The zinc cluster transcription factor Czf1 regulates cell wall architecture and integrity in Candida albicans

AbstractZinc cluster transcription factors are essential fungal specific regulators of gene expression. In the dimorphic pathogen Candida albicans, they control processes ranging from metabolism and stress adaptation to mating, virulence, and antifungal resistance. Here, we have identified the gene CaORF19.1604 as encoding a zinc cluster transcription factor that acts as a regulator of filament development. Hyperactivation of CaORF19.1604, which we have named RHA1 for Regulator of Hyphal Activity, leads to a wrinkled colony morphology under non-hyphal growth conditions, to pseudohyphal growth and filament formation, to invasiveness and enhanced biofilm formation.  Cells with activated Rha1 are sensitive to cell wall modifying agents such as Congo red and the echinocandin drug caspofungin but show normal sensitivity to fluconazole. RNA-sequencing-based transcriptional profiling of the activated Rha1 strain reveals the up-regulation of genes for core filamentation and cell-wall-adhesion-related proteins such as Als1, Als3, Ece1, and Hwp1. Upregulation is also seen for the genes for the hyphal-inducing transcription factors Brg1 and Ume6 and genes encoding several enzymes involved in arginine metabolism, while downregulation is seen for the hyphal repressor Nrg1. The deletion of BRG1 blocks the filamentation caused by activated Rha1, while null mutants of UME6 result in a partial block. Deletion of RHA1 can partially reduce healthy hyphal development triggered by environmental conditions such as Spider medium or serum at 37°C.In contrast to the limited effect of either single mutant, the double rha1 ume6 deletion strain is totally defective in both serum and Spider medium stimulated hyphal development. While the loss of Brg1 function blocks serum-stimulated hyphal development, this block can be significantly bypassed by Rha1 hyperactivity, and the combination of Rha1 hyperactivity and serum addition can generate significant polarization in even brg1 ume6 double mutants. Our results thus suggest that in response to external signals, Rha1 functions to facilitate the switch from an Nrg1 controlled yeast state to a Brg1/Ume6 regulated hyphal state.Author SummaryCandida albicans is the predominant human fungal pathogen, generating a mortality rate of 40% in systemically infected patients. The ability of Candida albicans to change its morphology is a determinant of its tissue penetration and invasion in response to variant host-related stimuli. The regulatory mechanism for filamentation includes a complex network of transcription factors that play roles in regulating hyphae associated genes. We identify here a new regulator of filamentation from the zinc cluster transcription factor family. We present evidence suggesting that this transcription factor assists the Nrg1/Brg1 switch regulating hyphal development.

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Functional Dissection of a Candida albicans Zinc Cluster Transcription Factor, the Multidrug Resistance Regulator Mrr1

Eukaryotic Cell ◽

10.1128/ec.05100-11 ◽

2011 ◽

Vol 10 (8) ◽

pp. 1110-1121 ◽

Cited By ~ 18

Author(s):

Sabrina Schubert ◽

Christina Popp ◽

P. David Rogers ◽

Joachim Morschhäuser

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Transcriptional Activation ◽

Efflux Pump ◽

Major Facilitator Superfamily ◽

Constitutive Activity ◽

Activation Domain ◽

Pathogenic Yeast ◽

Content Type ◽

Zinc Cluster

ABSTRACTThe overexpression of theMDR1gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to the widely used antimycotic agent fluconazole and other toxic compounds in the pathogenic yeastCandida albicans. The zinc cluster transcription factor Mrr1 controlsMDR1expression in response to inducing chemicals, and gain-of-function mutations inMRR1are responsible for the constitutiveMDR1upregulation in fluconazole-resistantC. albicansstrains. To understand how Mrr1 activity is regulated, we identified functional domains of this transcription factor. A hybrid protein consisting of the N-terminal 106 amino acids of Mrr1 and the transcriptional activation domain of Gal4 fromSaccharomyces cerevisiaeconstitutively inducedMDR1expression, demonstrating that the DNA binding domain is sufficient to target Mrr1 to theMDR1promoter. Using a series of C-terminal truncations and systematic internal deletions, we could show that Mrr1 contains multiple activation and inhibitory domains. One activation domain (AD1) is located in the C terminus of Mrr1. When fused to the tetracycline repressor TetR, this distal activation domain induced gene expression from a TetR-dependent promoter. The deletion of an inhibitory region (ID1) located near the distal activation domain resulted in constitutive activity of Mrr1. The additional removal of AD1 abolished the constitutive activity, but the truncated Mrr1 still could activate theMDR1promoter in response to the inducer benomyl. These results demonstrate that the activity of Mrr1 is regulated in multiple ways and provide insights into the function of an important mediator of drug resistance inC. albicans.

