Tetracycline-Inducible Gene Expression and Gene Deletion in
            <i>Candida albicans</i>

Park, Yang-Nim; Morschhäuser, Joachim

doi:10.1128/ec.4.8.1328-1342.2005

Cited by 176 publications

(164 citation statements)

References 46 publications

Supporting

Mentioning

162

Contrasting

Order By: Relevance

“…Plasmids pFL39-GAL-HSP104 and pFL39-GAL-HSP104-2KT are TRP1-based centromeric plasmids and have HSP104 under the control of the GAL1 promoter (8). Plasmid pRS314-rtHSP104 has HSP104, which is placed under the control of the tetracycline-inducible (TET-ON) expression system from pTET23 (27). To generate pRS314-rtHsp104, the tetracycline-inducible system was engineered into the centromeric pRS314 plasmid, and the PCR-amplified HSP104 coding region with the terminator region (positions Ϫ9 to ϩ2962) was fused to the TET-ON promoter for the regulation of Hsp104 expression.…”

Section: Methodsmentioning

confidence: 99%

Hsp104 Overexpression Cures Saccharomyces cerevisiae [ PSI ⁺ ] by Causing Dissolution of the Prion Seeds

et al. 2014

Self Cite

View full text Add to dashboard Cite

S accharomyces cerevisiae [PSI ϩ ], the infectious misfolded amyloid form of the protein Sup35, is one of the better-characterized yeast prions (1)(2)(3)(4)(5). Sup35 is an essential protein with three domains, an N-terminal Asn/Gln-rich aggregation-prone domain, a middle domain, and a C-terminal domain that mediates translation termination. In [psi Ϫ ] yeast cells, Sup35 is properly folded, whereas in [PSI ϩ ] yeast cells, most of the Sup35 is misfolded into amyloid seeds, resulting in significant readthrough at stop codons because of the absence of functional Sup35. The conversion of properly folded Sup35 into its misfolded amyloid conformation can occur either spontaneously or by seeding with misfolded Sup35. Once conversion occurs, the [PSI ϩ ] prion conformation continues to pass from generation to generation, with new seeds constantly being generated to maintain a steady-state level of seeds in the yeast population.New seeds are produced when preexisting seeds are severed by Hsp104, a member of the triple-A ATPase family (6). Hsp104 is composed of six identical subunits that form a hexameric ring. Threading of Sup35 molecules through the pore of the Hsp104 hexamer severs the prion seeds in an ATP-dependent reaction (7). Inactivation of the Hsp104 ATPase activity inhibits its severing activity, which in turn cures [PSI ϩ ] and other prions by causing the seeds to dilute out as the cells divide (3,(8)(9)(10)(11)(12)(13)(14).Surprisingly, unlike other yeast prions, the [PSI ϩ ] prion is also cured by overexpression of Hsp104 (3), and even though this was shown more than a decade ago, the mechanism of this curing is still not understood. However, it does appear that the mechanism of [PSI ϩ ] curing by Hsp104 overexpression is very different from the mechanism of curing by Hsp104 inactivation. First, the kinetics of [PSI ϩ ] curing when Hsp104 is overexpressed are very different from the kinetics of curing when Hsp104 is inactivated (8,15,16 One model that has been proposed for the curing of [PSI ϩ ] by the overexpression of Hsp104 is that it involves disaggregation or dissolution of the prion seeds (3,4,8). The role of Hsp104 in this dissolution process could be due to its severing activity, which reduces the size of the prion seeds and concomitantly increases the number of seeds. . This observation was supported by measuring the mobility of GFP-labeled Sup35 in the cytosol and also by measuring the soluble fraction of GFP-labeled Sup35 in cell lysates; both measurements showed an increase in the level of free Sup35. Therefore, in contrast to severing activity, trimming activity reduces the size of the prion seeds without increasing their number.Recently, two other models have been proposed for the curing of [PSI ϩ ] by Hsp104 overexpression. One model proposes that the overexpression of Hsp104 blocks fragmentation of the amyloid fiber (21). This model was based on the observation that when Hsp104 was overexpressed, rather than being recruited to the NMG fibrils by Ssa1, it bound directly to the amyloid ...

