Impact of the lectin chaperone calnexin on the stress response, virulence and proteolytic secretome of the fungal pathogen Aspergillus fumigatus

Calnexin is a membrane-bound lectin chaperone in the endoplasmic reticulum (ER) that is part of a quality control system that promotes the accurate folding of glycoproteins entering the secretory pathway. We have previously shown that ER homeostasis is important for virulence of the human fungal pathogen Aspergillus fumigatus, but the contribution of calnexin has not been explored. Here, we determined the extent to which A. fumigatus relies on calnexin for growth under conditions of environmental stress and for virulence. The calnexin gene, clxA, was deleted from A. fumigatus and complemented by reconstitution with the wild type gene. Loss of clxA altered the proteolytic secretome of the fungus, but had no impact on growth rates in either minimal or complex media at 37°C. However, the ΔclxA mutant was growth impaired at temperatures above 42°C and was hypersensitive to acute ER stress caused by the reducing agent dithiothreitol. In contrast to wild type A. fumigatus, ΔclxA hyphae were unable to grow when transferred to starvation medium. In addition, depleting the medium of cations by chelation prevented ΔclxA from sustaining polarized hyphal growth, resulting in blunted hyphae with irregular morphology. Despite these abnormal stress responses, the ΔclxA mutant remained virulent in two immunologically distinct models of invasive aspergillosis. These findings demonstrate that calnexin functions are needed for growth under conditions of thermal, ER and nutrient stress, but are dispensable for surviving the stresses encountered in the host environment.     

Turgor and net ion flux responses to activation of the osmotic MAP kinase cascade by fludioxonil in the filamentous fungus Neurospora crassa

The internal hydrostatic pressure (turgor) of the filamentous fungus Neurospora crassa is regulated at about 400–500 kiloPascals, primarily by an osmotic MAP kinase cascade which activates ion uptake from the extracellular medium and glycerol synthesis. In the absence of hyperosmotic stress, the phenylpyrrole fungicide fludioxonil activates the osmotic MAP kinase cascade, resulting in cell death. Turgor, the electrical potential and net ion fluxes were measured after treatment with fludioxonil. In wildtype, fludioxonil causes a hyperpolarization of the plasma membrane and net H⁺ efflux from the cell, consistent with activation of the H⁺-ATPase. At the same time, net K⁺ uptake occurs, and turgor increases (about 2-fold above normal levels). None of these changes are observed in the os–2 mutant (which lacks a functional MAP kinase, the last of the three kinases in the osmotic MAP kinase cascade). Tip growth ceases as hyperpolarization, net ion flux changes, and turgor increases begin. The inappropriate turgor increase is the probable cause of eventual lysis and death. The results corroborate a multi-pathway response to hyperosmotic stress that includes activation of plasma membrane transport. The relation to cell expansion (tip growth) is not direct. Increases in turgor due to ion transport might be expected to increase growth rate, but this does not occur. Instead, there must be a complex regulatory interplay between the growth and the turgor driving force, possibly mediated by regulation of cell wall extensibility.     

Enhanced activity of strobilurin and fludioxonil by using berberine and phenolic compounds to target fungal antioxidative stress response

AIMS: Identify natural products that effectively target antioxidative signal transduction/stress response systems [i.e., mitogen-activated protein kinase (MAPK) pathway, mitochondrial superoxide dismutase (Mn-SOD)] of fungi. Enhance activity of strobilurin or fludioxonil with discovered compounds. METHODS AND RESULTS: Enhancement of antifungal activity of strobilurins, inhibitors of complex III of the mitochondrial respiratory chain, was tested using berberine hemisulfate and different phenolic compounds. The Saccharomyces cerevisiae sod2Delta, a deletion mutant lacking Mn-SOD gene, was highly sensitive to berberine and veratraldehyde. Functional complementation analysis verified these compounds target Mn-SOD. Activity of strobilurin (25-50 micromol l(-1)) was elevated on most aspergilli and Penicillium expansum by co-application with berberine or veratraldehyde (2-4 mmol l(-1)). These compounds also prevented Aspergillus fumigatus MAPK mutants (sakADelta and mpkCDelta) from escaping toxicity of fludioxonil (a phenylpyrrole fungicide potentiated by the MAPK pathway), a typical phenotype of fungal MAPK mutants. CONCLUSIONS: Strobilurin activity or prevention of fungal escape from fludioxonil toxicity can be enhanced by co-application of certain alkaloids or phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural products can be used to target cellular stress response systems in fungal pathogens and serve as alternatives/additives to commercial antifungal agents.     

