Elucidating the fungal stress response by proteomics

JPROT-01456; No of Pages 13 JOURNAL OF P ROTEOM IC S XX ( 2013) X XX–X XX

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Review

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Elucidating the fungal stress response by proteomics☆

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Kristin Krolla,b , Vera Pähtza,b,c , Olaf Kniemeyera,b,c,⁎ a

Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology — Hans-Knöll-Institute (HKI), Beutenbergstrasse 11a, 07745 Jena, Germany b Friedrich Schiller University, Institute of Microbiology, Philosophenweg 12, 07743 Jena, Germany c Integrated Research and Treatment Center, Center for Sepsis Control and Care Jena, University Hospital (CSCC), 07747 Jena, Germany

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Keywords:

Fungal species need to cope with stress, both in the natural environment and during

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Fungi

interaction of human- or plant pathogenic fungi with their host. Many regulatory circuits

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Proteome

governing the fungal stress response have already been discovered. However, there are still

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Stress response

large gaps in the knowledge concerning the changes of the proteome during adaptation to

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Oxidative stress

environmental stress conditions. With the application of proteomic methods, particularly

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Hypoxia

2D-gel and gel-free, LC/MS-based methods, first insights into the composition and dynamic

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changes of the fungal stress proteome could be obtained. Here, we review the recent proteome

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data generated for filamentous fungi and yeasts. This article is part of a Special Issue entitled:

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Trends in Microbial Proteomics.

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Introduction . . . . . . . . . . . . . . . . . . . Nitrosative and oxidative stress . . . . . . . . 2.1. Oxygen limitation (hypoxia) . . . . . . . 3. Nitrogen limitation in fungal plant pathogens . 4. Amino acid starvation . . . . . . . . . . . . . . 5. Glucose limitation . . . . . . . . . . . . . . . . 6. Future perspectives of fungal stress proteomics Acknowledgement . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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☆ This article is part of a Special Issue entitled: Trends in Microbial Proteomics. ⁎ Corresponding author at: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology — Hans-Knöll-Institute (HKI), Beutenbergstrasse 11a, 07745 Jena, Germany. Tel.: +49 3641 532 1070; fax: +49 3641 532 0803. E-mail address: [email protected] (O. Kniemeyer). 1874-3919/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jprot.2013.06.001

Please cite this article as: Kroll K, et al, Elucidating the fungal stress response by proteomics, J Prot (2013), http://dx.doi.org/10.1016/ j.jprot.2013.06.001

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Fungi comprise a diverse lineage of eukaryotic organisms and some estimates suggest that around 1.5 million fungal species exist. Despite their genetic and morphological diversity they have several characteristics in common: Most fungi grow as tubular, elongated structures (filaments) and extend at their tip, whereas a smaller group grows as single cells (yeast cells). They have a heterotrophic growth style and live either on dead organic matter or are biotrophs and live in close association with plants, animals or prokaryotes [1,2]. Due to their sessile life, fungi have developed sophisticated responses to environmental stresses in the course of evolution. As a result, fungi have occupied many ecological niches including harsh environments such as glaciers or salterns [3]. Genetic studies and cell biology research on fungal model organisms, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa and Aspergillus nidulans, have led to the discovery of many global stress regulators and mechanisms conferring resistance to adverse environments. Based on these findings, the amount of knowledge related to the stress response in plant- and human pathogenic fungi has significantly increased over the last two decades. An environmental challenge, to which all aerobically growing organisms including fungi must adapt, is the exposure to reactive oxygen species (ROS) caused by partially reduced forms of molecular oxygen. Intensive research activity in the field of yeast genetics and molecular biology resulted in the elucidation of essential global regulators of the oxidative stress response. A good example being, the bZIP family transcription factor Yap1, which represents hereby the major regulator present in all fungal species [4]. In addition, two-component signal transduction systems contribute to sensing of oxidative stress and activating stress response factor, e.g. the transcription factor Skn7 [5]. Compared to the regulators of the oxidative stress response, very little is known regarding the transcriptional circuits associated with the detoxification of reactive nitrogen species (RNS). Phagocytic cells of the human innate immune system generate RNS to defeat pathogenic microorganisms. For this reason the NO response was studied in the human pathogenic yeast Candida albicans and the associated transcription factor, Cta4p, which was found to initiate the NO response [6]. The analogous response in baker's yeast is regulated by the transcription factor, Fzf1p [7]. Later, a negative regulator of NO stress, Cwt1p, was described for C. albicans [8]. Oxygen depletion is another stress factor fungi have to cope with in the environment and is at the battlefront of host– pathogen interaction. Data from the yeasts, S. cerevisiae and S. pombe, have shown that oxygen depletion is indirectly sensed by the drop in metabolite levels of haem and ergosterol, respectively, where the biosynthesis depends on the availability of molecular oxygen. Similar mechanisms of sensing hypoxia was also shown for the human-pathogenic fungi, Aspergillus fumigatus and Cryptococcus neoformans [9,10]. Low availability of nitrogen and carbon sources denotes another situation common to physiological scenarios in fungal cells. In fungi, transcription factors of the GATA family mediate a general control mechanism termed nitrogen metabolite

