Dataset Information

Mechanisms Underlying the Delayed Activation of the Cap1 Transcription Factor in Candida albicans following Combinatorial Oxidative and Cationic Stress Important for Phagocytic Potency.

ABSTRACT:

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Following phagocytosis, microbes are exposed to an array of antimicrobial weapons that include reactive oxygen species (ROS) and cationic fluxes. This is significant as combinations of oxidative and cationic stresses are much more potent than the corresponding single stresses, triggering the synergistic killing of the fungal pathogenCandida albicansby "stress pathway interference." Previously we demonstrated that combinatorial oxidative plus cationic stress triggers a dramatic increase in intracellular ROS levels compared to oxidative stress alone. Here we show that activation of Cap1, the major regulator of antioxidant gene expression inC. albicans, is significantly delayed in response to combinatorial stress treatments and to high levels of H2O2 Cap1 is normally oxidized in response to H2O2; this masks the nuclear export sequence, resulting in the rapid nuclear accumulation of Cap1 and the induction of Cap1-dependent genes. Here we demonstrate that following exposure of cells to combinatorial stress or to high levels of H2O2, Cap1 becomes trapped in a partially oxidized form, Cap1(OX-1) Notably, Cap1-dependent gene expression is not induced when Cap1 is in this partially oxidized form. However, while Cap1(OX-1)readily accumulates in the nucleus and binds to target genes following high-H2O2stress, the nuclear accumulation of Cap1(OX-1)following combinatorial H2O2and NaCl stress is delayed due to a cationic stress-enhanced interaction with the Crm1 nuclear export factor. These findings define novel mechanisms that delay activation of the Cap1 transcription factor, thus preventing the rapid activation of the stress responses vital for the survival ofC. albicanswithin the host.

Importance

Combinatorial stress-mediated synergistic killing represents a new unchartered area in the field of stress signaling. This phenomenon contrasts starkly with "stress cross-protection," where exposure to one stress protects against subsequent exposure to a different stress. Previously we demonstrated that the pathogenCandida albicansis acutely sensitive to combinations of cationic and oxidative stresses, because the induction of H2O2-responsive genes is blocked in the presence of cationic stress. We reveal that this is due to novel mechanisms that delay activation of the Cap1 AP-1-like transcription factor, the major regulator of the H2O2-induced regulon. Cap1 becomes trapped in a partially oxidized form following simultaneous exposure to oxidative and cationic stresses. In addition, cationic stress promotes the interaction of Cap1 with the Crm1 nuclear export factor, thus inhibiting its nuclear accumulation. These mechanisms probably explain the potency of neutrophils, which employ multiple stresses to kill fungal pathogens.

SUBMITTER: Kos I

PROVIDER: S-EPMC4817257 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Mechanisms Underlying the Delayed Activation of the Cap1 Transcription Factor in Candida albicans following Combinatorial Oxidative and Cationic Stress Important for Phagocytic Potency.

Kos Iaroslava I Patterson Miranda J MJ Znaidi Sadri S Kaloriti Despoina D da Silva Dantas Alessandra A Herrero-de-Dios Carmen M CM d'Enfert Christophe C Brown Alistair J P AJ Quinn Janet J

mBio 20160329 2

<h4>Unlabelled</h4>Following phagocytosis, microbes are exposed to an array of antimicrobial weapons that include reactive oxygen species (ROS) and cationic fluxes. This is significant as combinations of oxidative and cationic stresses are much more potent than the corresponding single stresses, triggering the synergistic killing of the fungal pathogenCandida albicansby "stress pathway interference." Previously we demonstrated that combinatorial oxidative plus cationic stress triggers a dramatic ...[more]

PMID: 27025253

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Project description:Immune cells exploit reactive oxygen species (ROS) and cationic fluxes to kill microbial pathogens, such as the fungus Candida albicans. Yet, C. albicans is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity of neutrophils? We show that simultaneous exposure to oxidative and cationic stresses is much more potent than the individual stresses themselves and that this combinatorial stress kills C. albicans synergistically in vitro. We also show that the high fungicidal activity of human neutrophils is dependent on the combinatorial effects of the oxidative burst and cationic fluxes, as their pharmacological attenuation with apocynin or glibenclamide reduced phagocytic potency to a similar extent. The mechanistic basis for the extreme potency of combinatorial cationic plus oxidative stress--a phenomenon we term stress pathway interference--lies with the inhibition of hydrogen peroxide detoxification by the cations. In C. albicans this causes the intracellular accumulation of ROS, the inhibition of Cap1 (a transcriptional activator that normally drives the transcriptional response to oxidative stress), and altered readouts of the stress-activated protein kinase Hog1. This leads to a loss of oxidative and cationic stress transcriptional outputs, a precipitous collapse in stress adaptation, and cell death. This stress pathway interference can be suppressed by ectopic catalase (Cat1) expression, which inhibits the intracellular accumulation of ROS and the synergistic killing of C. albicans cells by combinatorial cationic plus oxidative stress. Stress pathway interference represents a powerful fungicidal mechanism employed by the host that suggests novel approaches to potentiate antifungal therapy. Importance: The immune system combats infection via phagocytic cells that recognize and kill pathogenic microbes. Human neutrophils combat Candida infections by killing this fungus with a potent mix of chemicals that includes reactive oxygen species (ROS) and cations. Yet, Candida albicans is relatively resistant to these stresses in vitro. We show that it is the combination of oxidative plus cationic stresses that kills yeasts so effectively, and we define the molecular mechanisms that underlie this potency. Cations inhibit catalase. This leads to the accumulation of intracellular ROS and inhibits the transcription factor Cap1, which is critical for the oxidative stress response in C. albicans. This triggers a dramatic collapse in fungal stress adaptation and cell death. Blocking either the oxidative burst or cationic fluxes in human neutrophils significantly reduces their ability to kill this fungal pathogen, indicating that combinatorial stress is pivotal to immune surveillance.