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SAGA/ADA Complex Subunit Ada2 Is Required for Cap1- but Not Mrr1-Mediated Upregulation of the Candida albicans Multidrug Efflux PumpMDR1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03065-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5102-5110 ◽

Cited By ~ 16

Author(s):

Bernardo Ramírez-Zavala ◽

Selene Mogavero ◽

Eva Schöller ◽

Christoph Sasse ◽

P. David Rogers ◽

...

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Efflux Pump ◽

Wild Type ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Gain Of Function ◽

Content Type ◽

Zinc Cluster ◽

Fluconazole Resistant

ABSTRACTOverexpression of the multidrug efflux pumpMDR1is one mechanism by which the pathogenic yeastCandida albicansdevelops resistance to the antifungal drug fluconazole. The constitutive upregulation ofMDR1in fluconazole-resistant, clinicalC. albicansisolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested that Mrr1 activatesMDR1transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However,MDR1expression is also regulated by the bZIP transcription factor Cap1, which mediates the oxidative stress response inC. albicans. Here, we show that a hyperactive Mrr1 containing a gain-of-function mutation promotesMDR1overexpression independently of Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 causedMDR1overexpression in a wild-type strain but only weakly in mutants lackingADA2. In the presence of benomyl or H2O2, compounds that induceMDR1expression in an Mrr1- and Cap1-dependent fashion,MDR1was upregulated with the same efficiency in wild-type andada2Δ cells. These results indicate that Cap1, but not Mrr1, recruits Ada2 to theMDR1promoter to induce the expression of this multidrug efflux pump and that Ada2 is not required forMDR1overexpression in fluconazole-resistantC. albicansstrains containing gain-of-function mutations in Mrr1.

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Candida albicans Ecm33p Is Important for Normal Cell Wall Architecture and Interactions with Host Cells

Eukaryotic Cell ◽

10.1128/ec.5.1.140-147.2006 ◽

2006 ◽

Vol 5 (1) ◽

pp. 140-147 ◽

Cited By ~ 55

Author(s):

Raquel Martinez-Lopez ◽

Hyunsook Park ◽

Carter L. Myers ◽

Concha Gil ◽

Scott G. Filler

Keyword(s):

Endothelial Cells ◽

Cell Wall ◽

Candida Albicans ◽

Epithelial Cells ◽

Normal Cell ◽

Cell Wall Integrity ◽

Host Cells ◽

Epithelial Cell Line ◽

Oral Epithelial ◽

Cell Wall Architecture

ABSTRACT Candida albicans ECM33 encodes a glycosylphosphatidylinositol-linked cell wall protein that is important for cell wall integrity. It is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis. To identify potential mechanisms through which Ecm33p contributes to virulence, we investigated the interactions of C. albicans ecm33Δ mutants with endothelial cells and the FaDu oral epithelial cell line in vitro. The growth rate of blastospores of strains containing either one or no intact copies of ECM33 was 50% slower than that of strains containing two intact copies of ECM33. However, all strains germinated at the same rate, forming similar-length hyphae on endothelial cells and oral epithelial cells. Strains containing either one or no intact copies of ECM33 had modestly reduced adherence to both types of host cells, and a markedly reduced capacity to invade and damage these cells. Saccharomyces cerevisiae expressing C. albicans ECM33 did not adhere to or invade epithelial cells, suggesting that Ecm33p by itself does not act as an adhesin or invasin. Examination of ecm33Δ mutants by transmission electron microscopy revealed that the cell wall of these strains had an abnormally electron-dense outer mannoprotein layer, which may represent a compensatory response to reduced cell wall integrity. The hyphae of these mutants also had aberrant surface localization of the adhesin Als1p. Collectively, these results suggest that Ecm33p is required for normal cell wall architecture as well as normal function and expression of cell surface proteins in C. albicans.

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A Gain-of-Function Mutation in the Transcription Factor Upc2p Causes Upregulation of Ergosterol Biosynthesis Genes and Increased Fluconazole Resistance in a Clinical Candida albicans Isolate

Eukaryotic Cell ◽

10.1128/ec.00103-08 ◽

2008 ◽

Vol 7 (7) ◽

pp. 1180-1190 ◽

Cited By ~ 152

Author(s):

Nico Dunkel ◽

Teresa T. Liu ◽

Katherine S. Barker ◽

Ramin Homayouni ◽

Joachim Morschhäuser ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Candida Albicans ◽

Clinical Isolates ◽

Resistant Isolate ◽

Ergosterol Biosynthesis ◽

Gain Of Function ◽

Zinc Cluster ◽

Fluconazole Resistant ◽

Lanosterol Demethylase

ABSTRACT In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate the expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Overexpression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Overexpression of ERG11 leads to the increased production of lanosterol demethylase, which contributes to azole resistance in clinical isolates of C. albicans, but the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately upregulated with ERG11 in a fluconazole-resistant clinical isolate compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single-nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug-susceptible strain resulted in constitutive upregulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole-resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to the increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans.

