Christa Gregori

War1p, a Novel Transcription Factor Controlling Weak Acid Stress Response in Yeast

ABSTRACT The Saccharomyces cerevisiae ATP-binding cassette (ABC) transporter Pdr12p effluxes weak acids such as sorbate and benzoate, thus mediating stress adaptation. In this study, we identify a novel transcription factor, War1p, as the regulator of this stress adaptation through transcriptional induction of PDR12. Cells lacking War1p are weak acid hypersensitive, since they fail to induce Pdr12p. The nuclear Zn2Cys6 transcriptional regulator War1p forms homodimers and is rapidly phosphorylated upon sorbate stress. The appearance of phosphorylated War1p isoforms coincides with transcriptional activation of PDR12. Promoter deletion analysis identified a novel cis-acting weak acid response element (WARE) in the PDR12 promoter required for PDR12 induction. War1p recognizes and decorates the WARE both in vitro and in vivo, as demonstrated by band shift assays and in vivo footprinting. Importantly, War1p occupies the WARE in the presence and absence of stress, demonstrating constitutive DNA binding in vivo. Our results suggest that weak acid stress triggers phosphorylation and perhaps activation of War1p. In turn, War1p activation is necessary for the induction of PDR12 through a novel signal transduction event that elicits weak organic acid stress adaptation.

Candida glabrata environmental stress response involves Saccharomyces cerevisiae Msn2/4 orthologous transcription factors

We determined the genome‐wide environmental stress response (ESR) expression profile of Candida glabrata, a human pathogen related to Saccharomyces cerevisiae. Despite different habitats, C. glabrata, S. cerevisiae, Schizosaccharomyces pombe and Candida albicans have a qualitatively similar ESR. We investigate the function of the C. glabrata syntenic orthologues to the ESR transcription factor Msn2. The C. glabrata orthologues CgMsn2 and CgMsn4 contain a motif previously referred to as HD1 (homology domain 1) also present in Msn2 orthologues from fungi closely related to S. cerevisiae. We show that regions including this motif confer stress‐regulated intracellular localization when expressed in S. cerevisiae. Site‐directed mutagenesis confirms that nuclear export of CgMsn2 in C. glabrata requires an intact HD1. Transcript profiles of CgMsn2/4 mutants and CgMsn2 overexpression strains show that they regulate a part of the CgESR. CgMsn2 complements a S. cerevisiae msn2 null mutant and in stressed C. glabrata cells, rapidly translocates from the cytosol to the nucleus. CgMsn2 is required for full resistance against severe osmotic stress and rapid and full induction of trehalose synthesis genes (TPS1, TPS2). Constitutive activation of CgMsn2 is detrimental for C. glabrata. These results establish an Msn2‐regulated general stress response in C. glabrata.

MAPK Hog1 closes the S. cerevisiae glycerol channel Fps1 by phosphorylating and displacing its positive regulators

The aquaglyceroprin Fps1 is responsible for glycerol transport in yeast in response to changes in extracellular osmolarity. Control of Fps1 channel activity in response to hyperosmotic shock involves a redundant pair of regulators, Rgc1 (regulator of the glycerol channel 1) and Rgc2, and the MAPK Hog1 (high-osmolarity glycerol response 1). However, the mechanism by which these factors influence channel activity is unknown. We show that Rgc2 maintains Fps1 in the open channel state in the absence of osmotic stress by binding to its C-terminal cytoplasmic domain. This interaction involves a tripartite pleckstrin homology (PH) domain within Rgc2 and a partial PH domain within Fps1. Activation of Hog1 in response to hyperosmotic shock induces the rapid eviction of Rgc2 from Fps1 and consequent channel closure. Hog1 was recruited to the N-terminal cytoplasmic domain of Fps1, which it uses as a platform from which to multiply phosphorylate Rgc2. Thus, these results reveal the mechanism by which Hog1 regulates Fps1 in response to hyperosmotic shock.

The High-Osmolarity Glycerol Response Pathway in the Human Fungal Pathogen Candida glabrata Strain ATCC 2001 Lacks a Signaling Branch That Operates in Baker's Yeast