repression. It means that during growth on easily assimilated nitrogen sources, e.g. ammonium, the expression of genes involved in the utilisation of alternative N-sources is repressed. Also the TOR signalling pathway was reported to control gene regulation upon nitrogen limitation in yeast [11]. Although there are great similarities in the general principle of regulating the N-metabolism, the involved transcriptional regulators differ significantly among fungal species [12–14]. The regulatory response of yeast and filamentous fungi to amino acid depletion is known as general amino acid control or cross-pathway control, respectively. Here, the transcriptional activator GCN4p (CpcA/Cpc1) induces the expression of most amino acid biosynthetic enzymes in response to starvation of amino acids [15,16]. It is worthy to note that this stress response contributes to the survival of human-pathogenic fungi in the host [16,17]. Similarly, carbon source limitation is probably an important state of fungal cells in the infected host. The glucose repression pathway, which controls the preferential utilisation of glucose, can be found in most fungi. Other complex regulatory mechanisms to sense and respond to fluctuating levels of glucose differ from fungus to fungus [18,19]. Altogether, a vast amount of knowledge on the molecular mechanisms of adaptation to harsh stress conditions in fungi has been accumulated. Besides, numerous studies in a variety of fungi have been performed on the changes of the transcriptome in response to environmental stresses or during host–pathogen interaction [20]. In contrast, the knowledge about the impact of stress on the fungal proteome is limited. Most conducted studies on the fungal stress proteome were based on classical 2D-gel electrophoresis [21–25]. However, modern proteomic technologies hold great potential for deciphering regulatory circuits and the role of posttranslational regulatory mechanisms in fungal stress response. The aim of this review is to provide an overview about proteome-related studies of the fungal stress response with emphasis on oxidative and nitrosative stress, hypoxia and nutrient depletion. Finally, the trends and perspectives of proteomic technologies and their future impact on fungal research will be discussed.

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Introduction

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Nitrosative and oxidative stress

In low amounts, reactive oxygen species (ROS) are generated continuously as side products of aerobic respiration in the mitochondria and, although potentially cytotoxic, function as important signal molecules in cellular processes [26,27]. In addition, following pathogen infection, phagocytic cells, which are crucial in fighting infections in vertebrates, release ROS by activating the NADPH oxidase. This enzyme complex catalyses the NADPH-dependent conversion of molecular oxygen (O2) to reactive superoxide (O−2) [28]. Interestingly, a lack of functional NADPH oxidase activity leads to an enhanced susceptibility towards fungal and bacterial infections in humans [29–31]. However, there is still some controversy about whether reactive oxygen species are one of the main factors responsible for killing microbes [32]. Nevertheless, it is evident that they play, at least indirectly, an important role in the battle against microbial infections [30,33].