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Role of Transcription Factor CaNdt80p in Cell Separation, Hyphal Growth, and Virulence in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00325-09 ◽

2010 ◽

Vol 9 (4) ◽

pp. 634-644 ◽

Cited By ~ 47

Author(s):

Adnane Sellam ◽

Christopher Askew ◽

Elias Epp ◽

Faiza Tebbji ◽

Alaka Mullick ◽

...

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Candida Albicans ◽

Cell Separation ◽

Hyphal Growth ◽

Functional Domain ◽

Transcription Factor Family ◽

Content Type ◽

Genes Encoding

ABSTRACT The NDT80/PhoG transcription factor family includes ScNdt80p, a key modulator of the progression of meiotic division in Saccharomyces cerevisiae. In Candida albicans, a member of this family, CaNdt80p, modulates azole sensitivity by controlling the expression of ergosterol biosynthesis genes. We previously demonstrated that CaNdt80p promoter targets, in addition to ERG genes, were significantly enriched in genes related to hyphal growth. Here, we report that CaNdt80p is indeed required for hyphal growth in response to different filament-inducing cues and for the proper expression of genes characterizing the filamentous transcriptional program. These include noteworthy genes encoding cell wall components, such as HWP1, ECE1, RBT4, and ALS3. We also show that CaNdt80p is essential for the completion of cell separation through the direct transcriptional regulation of genes encoding the chitinase Cht3p and the cell wall glucosidase Sun41p. Consistent with their hyphal defect, ndt80 mutants are avirulent in a mouse model of systemic candidiasis. Interestingly, based on functional-domain organization, CaNdt80p seems to be a unique regulator characterizing fungi from the CTG clade within the subphylum Saccharomycotina. Therefore, this study revealed a new role of the novel member of the fungal NDT80 transcription factor family as a regulator of cell separation, hyphal growth, and virulence.

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The zinc cluster transcription factor Tac1p regulates PDR16 expression in Candida albicans

Molecular Microbiology ◽

10.1111/j.1365-2958.2007.05931.x ◽

2007 ◽

Vol 66 (2) ◽

pp. 440-452 ◽

Cited By ~ 67

Author(s):

Sadri Znaidi ◽

Xavier De Deken ◽

Sandra Weber ◽

Tracey Rigby ◽

André Nantel ◽

...

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Zinc Cluster

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The zinc cluster transcription factor Rha1 is a positive filamentation regulator in Candida albicans

Genetics ◽

10.1093/genetics/iyab155 ◽

2021 ◽

Author(s):

Raha Parvizi Omran ◽

Bernardo Ramírez-Zavala ◽

Walters Aji Tebung ◽

Shuangyan Yao ◽

Jinrong Feng ◽

...

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Transcription Factors ◽

Systemic Infection ◽

Hyphal Growth ◽

Growth Conditions ◽

Hyphal Development ◽

Zinc Cluster ◽

Function Blocks ◽

External Signals

Abstract Zinc cluster transcription factors are essential fungal regulators of gene expression. In the pathogen Candida albicans, the gene orf19.1604 encodes a zinc cluster transcription factor regulating filament development. Hyperactivation of orf19.1604, which we have named RHA1 for Regulator of Hyphal Activity, generates wrinkled colony morphology under non-hyphal growth conditions, triggers filament formation, invasiveness, and enhanced biofilm formation and causes reduced virulence in the mouse model of systemic infection. The strain expressing activated Rha1 shows up-regulation of genes required for filamentation and cell-wall-adhesion-related proteins. Increased expression is also seen for the hyphal-inducing transcription factors Brg1 and Ume6, while the hyphal repressor Nrg1 is downregulated. Inactivation of RHA1 reduces filamentation under a variety of filament-inducing conditions. In contrast to the partial effect of either single mutant, the double rha1 ume6 mutant strain is highly defective in both serum- and Spider-medium-stimulated hyphal development. While the loss of Brg1 function blocks serum-stimulated hyphal development, this block can be significantly bypassed by Rha1 hyperactivity, and the combination of Rha1 hyperactivity and serum addition can generate significant polarization even in brg1 ume6 double mutants. Thus, in response to external signals, Rha1 functions with other morphogenesis regulators including Brg1 and Ume6, to mediate filamentation.

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