Fungicide activity through activation of a fungal signalling pathway

Fungicides generally inhibit enzymatic reactions involved in fungal cellular biosynthesis. Here we report, for the first time, an example of fungicidal effects through hyperactivation of a fungal signal transduction pathway. The OSC1 gene, encoding a MAP kinase (MAPK) related to yeast Hog1, was isolated from the fungal pathogen Colletotrichum lagenarium that causes cucumber anthracnose. The osc1 knockout mutants were sensitive to high osmotic stress and showed increased resistance to the fungicide fludioxonil, indicating that Osc1 is involved in responses to hyperosmotic stress and sensitivity to fludioxonil. The Osc1 MAPK is phosphorylated under high osmotic conditions, indicating activation of Osc1 by high osmotic stress. Importantly, fludioxonil treatment also activates phosphorylation of Osc1, suggesting that improper activation of Osc1 by fludioxonil has negative effects on fungal growth. In the presence of fludioxonil, the wild-type fungus was not able to infect the host plant because of a failure of appressorium-mediated penetration, whereas osc1 mutants successfully infected plants. Analysis using a OSC1-GFP fusion gene indicated that Osc1 is rapidly translocated to the nucleus in appressorial cells after the addition of fludioxonil, suggesting that fludioxonil impairs the function of infection structures by activation of Osc1. Furthermore, fludioxonil activates Hog1-type MAPKs in the plant pathogenic fungi Cochliobolus heterostrophus and Botrytis cinerea. These results strongly suggest that fludioxonil acts as a fungicide, in part, through activation of the MAPK cascade in fungal pathogens.     

Approaching the secrets of N-glycosylation in Aspergillus fumigatus: characterization of the AfOch1 protein

The mannosyltransferase Och1 is the key enzyme for synthesis of elaborated protein N-glycans in yeast. In filamentous fungi genes implicated in outer chain formation are present, but their function is unclear. In this study we have analyzed the Och1 protein of Aspergillus fumigatus. We provide first evidence that poly-mannosylated N-glycans exist in A. fumigatus and that their synthesis requires AfOch1 activity. This implies that AfOch1 plays a similar role as S. cerevisiae ScOch1 in the initiation of an N-glycan outer chain. A Δafoch1 mutant showed normal growth under standard and various stress conditions including elevated temperature, cell wall and oxidative stress. However, sporulation of this mutant was dramatically reduced in the presence of high calcium concentrations, suggesting that certain proteins engaged in sporulation require N-glycan outer chains to be fully functional. A characteristic feature of AfOch1 and Och1 homologues from other filamentous fungi is a signal peptide that clearly distinguishes them from their yeast counterparts. However, this difference does not appear to have consequences for its localization in the Golgi. Replacing the signal peptide of AfOch1 by a membrane anchor had no impact on its ability to complement the sporulation defect of the Δafoch1 strain. The mutant triggered a normal cytokine response in infected murine macrophages, arguing against a role of outer chains as relevant Aspergillus pathogen associated molecular patterns. Infection experiments provided no evidence for attenuation in virulence; in fact, according to our data the Δafoch1 mutant may even be slightly more virulent than the control strains.     

