Blocking Polyphosphate Mobilization Inhibits Pho4 Activation and Virulence in the Pathogen Candida albicans.

The ability of pathogenic fungi to obtain essential nutrients from the host is vital for virulence. In Candida albicans, acquisition of the macronutrient phosphate is regulated by the Pho4 transcription factor and is important for both virulence and resistance to host-encountered stresses. All cells store phosphate in the form of polyphosphate (polyP), a ubiquitous polymer comprising tens to hundreds of phosphate residues. Release of phosphate from polyP is one of the first responses evoked in response to phosphate starvation, and here, we sought to explore the importance of polyP mobilization in the pathobiology of C. albicans. We found that two polyphosphatases, Ppn1 and Ppx1, function redundantly to release phosphate from polyP in C. albicans. Strikingly, we reveal that blocking polyP mobilization prevents the activation of the Pho4 transcription factor: following Pi starvation, Pho4 fails to accumulate in the nucleus and induce Pi acquisition genes in ppn1Δ ppx1Δ cells. Consequently, ppn1Δ ppx1Δ cells display impaired resistance to the same range of stresses that require Pho4 for survival. In addition, cells lacking both polyphosphatases are exquisitely sensitive to DNA replication stress, indicating that polyP mobilization is needed to support the phosphate-demanding process of DNA replication. Blocking polyP mobilization also results in significant morphological defects, as ppn1Δ ppx1Δ cells form large pseudohypha-like cells that are resistant to serum-induced hypha formation. Thus, polyP mobilization impacts key processes important for the pathobiology of C. albicans, and consistent with this, we found that blocking this process attenuates the virulence of this important human fungal pathogen. IMPORTANCE Acquisition of the essential macronutrient phosphate is important for the virulence of Candida albicans, a major human fungal pathogen. All cells store phosphate as polyphosphate (polyP), which is rapidly mobilized when phosphate is limiting. Here, we identified the major phosphatases involved in releasing phosphate from polyP in C. albicans. By blocking this process, we found that polyP mobilization impacts many process that contribute to C. albicans pathogenesis. Notably, we found that blocking polyP mobilization inhibits activation of the Pho4 transcription factor, the master regulator of phosphate acquisition. In addition, cell cycle progression, stress resistance, morphogenetic switching, and virulence are all impaired in cells that cannot mobilize polyP. This study therefore provides new insight into the importance of polyP mobilization in promoting the virulence of C. albicans. As phosphate homeostasis strategies differ between fungal pathogen and host, this offers promise for the future development of antifungals.

Redox Regulation, Rather than Stress-Induced Phosphorylation, of a Hog1 Mitogen-Activated Protein Kinase Modulates Its Nitrosative-Stress-Specific Outputs.

In all eukaryotic kingdoms, mitogen-activated protein kinases (MAPKs) play critical roles in cellular responses to environmental cues. These MAPKs are activated by phosphorylation at highly conserved threonine and tyrosine residues in response to specific inputs, leading to their accumulation in the nucleus and the activation of their downstream targets. A specific MAP kinase can regulate different downstream targets depending on the nature of the input signal, thereby raising a key question: what defines the stress-specific outputs of MAP kinases? We find that the Hog1 MAPK contributes to nitrosative-stress resistance in Candida albicans even though it displays minimal stress-induced phosphorylation under these conditions. We show that Hog1 becomes oxidized in response to nitrosative stress, accumulates in the nucleus, and regulates the nitrosative stress-induced transcriptome. Mutation of specific cysteine residues revealed that C156 and C161 function together to promote stress resistance, Hog1-mediated nitrosative-stress-induced gene expression, resistance to phagocytic killing, and C. albicans virulence. We propose that the oxidation of Hog1, rather than its phosphorylation, contributes to the nitrosative-stress-specific responses of this MAP kinase.IMPORTANCE Mitogen-activated protein kinases play key roles in the responses of eukaryotic cells to extracellular signals and are critical for environmental-stress resistance. The widely accepted paradigm is that MAP kinases are activated by phosphorylation, which then triggers their nuclear accumulation and the activation of target proteins and genes that promote cellular adaptation. Our data suggest that alternative forms of posttranslational modification can modulate MAP kinase functionality in Candida albicans We demonstrate that Hog1 is not significantly phosphorylated in response to nitrosative stress, yet it displays nuclear accumulation and contributes to the global transcriptional response to this stress, as well as promoting nitrosative-stress resistance. Instead, nitrosative stress triggers changes in the redox status of Hog1. We also show that specific Hog1 cysteine residues influence its activation of stress genes. Therefore, alternative posttranslational modifications appear to regulate the stress-specific outputs of MAP kinases.

Phosphate Acquisition and Virulence in Human Fungal Pathogens.

The ability of pathogenic fungi to acquire essential macro and micronutrients during infection is a well-established virulence trait. Recent studies in the major human fungal pathogens Candida albicans and Cryptococcus neoformans have revealed that acquisition of the essential macronutrient, phosphate, is essential for virulence. The phosphate sensing and acquisition pathway in fungi, known as the PHO pathway, has been extensively characterized in the model yeast Saccharomyces cerevisiae. In this review, we highlight recent advances in phosphate sensing and signaling mechanisms, and use the S. cerevisiae PHO pathway as a platform from which to compare the phosphate acquisition and storage strategies employed by several human pathogenic fungi. We also explore the multi-layered roles of phosphate acquisition in promoting fungal stress resistance to pH, cationic, and oxidative stresses, and describe emerging roles for the phosphate storage molecule polyphosphate (polyP). Finally, we summarize the recent studies supporting the necessity of phosphate acquisition in mediating the virulence of human fungal pathogens, highlighting the concept that this requirement is intimately linked to promoting resistance to host-imposed stresses.