ABSTRACT The high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway mediates adaptation to high-osmolarity stress in the yeast Saccharomyces cerevisiae. Here we investigate the function of HOG in the human opportunistic fungal pathogen Candida glabrata. C. glabrata sho1Δ (Cgsho1Δ) deletion strains from the sequenced ATCC 2001 strain display severe growth defects under hyperosmotic conditions, a phenotype not observed for yeast sho1Δ mutants. However, deletion of CgSHO1 in other genetic backgrounds fails to cause osmostress hypersensitivity, whereas cells lacking the downstream MAP kinase Pbs2 remain osmosensitive. Notably, ATCC 2001 Cgsho1Δ cells also display methylglyoxal hypersensitivity, implying the inactivity of the Sln1 branch in ATCC 2001. Genomic sequencing of CgSSK2 in different C. glabrata backgrounds demonstrates that ATCC 2001 harbors a truncated and mutated Cgssk2-1 allele, the only orthologue of yeast SSK2/SSK22 genes. Thus, the osmophenotype of ATCC 2001 is caused by a point mutation in Cgssk2-1, which debilitates the second HOG pathway branch. Functional complementation experiments unequivocally demonstrate that HOG signaling in yeast and C. glabrata share similar functions in osmostress adaptation. In contrast to yeast, however, Cgsho1Δ mutants display hypersensitivity to weak organic acids such as sorbate and benzoate. Hence, CgSho1 is also implicated in modulating weak acid tolerance, suggesting that HOG signaling in C. glabrata mediates the response to multiple stress conditions.

Weak Organic Acids Trigger Conformational Changes of the Yeast Transcription Factor War1 in Vivo to Elicit Stress Adaptation

The Saccharomyces cerevisiae zinc cluster regulator War1 mediates an essential transcriptional and adaptive response to weak organic acid stress. Here we investigate the mechanism of War1 activation upon weak acid stress. We identified several gain-of-function WAR1 alleles mapping to the central War1 region. These mutations constitutively increase levels of the plasma membrane ABC transporter Pdr12, the main War1 target mediating stress adaptation. Functional analysis of War1 reveals that the central region and its C-terminal activation domain are required for function. Notably, the native DNA-binding and dimerization domains appear dispensable for War1 activity, because they can be replaced by a LexA DNA-binding domain. Chromatin immunoprecipitation demonstrates elevated promoter affinity of activated War1, because its PDR12 promoter association increases upon stress. Hyperactive WAR1 alleles have constitutively high PDR12 promoter association. Furthermore, fluorescence resonance energy transfer of functional CFP-War1-YFP proteins also demonstrates conformational changes of stress-activated War1 in vivo. Our results suggest a mechanism whereby War1 activation is accompanied by conformational changes enhancing promoter association, thus initiating the adaptation process.

Efg1 Controls Caspofungin-Induced Cell Aggregation of Candida albicans through the Adhesin Als1

ABSTRACT Echinocandin drugs such as caspofungin (CASP), micafungin, and anidulafungin inhibit fungal cell wall biogenesis by blocking Fks1-mediated β-glucan deposition into the cell surface. Candins have become suitable drugs to treat life-threatening diseases caused by several fungal species, including Candida albicans, that are pathogenic for humans. Here, we present the discovery of a novel CASP-induced flocculation phenotype of C. albicans, which formed large cell aggregates in the presence of CASP. High concentrations of sugars such as mannose or glucose inhibit CASP-induced flocculation and improve survival of C. albicans cells exposed to CASP. Notably, exposure of C. albicans cells to CASP triggers Efg1-dependent expression of the adhesin ALS1 and induces invasive growth on agar plates. Indeed, cells lacking either Efg1 or Als1 show strongly diminished CASP-induced flocculation, and the absence of Efg1 leads to marked CASP hypersensitivity. On the other hand, CASP-induced invasive growth is enhanced in cells lacking Efg1. Hence, CASP stress drives an Efg1-dependent response, indicating that this multifunctional transcriptional regulator, which is otherwise involved in filamentation, white-to-opaque switching, and virulence, also modulates cell wall remodeling upon CASP challenge. Taken together, our data suggest that CASP-induced cell wall damage activates Efg1 in parallel with the known cell integrity stress signaling pathway to coordinate cell wall remodeling.

Sorbic acid stress activates the Candida glabrata high osmolarity glycerol MAP kinase pathway.

Weak organic acids such as sorbic acid are important food preservatives and powerful fungistatic agents. These compounds accumulate in the cytosol and disturb the cellular pH and energy homeostasis. Candida glabrata is in many aspects similar to Saccharomyces cerevisiae. However, with regard to confrontation to sorbic acid, two of the principal response pathways behave differently in C. glabrata. In yeast, sorbic acid stress causes activation of many genes via the transcription factors Msn2 and Msn4. The C. glabrata homologs CgMsn2 and CgMsn4 are apparently not activated by sorbic acid. In contrast, in C. glabrata the high osmolarity glycerol (HOG) pathway is activated by sorbic acid. Here we show that the MAP kinase of the HOG pathway, CgHog1, becomes phosphorylated and has a function for weak acid stress resistance. Transcript profiling of weak acid treated C. glabrata cells suggests a broad and very similar response pattern of cells lacking CgHog1 compared to wild type which is over lapping with but distinct from S. cerevisiae. The PDR12 gene was the highest induced gene in both species and it required CgHog1 for full expression. Our results support flexibility of the response cues for general stress signaling pathways, even between closely related yeasts, and functional extension of a specific response pathway.