Project description:Cap1p, a transcription factor of the basic region-leucine zipper family, controls the oxidative stress response in Candida albicans. It was shown that alteration of the C-terminal cysteine-rich domain (CRD) of Cap1p results in nuclear retention and constitutive transcriptional activation. To further characterize the function of Cap1p in C. albicans, we used genome-wide location profiling (ChIP-on-chip), allowing the identification of Cap1p-transcriptional targets in vivo. Location profiling using a tiled-oligonucleotide DNA microarray identified 89 targets that were bound by Cap1p-HA3 or Cap1p-CSE-HA3 (binding ratio â¥ 2-fold, P â¤ 0.01). Strikingly, Cap1p binding was not only detected at the promoter region of its target genes but also at their 3'-end and within their open-reading frame. Overrepresented functional groups of Cap1p targets (P â¤ 0.02) included notably 11 genes involved in response to oxidative stress (CAP1, GLR1, TRX1, others), 13 genes involved in response to drug (PDR16, MDR1, FLU1, others) and 3 genes involved in regulation of nitrogen utilization (orf19.2693, orf19.3121 and GST3). Bioinformatic analyses suggested that Cap1p binds to the DNA motif 5'- MTKASTMA. Transcriptome analyses showed that increased expression of most of Cap1p targets accompanies Cap1p binding at these targets, indicating that Cap1p is a transcriptional activator. We conclude that, in addition to protecting the cell against oxidative stress, Cap1p appears to have other functions including drug resistance and the regulation of nitrogen utilization. The atypical binding pattern of Cap1p suggests that this transcription factor may associate with the transcriptional or the chromatin remodeling machinery to exert its activity. We performed three gene expression profile comparisons: (1) benomyl-induced gene expression in a strain containing wildtype CAP1 (CJD21/PMK-CAP1 +/- benomyl), (2) benomyl-induced gene expression in a strain where CAP1 is disrupted (CJD21/PMK +/- benomyl), and (3) gene expression in a strain containing a hyperactive allele of CAP1 compared to a strain containing wildtype CAP1 (CJD21/PMK-CAP1-CSE vs. CJD21/PMK-CAP1). There were three biological replicates (each drug-treated and untreated strain was grown/treated in three independent experiments). Since our Affymetrix platform requires each sample is hybridized on a separate chip, there is no dye-swapping. For each benomyl-treatment experiment (comparisons 1 and 2 above), the untreated (diluent-treated) samples are the reference samples. For comparison 3, the CJD21-PMK-CAP1 sample is the reference sample.

Project description:Cap1p, a transcription factor of the basic region-leucine zipper family, regulates oxidative stress response (OSR) in Candida albicans. Alteration of its C-terminal cysteine-rich domain (CRD) results in Cap1p nuclear retention and transcriptional activation. To better understand the function of Cap1p in C. albicans, we used genome-wide location profiling (ChIP-on-chip) to identify its transcriptional targets in vivo. A triple-hemagglutinin epitope was introduced at the C-terminus of wild-type Cap1p (Cap1p-HA3) or hyperactive Cap1p with an altered CRD (Cap1p-CSE-HA3). Location profiling using whole-genome oligonucleotide tiling microarrays identified 89 targets bound by Cap1p-HA3 or Cap1p-CSE-HA3 (binding ratio â?¥ 2-fold, P â?¤ 0.01). Strikingly, Cap1p binding was not only detected at the promoter region of its target genes but also at their 3'-end and within their open-reading frame, suggesting that Cap1p may associate with the transcriptional or the chromatin remodeling machinery to exert its activity. Overrepresented functional groups of the Cap1p targets (P â?¤ 0.02) included 11 genes involved in OSR (CAP1, GLR1, TRX1, SOD1, CAT1, others), 13 genes involved in response to drugs (PDR16, MDR1, FLU1, YCF1, FCR1, others), 4 genes involved in phospholipid transport (PDR16, GIT1, RTA2, orf19.932) and 3 genes involved in the regulation of nitrogen utilization (GST3, orf19.2693, orf19.3121), suggesting that Cap1p has other cellular functions in addition to OSR. Bioinformatic analyses of the bound sequences suggest that Cap1p recognizes the DNA motif 5'- MTKASTMA. Finally, transcriptome analyses showed that increased expression generally accompanies Cap1p binding at its targets, indicating that Cap1p functions as a transcriptional activator. Processing of raw data from 3 independent replicate samples for each Cap1 dataset was conducted using Tilescope (http://tilescope.gersteinlab.org:8080/mosaic/pipeline.html). Quantile normalization was applied to the data. The parameters used were as follows: window size of 400 bp, MaxGap of 60 bp and MinRun of 120 bp. The replicate data were combined and peak-finding (i.e. Cap1p-binding sites) was determined using the following criteria: pseudo-median signal threshold of â?¥ 2-fold and p-value cut-off of â?¤ 0.01.

Dataset Information

Mechanisms Underlying the Delayed Activation of the Cap1 Transcription Factor in Candida albicans following Combinatorial Oxidative and Cationic Stress Important for Phagocytic Potency.

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Importance

Publications

Mechanisms Underlying the Delayed Activation of the Cap1 Transcription Factor in Candida albicans following Combinatorial Oxidative and Cationic Stress Important for Phagocytic Potency.

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