Deborah A. Smith

Thioredoxin Regulates Multiple Hydrogen Peroxide-Induced Signaling Pathways in Candida albicans

ABSTRACT The ability of the major systemic fungal pathogen of humans, Candida albicans, to sense and respond to reactive oxygen species (ROS), such as H2O2 generated by the host immune system, is required for survival in the host. However, the intracellular signaling mechanisms underlying such responses are poorly understood. Here, we show that thioredoxin (Trx1), in addition to its antioxidant activity, plays a central role in coordinating the response of C. albicans to ROS by regulating multiple pathways. In particular, Trx1 function is important for H2O2-induced phosphorylation of the Hog1 stress-activated protein kinase and to reverse H2O2-induced oxidation and activation of the AP-1 like transcription factor Cap1. Furthermore, Trx1 regulates H2O2-induced hyperpolarized bud growth in a mechanism that involves activation of the Rad53 checkpoint kinase. Consistent with its key roles in responses to ROS, cells lacking Trx1 displayed significantly attenuated virulence in a murine model of C. albicans systemic infection. Collectively, our data indicate that Trx1 has a multifaceted role in H2O2 signaling and promotes C. albicans survival in the host.

Stress signalling to fungal stress‐activated protein kinase pathways

The ability of microorganisms to survive and thrive within hostile environments depends on rapid and robust stress responses. Stress-activated protein kinase (SAPK) pathways are important stress-signalling modules found in all eukaryotes, including eukaryotic microorganisms such as fungi. These pathways consist of a SAPK that is activated by phosphorylation through a kinase cascade, and once activated, the SAPK phosphorylates a range of cytoplasmic and nuclear target substrates, which determine the appropriate response. However, despite their conservation in fungi, mechanisms that have evolved to relay stress signals to the SAPK module in different fungi have diverged significantly. Here, we present an overview of the diverse strategies used in the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the pathogenic fungus Candida albicans, to sense and transduce stress signals to their respective SAPKs.

Ybp1 and Gpx3 Signaling in Candida albicans Govern Hydrogen Peroxide-Induced Oxidation of the Cap1 Transcription Factor and Macrophage Escape

AIMS As Candida albicans is the major fungal pathogen of humans, there is an urgent need to understand how this pathogen evades toxic reactive oxygen species (ROS) generated by the host immune system. A key regulator of antioxidant gene expression, and thus ROS resistance, in C. albicans is the AP-1-like transcription factor Cap1. Despite this, little is known regarding the intracellular signaling mechanisms that underlie the oxidation and activation of Cap1. Therefore, the aims of this study were; (i) to identify the regulatory proteins that govern Cap1 oxidation, and (ii) to investigate the importance of Cap1 oxidation in C. albicans pathogenesis. RESULTS In response to hydrogen peroxide (H2O2), but not glutathione-depleting/modifying oxidants, Cap1 oxidation, nuclear accumulation, phosphorylation, and Cap1-dependent gene expression, is mediated by a glutathione peroxidase-like enzyme, which we name Gpx3, and an orthologue of the Saccharomyces cerevisiae Yap1 binding protein, Ybp1. In addition, Ybp1 also functions to stabilise Cap1 and this novel function is conserved in S. cerevisiae. C. albicans cells lacking Cap1, Ybp1, or Gpx3, are unable to filament and thus, escape from murine macrophages after phagocytosis, and also display defective virulence in the Galleria mellonella infection model. INNOVATION Ybp1 is required to promote the stability of fungal AP-1-like transcription factors, and Ybp1 and Gpx3 mediated Cap1-dependent oxidative stress responses are essential for the effective killing of macrophages by C. albicans. CONCLUSION Activation of Cap1, specifically by H2O2, is a prerequisite for the subsequent filamentation and escape of this fungal pathogen from the macrophage.

A proteomic analysis of the salt, cadmium and peroxide stress responses in Candida albicans and the role of the Hog1 stress-activated MAPK in regulating the stress-induced proteome.