show abstract

Section: Methodsmentioning

confidence: 99%

Hsp104 Overexpression Cures Saccharomyces cerevisiae [ PSI ⁺ ] by Causing Dissolution of the Prion Seeds

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…Both a/a tec1⌬/tec1⌬ (A) and (B) were complemented by integrating wild-type TEC1 back into its native locus, placing it under the control of its own promoter. To generate the plasmid pTEC1c, used for complementation of a/a tec1⌬/tec1⌬ (A) and (B), the CaSAT1 gene in the plasmid pNIMI (29) was flanked with the promoter and coding sequences of TEC1 and the 3= region of TEC1, which were amplified by PCR with the primer pairs TEC1-F1/-R1 and TEC1-F2/-R2, respectively (see Table S1 in the supplemental material for a list of all primers used in this study). The genomic DNA of the SC5314 strain served as the template for PCR amplification of the TEC1 gene.…”

Section: Methodsmentioning

confidence: 99%

Role of Tec1 in the Development, Architecture, and Integrity of Sexual Biofilms of Candida albicans

et al. 2015

Self Cite

View full text Add to dashboard Cite

MTL-homozygous (a/a or ␣/␣) white cells form a complex sexual biofilm that exhibits the same architecture as that of MTLheterozygous (a/␣) pathogenic biofilms. However, the former is regulated by the mitogen-activated protein (MAP) kinase pathway, while the latter is regulated by the Ras1/cyclic AMP (cAMP) pathway. We previously demonstrated that in the formation of an MTL-homozygous, mature (48 h) sexual biofilm in RPMI 1640 medium, the MAP kinase pathway targets Tec1 rather than Cph1, the latter of which is the target of the same pathway, but for the opaque cell mating response. Here we continued our analysis of the role of Tec1 by comparing the effects of deleting TEC1 on initial adhesion to silicone elastomer, high-resolution confocal microscopy assessments of the stages and cellular phenotypes during the 48 h of biofilm development, human white cell penetration, and biofilm fragility. We show that although Tec1 plays only a minor role in initial adhesion to the silicone elastomer, it does play a major role in the growth of the basal yeast cell polylayer, vertical extension of hyphae and matrix deposition in the upper portion of the biofilm, final biofilm thickness, penetrability of human white blood cells, and final biofilm integrity (i.e., resistance to fluid flow). These results provide a more detailed description of normal biofilm development and architecture and confirm the central role played by the transcription factor Tec1 in the biofilm model employed here.C andida albicans forms biofilms with different functional characteristics, depending upon the configuration of the MTL locus (1-4). After 48 h in pH-stabilized RPMI 1640 medium at 37°C in air, a/␣ cells form a biofilm on silicone elastomer that is approximately 100 m thick and composed of a basal yeast cell polylayer (ϳ20 m thick) and an extensive upper layer of vertically oriented hyphae (ϳ80 m thick) that are uniformly distributed and embedded in a dense extracellular matrix. These a/␣ biofilms are firmly attached to the silicone elastomer substratum, highly resistant to penetration by phagocytic human white blood cells, resistant to drugs, such as fluconazole, and impermeable to low-and high-molecular-weight molecules (5). Under exactly the same conditions, a/a and ␣/␣ cells in the white phase of the whiteopaque transition (6) form biofilms that have the same architecture and integrity as those of a/␣ biofilms but differ in that they are readily penetrated by human phagocytic white blood cells, susceptible to fluconazole, and permeable to low-and high-molecularweight molecules (1, 5). MTL-heterozygous and MTL-homozygous biofilms also differ in the capacity to support mating (2, 7). MTL-homozygous biofilms support mating of seeded minority opaque cells at 10 to over 100 times the frequency of that of MTLheterozygous biofilms (7). Because of the differences in the latter pathogenic and mating characteristics, biofilms formed by a/␣ cells, which represent the predominant MTL genotype colonizing hosts (8-12), were deemed "pathogenic," while those ...