Targeting antioxidative signal transduction and stress response system: control of pathogenic Aspergillus with phenolics that inhibit mitochondrial function

AIMS: The aim of this study was to show whether antioxidative response systems are potentially useful molecular targets for control of Aspergillus fumigatus and Aspergillus flavus. Selected phenolic agents are used in target-gene-based bioassays to determine their impact on mitochondrial respiration. METHODS AND RESULTS: Vanillyl acetone, vanillic acid, vanillin, cinnamic acid, veratraldehyde, m-coumaric acid (phenolic agents to which Saccharomyces cerevisiae sod2delta mutant showed sensitivity), carboxin (inhibits complex II of the mitochondrial respiratory chain), strobilurins/antimycin A (inhibits complex III of the mitochondrial respiratory chain) and fludioxonil/fenpiclonil [antifungals potentiated by mitogen-activated protein kinase (MAPK)] were examined in A. fumigatus, A. flavus and S. cerevisiae. Individual or combined application of phenolics with inhibitors of mitochondrial respiration showed some of the phenolics effectively inhibited fungal growth. Target-gene bioassays were performed using a sakAdelta (MAPK deletion) strain of A. fumigatus and a complementation analysis using the mitochondrial superoxide dismutase (Mn-SOD) gene (sodA) of A. flavus in the ortholog mutant, sod2delta, of S. cerevisiae. The results demonstrated that mitochondrial antioxidative stress system plays important roles in fungal response to antifungal agents tested. CONCLUSIONS: Antioxidative response systems of fungi can be an efficient molecular target of phenolics for pathogen control. Combined application of phenolics with inhibitors of mitochondrial respiration can effectively suppress the growth of fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural compounds that do not pose any significant medical or environmental risks could serve as useful alternatives or additives to conventional antifungals. Identifying the antioxidative response systems in other pathogens could improve methods for fungal control.     

Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue

Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway.     

Identification of OS-2 MAP kinase-dependent genes induced in response to osmotic stress, antifungal agent fludioxonil, and heat shock in Neurospora crassa

Two-component signal transduction comprising of OS-1 (histidine kinase), OS-4 (MAPKK kinase), OS-5 (MAPK kinase), and OS-2 (MAP kinase) plays an important role in osmotic regulation in Neurospora crassa. To identify the genes regulated downstream of OS-2 MAP kinase, quantitative real-time RT-PCR analysis was conducted in selected genes based on Hog1 MAP kinase regulated genes in yeast. In response to osmotic stress and fludioxonil, expression of six genes that for glycerol synthesis (gcy-1, gcy-3, and dak-1), gluconeogenesis (fbp-1 and pck-1), and catalase (ctt-1) was activated in the wild-type strain, but not in the os-2 mutant. A heat shock treatment also induced their expression in the same way. Consisting with the gene expression, the enzyme activity of glycerol dehydrogenase, but not glycerol-3-phosphate dehydrogenase, was increased in response to osmotic stress and fludioxonil in the wild-type strain. OS-2 was phosphorylated by the OS-1 cascade in response to relatively low osmotic stress and fludioxonil. However, OS-2 phosphorylation by heat shock and a higher osmotic stress was found in the os-1 mutant normally but not in the os-4 and os-5 mutants. These results suggested that non-OS-1 signaling activates OS-2 in an OS-4-dependent manner in such conditions.     

Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway

The adaptive response to hyperosmotic stress in yeast, termed the high osmolarity glycerol (HOG) response, is mediated by two independent upstream pathways that converge on the Pbs2 MAP kinase kinase (MAPKK), leading to the activation of the Hog1 MAP kinase. One branch is dependent on the Sho1 transmembrane protein, whose primary role was found to be the binding and translocation of the Pbs2 MAPKK to the plasma membrane, and specifically to sites of polarized growth. The yeast PAK homolog Ste20 is essential for the Sho1-dependent activation of the Hog1 MAP kinase in response to severe osmotic stress. This function of Ste20 in the HOG pathway requires binding of the small GTPase Cdc42. Overexpression of Cdc42 partially complements the osmosensitivity of ste20Delta mutants, perhaps by activating another PAK-like kinase, while a dominant-negative Cdc42 mutant inhibited signaling through the SHO1 branch of the HOG pathway. Since activated Cdc42 translocates Ste20 to sites of polarized growth, the upstream and downstream elements of the HOG pathway are brought together through the membrane targeting function of Sho1 and Cdc42.     