Stress responses are important for the virulence of the major fungal pathogen of humans, Candida albicans. In this study we employed a 2‐DE approach to examine the impact of exposure to peroxide (5 mM H2O2), salt (300 mM NaCl) or cadmium stress (0.5 mM Cd2+) upon the C. albicans proteome. Highly reproducible changes in the C. albicans proteome were observed in response to each stress condition. Significantly more proteins were up‐regulated in response to cadmium (77) than to the salt (35) or peroxide stresses (35). These proteomic changes displayed minimal overlap with those observed in the transcriptome under equivalent conditions and, importantly, revealed functional categories that respond to stress at the protein level but not the transcript level. Six proteins were up‐regulated by all three conditions: Adh1, Atp2, Cip1, Eft2, Ssa1 and Ssb1, which is consistent with the concept that a core stress response exists in C. albicans. This is the first time that a fungal core stress response has been defined at the proteomic level. We have also shown that the Hog1 stress‐activated mitogen‐activated protein kinase, which is activated in response to the stresses examined in this study, makes a major contribution to the C. albicans stress proteome.

Mechanisms Underlying the Exquisite Sensitivity of Candida albicans to Combinatorial Cationic and Oxidative Stress That Enhances the Potent Fungicidal Activity of Phagocytes

ABSTRACT 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. 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.

MAPKKK-independent Regulation of the Hog1 Stress-activated Protein Kinase in Candida albicans

The Hog1 stress-activated protein kinase regulates both stress responses and morphogenesis in Candida albicans and is essential for the virulence of this major human pathogen. Stress-induced Hog1 phosphorylation is regulated by the upstream MAPKK, Pbs2, which in turn is regulated by the MAPKKK, Ssk2. Here, we have investigated the role of phosphorylation of Hog1 and Pbs2 in Hog1-mediated processes in C. albicans. Mutation of the consensus regulatory phosphorylation sites of Hog1 (Thr-174/Tyr-176) and Pbs2 (Ser-355/Thr-359), to nonphosphorylatable residues, resulted in strains that phenocopied hog1Δ and pbs2Δ cells. Consistent with this, stress-induced phosphorylation of Hog1 was abolished in cells expressing nonphosphorylatable Pbs2 (Pbs2AA). However, mutation of the consensus sites of Pbs2 to phosphomimetic residues (Pbs2DD) failed to constitutively activate Hog1. Furthermore, Ssk2-independent stress-induced Hog1 activation was observed in Pbs2DD cells. Collectively, these data reveal a previously uncharacterized MAPKKK-independent mechanism of Hog1 activation in response to stress. Although Pbs2DD cells did not exhibit high basal levels of Hog1 phosphorylation, overexpression of an N-terminal truncated form of Ssk2 did result in constitutive Hog1 activation, which was further increased upon stress. Significantly, both Pbs2AA and Pbs2DD cells displayed impaired stress resistance and attenuated virulence in a mouse model of disease, whereas only Pbs2AA cells exhibited the morphological defects associated with loss of Hog1 function. This indicates that Hog1 mediates C. albicans virulence by conferring stress resistance rather than regulating morphogenesis.

Two-Component Mediated Peroxide Sensing and Signal Transduction in Fission Yeast

Two-component related proteins play a major role in regulating the oxidative stress response in the fission yeast, Schizosaccharomyces pombe. For example, the peroxide-sensing Mak2 and Mak3 histidine kinases regulate H(2)O(2)-induced activation of the Sty1 stress-activated protein kinase pathway, and the Skn7-related response regulator transcription factor, Prr1, is essential for activation of the core oxidative stress response genes. Here, we investigate the mechanism by which the S. pombe two-component system senses H(2)O(2), and the potential role of two-component signaling in the regulation of Prr1. Significantly, we demonstrate that PAS and GAF domains present in the Mak2 histidine kinase are essential for redox-sensing and activation of Sty1. In addition, we find that Prr1 is required for the transcriptional response to a wide range of H(2)O(2) concentrations and, furthermore, that two-component regulation of Prr1 is specifically required for the response of cells to high levels of H(2)O(2). Significantly, this provides the first demonstration that the conserved two-component phosphorylation site on Skn7-related proteins influences resistance to oxidative stress and oxidative stress-induced gene expression. Collectively, these data provide new insights into the two-component mediated sensing and signaling mechanisms underlying the response of S. pombe to oxidative stress.