show abstract

“…TAP tag plasmids Ip21 and Ip22 contained C.m.LEU2 and C.d.HIS1 marker genes, respectively (37), and the ACT1 terminator region (44) and the BamHI-EcoRV TAP tag fragment from pPK335 (45). The SFU1-TAP tagging cassettes were generated by PCR using primers ONC257/ONC258 and Ip21 and Ip22 as template.…”

Section: Methodsmentioning

confidence: 99%

Cap2-HAP Complex Is a Critical Transcriptional Regulator That Has Dual but Contrasting Roles in Regulation of Iron Homeostasis in Candida albicans

Singh¹,

Prasad²,

Sinha³

et al. 2011

Journal of Biological Chemistry

100

211

View full text Add to dashboard Cite

Iron homeostasis is highly regulated in organisms across evolutionary time scale as iron is essential for various cellular processes. In a computational screen, we identified the Yap/bZIP domain family in Candida clade genomes. Cap2/Hap43 is essential for C. albicans growth under iron-deprivation conditions and for virulence in mouse. Cap2 has an amino-terminal bipartite domain comprising a fungal-specific Hap4-like domain and a bZIP domain. Our mutational analyses showed that both the bZIP and Hap4-like domains perform critical and independent functions for growth under iron-deprivation conditions. Transcriptome analysis conducted under iron-deprivation conditions identified about 16% of the C. albicans ORFs that were differentially regulated in a Cap2-dependent manner. Microarray data also suggested that Cap2 is required to mobilize iron through multiple mechanisms; chiefly by activation of genes in three iron uptake pathways and repression of iron utilizing and iron storage genes. The expression of HAP2, HAP32, and HAP5, core components of the HAP regulatory complex was induced in a Cap2-dependent manner indicating a feed-forward loop. In a feed-back loop, Cap2 repressed the expression of Sfu1, a negative regulator of iron uptake genes. Cap2 was coimmunoprecipitated with Hap5 from cell extracts prepared from iron-deprivation conditions indicating an in vivo association. ChIP assays demonstrated Hap32-dependent recruitment of Hap5 to the promoters of FRP1 (Cap2-induced) and ACO1 (Cap2-repressed). Together our data indicates that the Cap2-HAP complex functions both as a positive and a negative regulator to maintain iron homeostasis in C. albicans.

show abstract

Tetracycline-Inducible Gene Expression and Gene Deletion in Candida albicans

Cited by 176 publications

References 46 publications

Hsp104 Overexpression Cures Saccharomyces cerevisiae [ PSI ⁺ ] by Causing Dissolution of the Prion Seeds

Hsp104 Overexpression Cures Saccharomyces cerevisiae [ PSI ⁺ ] by Causing Dissolution of the Prion Seeds

Role of Tec1 in the Development, Architecture, and Integrity of Sexual Biofilms of Candida albicans

Cap2-HAP Complex Is a Critical Transcriptional Regulator That Has Dual but Contrasting Roles in Regulation of Iron Homeostasis in Candida albicans

Contact Info

Product

Resources

About

Tetracycline-Inducible Gene Expression and Gene Deletion in Candida albicans

Cited by 176 publications

References 46 publications

Hsp104 Overexpression Cures Saccharomyces cerevisiae [ PSI + ] by Causing Dissolution of the Prion Seeds

Hsp104 Overexpression Cures Saccharomyces cerevisiae [ PSI + ] by Causing Dissolution of the Prion Seeds

Role of Tec1 in the Development, Architecture, and Integrity of Sexual Biofilms of Candida albicans

Cap2-HAP Complex Is a Critical Transcriptional Regulator That Has Dual but Contrasting Roles in Regulation of Iron Homeostasis in Candida albicans

Contact Info

Product

Resources

About

Hsp104 Overexpression Cures Saccharomyces cerevisiae [ PSI ⁺ ] by Causing Dissolution of the Prion Seeds

Hsp104 Overexpression Cures Saccharomyces cerevisiae [ PSI ⁺ ] by Causing Dissolution of the Prion Seeds