Elevated catalase expression in a fungal pathogen is a double-edged sword of iron.

Most fungal pathogens of humans display robust protective oxidative stress responses that contribute to their pathogenicity. The induction of enzymes that detoxify reactive oxygen species (ROS) is an essential component of these responses. We showed previously that ectopic expression of the heme-containing catalase enzyme in Candida albicans enhances resistance to oxidative stress, combinatorial oxidative plus cationic stress, and phagocytic killing. Clearly ectopic catalase expression confers fitness advantages in the presence of stress, and therefore in this study we tested whether it enhances fitness in the absence of stress. We addressed this using a set of congenic barcoded C. albicans strains that include doxycycline-conditional tetON-CAT1 expressors. We show that high basal catalase levels, rather than CAT1 induction following stress imposition, reduce ROS accumulation and cell death, thereby promoting resistance to acute peroxide or combinatorial stress. This conclusion is reinforced by our analyses of phenotypically diverse clinical isolates and the impact of stochastic variation in catalase expression upon stress resistance in genetically homogeneous C. albicans populations. Accordingly, cat1Δ cells are more sensitive to neutrophil killing. However, we find that catalase inactivation does not attenuate C. albicans virulence in mouse or invertebrate models of systemic candidiasis. Furthermore, our direct comparisons of fitness in vitro using isogenic barcoded CAT1, cat1Δ and tetON-CAT1 strains show that, while ectopic catalase expression confers a fitness advantage during peroxide stress, it confers a fitness defect in the absence of stress. This fitness defect is suppressed by iron supplementation. Also high basal catalase levels induce key iron assimilatory functions (CFL5, FET3, FRP1, FTR1). We conclude that while high basal catalase levels enhance peroxide stress resistance, they place pressure on iron homeostasis through an elevated cellular demand for iron, thereby reducing the fitness of C. albicans in iron-limiting tissues within the host.

Pho4 mediates phosphate acquisition in Candida albicans and is vital for stress resistance and metal homeostasis.

During interactions with its mammalian host, the pathogenic yeast Candida albicans is exposed to a range of stresses such as superoxide radicals and cationic fluxes. Unexpectedly, a nonbiased screen of transcription factor deletion mutants revealed that the phosphate-responsive transcription factor Pho4 is vital for the resistance of C. albicans to these diverse stresses. RNA-Seq analysis indicated that Pho4 does not induce stress-protective genes directly. Instead, we show that loss of Pho4 affects metal cation toxicity, accumulation, and bioavailability. We demonstrate that pho4Δ cells are sensitive to metal and nonmetal cations and that Pho4-mediated polyphosphate synthesis mediates manganese resistance. Significantly, we show that Pho4 is important for mediating copper bioavailability to support the activity of the copper/zinc superoxide dismutase Sod1 and that loss of Sod1 activity contributes to the superoxide sensitivity of pho4Δ cells. Consistent with the key role of fungal stress responses in countering host phagocytic defenses, we also report that C. albicans pho4Δ cells are acutely sensitive to macrophage-mediated killing and display attenuated virulence in animal infection models. The novel connections between phosphate metabolism, metal homeostasis, and superoxide stress resistance presented in this study highlight the importance of metabolic adaptation in promoting C. albicans survival in the host.

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.

Non-lytic expulsion/exocytosis of Candida albicans from macrophages.

Candida albicans is an opportunistic pathogen and is recognised and phagocytosed by macrophages. Using live-cell imaging, non-lytic expulsion/exocytosis of C. albicans from macrophages is demonstrated for the first time. Following complete expulsion, both the phagocyte and pathogen remain intact and viable. Partial engulfment of hyphal C. albicans without macrophage lysis is also demonstrated. These observations underpin the complexity of interactions between C. albicans and innate immune cells.

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 H(2)O(2)), salt (300 mM NaCl) or cadmium stress (0.5 mM Cd(2+)) 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.

Nitrosative and oxidative stress responses in fungal pathogenicity.

Fungal pathogenicity has arisen in polyphyletic manner during evolution, yielding fungal pathogens with diverse infection strategies and with differing degrees of evolutionary adaptation to their human host. Not surprisingly, these fungal pathogens display differing degrees of resistance to the reactive oxygen and nitrogen species used by human cells to counteract infection. Furthermore, whilst evolutionarily conserved regulators, such as Hog1, are central to such stress responses in many fungal pathogens, species-specific differences in their roles and regulation abound. In contrast, there is a high degree of commonality in the cellular responses to reactive oxygen and nitrogen species evoked in evolutionarily divergent fungal pathogens.

A 2-Cys peroxiredoxin regulates peroxide-induced oxidation and activation of a stress-activated MAP kinase.

Oxidative stress-induced cell damage is an important component of many diseases and ageing. In eukaryotes, activation of JNK/p38 stress-activated protein kinase (SAPK) signaling pathways is critical for the cellular response to stress. 2-Cys peroxiredoxins (2-Cys Prx) are highly conserved, extremely abundant antioxidant enzymes that catalyze the breakdown of peroxides to protect cells from oxidative stress. Here we reveal that Tpx1, the single 2-Cys Prx in Schizosaccharomyces pombe, is required for the peroxide-induced activation of the p38/JNK homolog, Sty1. Tpx1 activates Sty1, downstream of previously identified redox sensors, by a mechanism that involves formation of a peroxide-induced disulphide complex between Tpx1 and Sty1. We have identified conserved cysteines in Tpx1 and Sty1 that are essential for normal peroxide-induced Tpx1-Sty1 disulphide formation and Tpx1-dependent regulation of peroxide-induced Sty1 activation. Thus we provide new insight into the response of SAPKs to diverse stimuli by revealing a mechanism for SAPK activation specifically by oxidative stress.