Blocking two-component signalling enhances Candida albicans virulence and reveals adaptive mechanisms that counteract sustained SAPK activation

The Ypd1 phosphorelay protein is a central constituent of fungal two-component signal transduction pathways. Inhibition of Ypd1 in Saccharomyces cerevisiae and Cryptococcus neoformans is lethal due to the sustained activation of the ‘p38-related’ Hog1 stress-activated protein kinase (SAPK). As two-component signalling proteins are not found in animals, Ypd1 is considered to be a prime antifungal target. However, a major fungal pathogen of humans, Candida albicans, can survive the concomitant sustained activation of Hog1 that occurs in cells lacking YPD1. Here we show that the sustained activation of Hog1 upon Ypd1 loss is mediated through the Ssk1 response regulator. Moreover, we present evidence that C. albicans survives SAPK activation in the short-term, following Ypd1 loss, by triggering the induction of protein tyrosine phosphatase-encoding genes which prevent the accumulation of lethal levels of phosphorylated Hog1. In addition, our studies reveal an unpredicted, reversible, mechanism that acts to substantially reduce the levels of phosphorylated Hog1 in ypd1Δ cells following long-term sustained SAPK activation. Indeed, over time, ypd1Δ cells become phenotypically indistinguishable from wild-type cells. Importantly, we also find that drug-induced down-regulation of YPD1 expression actually enhances the virulence of C. albicans in two distinct animal infection models. Investigating the underlying causes of this increased virulence, revealed that drug-mediated repression of YPD1 expression promotes hyphal growth both within murine kidneys, and following phagocytosis, thus increasing the efficacy by which C. albicans kills macrophages. Taken together, these findings challenge the targeting of Ypd1 proteins as a general antifungal strategy and reveal novel cellular adaptation mechanisms to sustained SAPK activation.

Identification of a Novel Response Regulator, Crr1, That Is Required for Hydrogen Peroxide Resistance in Candida albicans

Candida albicans colonises numerous niches within humans and thus its success as a pathogen is dependent on its ability to adapt to diverse growth environments within the host. Two component signal transduction is a common mechanism by which bacteria respond to environmental stimuli and, although less common, two component-related pathways have also been characterised in fungi. Here we report the identification and characterisation of a novel two component response regulator protein in C. albicans which we have named CRR1 (Candida Response Regulator 1). Crr1 contains a receiver domain characteristic of response regulator proteins, including the conserved aspartate that receives phosphate from an upstream histidine kinase. Significantly, orthologues of CRR1 are present only in fungi belonging to the Candida CTG clade. Deletion of the C. albicans CRR1 gene, or mutation of the predicted phospho-aspartate, causes increased sensitivity of cells to the oxidising agent hydrogen peroxide. Crr1 is present in both the cytoplasm and nucleus, and this localisation is unaffected by oxidative stress or mutation of the predicted phospho-aspartate. Furthermore, unlike the Ssk1 response regulator, Crr1 is not required for the hydrogen peroxide-induced activation of the Hog1 stress-activated protein kinase pathway, or for the virulence of C. albicans in a mouse model of systemic disease. Taken together, our data suggest that Crr1, a novel response regulator restricted to the Candida CTG clade, regulates the response of C. albicans cells to hydrogen peroxide in a Hog1-independent manner that requires the function of the conserved phospho-aspartate.

Identification of domains responsible for signal recognition and transduction within the QUTR transcription repressor protein.

QUTR (qutR-encoded transcription-repressing protein) is a multi-domain repressor protein active in the signal-transduction pathway that regulates transcription of the quinic acid utilization (qut) gene cluster in Aspergillus nidulans. In the presence of quinate, production of mRNA from the eight genes of the qut pathway is stimulated by the activator protein QUTA (qutA-encoded transcription-activating protein). Mutations in the qutR gene alter QUTR function such that the transcription of the qut gene cluster is permanently on (constitutive phenotype) or is insensitive to the presence of quinate (super-repressed phenotype). These mutant phenotypes imply that the QUTR protein plays a key role in signal recognition and transduction, and we have used deletion analysis to determine which regions of the QUTR protein are involved in these functions. We show that the QUTR protein recognizes and binds to the QUTA protein in vitro and that the N-terminal 88 amino acids of QUTR are sufficient to inactivate QUTA function in vivo. Deletion analysis and domain-swap experiments imply that the two C-terminal domains of QUTR are mainly involved in signal recognition.