A unique fungal two-component system regulates stress responses, drug sensitivity, sexual development, and virulence of Cryptococcus neoformans

The stress-activated mitogen-activated protein kinase (MAPK) pathway is widely used by eukaryotic organisms as a central conduit via which cellular responses to the environment effect growth and differentiation. The basidiomycetous human fungal pathogen Cryptococcus neoformans uniquely uses the stress-activated Pbs2-Hog1 MAPK system to govern a plethora of cellular events, including stress responses, drug sensitivity, sexual reproduction, and virulence. Here, we characterized a fungal "two-component" system that controls these fundamental cellular functions via the Pbs2-Hog1 MAPK cascade. A typical response regulator, Ssk1, modulated all Hog1-dependent phenotypes by controlling Hog1 phosphorylation, indicating that Ssk1 is the major upstream signaling component of the Pbs2-Hog1 pathway. A second response regulator, Skn7, governs sensitivity to Na+ ions and the antifungal agent fludioxonil, negatively controls melanin production, and functions independently of Hog1 regulation. To control these response regulators, C. neoformans uses multiple sensor kinases, including two-component-like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. Our findings highlight unique adaptations of this global two-component MAPK signaling cascade in a ubiquitous human fungal pathogen.     

Regulation of caspase 9 through phosphorylation by protein kinase C zeta in response to hyperosmotic stress

Caspase 9 is a critical component of the mitochondrial or intrinsic apoptotic pathway and is activated by Apaf-1 following release of cytochrome c from mitochondria in response to a variety of stimuli. Caspase 9 cleaves and activates effector caspases, mainly caspase 3, leading to the demise of the cell. Survival signaling pathways can impinge on this pathway to restrain apoptosis. Here, we have identified Ser144 of human caspase 9as an inhibitory site that is phosphorylated in a cell-free system and in cells in response to the protein phosphatase inhibitor okadaic acid. Inhibitor sensitivity and interactions with caspase 9 indicate that the predominant kinase that targets Ser144 is the atypical protein kinase C isoform zeta (PKCzeta). Prevention of Ser144 phosphorylation by inhibition of PKCzeta or mutation of caspase 9 promotes caspase 3 activation. Phosphorylation of serine 144 in cells is also induced by hyperosmotic stress, which activates PKCzeta and regulates its interaction with caspase 9, but not by growth factors, phorbol ester, or other cellular stresses. These results indicate that phosphorylation and inhibition of caspase 9 by PKCzeta restrain the intrinsic apoptotic pathway during hyperosmotic stress. This work provides further evidence that caspase 9 acts as a focal point for multiple protein kinase signaling pathways that regulate apoptosis.     

Preferential phosphorylation of focal adhesion kinase tyrosine 861 is critical for mediating an anti-apoptotic response to hyperosmotic stress

The results presented here demonstrate that focal adhesion kinase (FAK) Tyr-861 is the predominant tyrosine phosphorylation site stimulated by hyperosmotic stress in a variety of cell types, including epithelial cell lines (ileum-derived IEC-18, colon-derived Caco2, and stomach-derived NCI-N87), FAK null fibroblasts re-expressing FAK, and Src family kinase triple null fibroblasts (SYF cells) in which c-Src has been restored (YF cells). We show that hyperosmotic stress-stimulated FAK phosphorylation in epithelial cells is inhibited by Src family kinase inhibitors PP2 and SU6656 and that it does not occur in SYF cells. Unexpectedly, hyperosmotic stress-induced phosphorylation of FAK at Tyr-397, Tyr-576, and most dramatically at Tyr-861 was completely insensitive to the F-actin-disrupting agents, latrunculin A and cytochalasin D. Finally, we show that in FAK null cells exposed to hyperosmotic stress or growth factor withdrawal, re-expression of wild type FAK restored cell survival, whereas re-expression of FAK mutated from tyrosine to phenylalanine at position 861 (FAKY861F) did not. Our results indicate that FAK Tyr-861 phosphorylation is required for mammalian cell survival of hyperosmotic stress. Furthermore, the results suggest that FAK is an upstream regulator (rather than downstream effector) of F-actin reorganization in response to hyperosmotic stress. We propose that FAK/c-Src bipartite enzyme is a sensor of cytoplasmic shrinkage, and that the phosphorylation on FAK Tyr-861 by Src and subsequent reorganization of F-actin can initiate an anti-apoptotic signaling pathway that protects cells from hyperosmotic stress.