mutant.
2010-05-25 | E-GEOD-10499 | biostudies-arrayexpress
Project description:Coordinated ribosomal protein (RP) gene expression is crucial for cellular viability, but the transcriptional network controlling this regulon has only been well characterized in the yeast Saccharomyces cerevisiae. We have used whole-genome transcriptional and location profiling to establish that, in Candida albicans, the RP regulon is controlled by the Myb-domain protein Tbf1 working in conjunction with Cbf1. These two factors bind both the promoters of RP genes and the rDNA locus; Tbf1 activates transcription at these loci and is essential. Orthologs of Tbf1 bind TTAGGG telomeric repeats in most eukaryotes, and TTAGGG cis-elements are present upstream of RP genes in plants and fungi, suggesting that Tbf1 was involved in both functions in ancestral eukaryotes. In all Hemiascomycetes, Rap1 substituted Tbf1 at telomeres and in the S. cerevisiae lineage this substitution also occurred independently at RP genes, illustrating the extreme adaptability and flexibility of transcriptional regulatory networks. Keywords: ChIP-CHIP Two independent biological replicates of ChIP-CHIP of Tbf1-HA and Cbf1-HA.
2010-05-25 | E-GEOD-10458 | biostudies-arrayexpress