A genetic screen identifies mutations in the yeast WAR1 gene, linking transcription factor phosphorylation to weak‐acid stress adaptation

Exposure of the yeast Saccharomyces cerevisiae to weak organic acids such as the food preservatives sorbate, benzoate and propionate leads to the pronounced induction of the plasma membrane ATP‐binding cassette (ABC) transporter, Pdr12p. This protein mediates efflux of weak acid anions, which is essential for stress adaptation. Recently, we identified War1p as the dedicated transcriptional regulator required for PDR12 stress induction. Here, we report the results from a genetic screen that led to the isolation of two war1 alleles encoding mutant variants, War1‐28p and War1‐42p, which are unable to support cell growth in the presence of sorbate. DNA sequencing revealed that War1‐28 encodes a truncated form of the transcriptional regulator, and War1‐42 carries three clustered mutations near the C‐terminal activation domain. Although War1‐42 is expressed and properly localized in the nucleus, the War1‐42p variant fails to bind the weak‐acid‐response elements in the PDR12 promoter, as shown by in vivo footprinting. Importantly, in contrast with wild‐type War1p, War1‐42p is also no longer phosphorylated upon weak‐acid challenge, demonstrating that phosphorylation of War1p, its activation and DNA binding are tightly linked processes that are essential for adaptation to weak‐acid stress.

Functional Genomics of Drug-Induced Ion Homeostasis Identifies a Novel Regulatory Crosstalk of Iron and Zinc Regulons in Yeast

Pyrrolidine dithiocarbamate (PDTC), a known inhibitor of NFκB activation, has antioxidative as well as antiviral activities. PDTC is effective against several virus families, indicating that its antiviral mechanism targets host rather than viral functions. To investigate its mode of action, we used bakers yeast as a simple eukaryotic model system and two types of genome-wide analysis. First, expression profiling using whole-genome DNA microarrays identifies more than 200 genes differentially regulated upon PDTC exposure. Interestingly, the Aft1-dependent iron regulon is a main target of PDTC, indicating a lack of iron availability. Moreover, the PDTC-caused zinc influx triggers a strong regulatory effect on zinc transporters due to the cytoplasmic zinc excess. Second, phenotypic screening the EUROSCARF collection for PDTC hypersensitivity identifies numerous mutants implicated in vacuolar maintenance, acidification as well as in transport, mitochondrial organization, and translation. Notably, the screening data indicate significant overlaps of PDTC-sensitive genes and those mediating zinc tolerance. Hence, we show that PDTC induces cytoplasmic zinc excess, eliciting vacuolar detoxification, which in turn, disturbs iron homeostasis and activates the iron-dependent regulator Aft1. Our work reveals a complex crosstalk in yeast ion homeostasis and the underlying regulatory networks.

Weak Organic Acid Stress Triggers Hyperphosphorylation of the Yeast Zinc-Finger Transcription Factor War1 and Dampens Stress Adaptation

Exposure of Saccharomyces cerevisiae to weak organic acids such as sorbate, propionate, or benzoate rapidly induces the plasma membrane ABC transporter Pdr12, requiring the Zn(II)(2)Cys(6) zinc-finger transcription factor War1. Weak acid stress rapidly triggers War1 phosphorylation but its role for War1 function is not clear yet. Here, we provide new insights into sorbate-induced phosphorylation of War1. A War1 zinc-finger mutant is still hyperphosphorylated in response to sorbate stress, indicating that War1 phosphorylation occurs independently of DNA recruitment. To map and identify phosphoresidues, War1 purified from stressed and unstressed cells was subjected to semiquantitative phosphopeptide mass spectrometry analysis. Remarkably, we show that weak acid stress causes a dramatic hyperphosphorylation of several already prephosphorylated residues. WAR1 alleles harboring combinations of mutations identified phosphoresidues were generated, some of which display altered gel mobility. Certain mutational combinations almost completely abolish stress-induced gel-shift, suggesting alternative phosphorylation. Surprisingly, PDR12 expression levels are similar in these mutants, demonstrating that War1 phosphorylation is not required for PDR12 induction. Strikingly, absence of hyperphosphorylation in response to stress leads to a faster stress adaptation, suggesting that phosphorylation might play a role in stabilizing War1 activity on the promoter elements, hence changing the dynamics and kinetics of the stress response.