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different time points between 5 and 60 min after stress treatment was performed to visualise the distinct protein synthesis pattern. 52 different proteins seemed to be associated with the characteristic proteome signature for hydrogen peroxide-induced stress. Proteins like catalase Cta1p, glutathione reductase Glr1p, or thioredoxin reductase Trr1p, which are typical members of the antioxidative system of C. albicans, ranked among the 30 most upregulated proteins. Additionally, other proteins were highly expressed upon H2O2 exposure, e.g. the Ipf2431 protein (Tsa1p), which has a strong sequence similarity to the thioredoxin-dependent peroxidases of the Tsa/ AhPC protein family [41] and the cadmium induced protein Cip1p [42,43]. The Ipf2431 protein was also shown to be important under several stress responses in other fungi [35,36,44–46]. A contribution of Cip1p in the oxidative stress response was also reported in other studies [41,43,47]. Furthermore, C. albicans increased the expression of a large set of oxidoreductases and chaperones as parts of the hydrogen peroxide response machinery, probably to maintain the redox homeostasis and to stabilize protein structure. To investigate an additional oxidative stress-inducing agent, Kusch et al. [40] used the thiol oxidizing agent diamide, which caused a characteristic proteome signature of 45 down- and upregulated proteins [40]. Interestingly, the identified proteins with an increased expression upon diamide treatment were almost identical to the proteins upregulated under hydrogen peroxide exposure. In order to find new candidates of the oxidative stress regulator, Cap1p regulon, Kusch et al. [40] performed additional 2-DE analyses of strains deleted in the cap1p gene. This approach revealed 12 proteins, which are controlled by Cap1p [40]. In 2009, Yin et al. investigated the impact of hydrogen peroxide treatment (5 mM H2O2), osmotic (300 mM NaCl) or cadmium stress (0.5 mM Cd2+) on the C. albicans proteome after 60 min of exposure [43]. Only a few of the 35 upregulated proteins, namely the thiol-specific peroxiredoxin Ahp1, the core stress protein Cip1, and a putative NADH-dependent flavin oxidoreductase Oye32, represented enzymes involved in redox regulation. The biggest group, among the upregulated proteins, comprised of proteins that were predicted as putative chaperones and proteins with roles in proteolysis. When comparing the two different studies from Kusch et al. [40] and Yin et al. [43], the abundance of only six proteins increased in both studies, including three proteins involved in cell redox homeostasis (Ahp1, Cip1, Oye32) and three proteins involved in protein folding and degradation (Hsp78, orf19.251, Ssa1). These variations in the proteomic response to oxidative stress may be explained by the different conditions used in the two studies: the hydrogen peroxide concentrations (1 versus 5 mM) as well as the incubation times differed (time scale experiment with different time points 5, 15, 30 and 60 min versus an end point experiment with 60 min). A proteome study from 2007 revealed several similarities between the oxidative stress response of the human-pathogenic mould, A. fumigatus and C. albicans [48]. After an exposure of 45 min with 2 mM hydrogen peroxide, 44 protein spots displayed a change in their abundance. In particular enzymes with antioxidant functions, heat shock proteins, and proteins involved in the biosynthesis of trehalose were increased under this condition. AspF3 was the most highly induced protein. It putatively exhibits a thioredoxin peroxidase activity and shows

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Besides ROS, immune cells, in particular phagocytic cells, produce reactive nitrogen species (RNS) that are formed by another enzyme, the nitric oxide synthase (RNS release). Because of their reactivity, ROS and RNS are known to destroy many cellular compounds. Additionally, ROS such as superoxides can react with NO radicals (NOU) to form peroxynitrite (ONOO−), which is a strong oxidant and capable of carbonylating proteins and oxidizing DNA [34]. The capacity to survive the attack of phagocytic cells, like macrophages and neutrophils, is one of the main achievements of fungal pathogens. A proteomic approach to investigate the nitrosative stress response has only been applied in C. neoformans thus far [35]. This basidiomycete yeast-like fungus with worldwide distribution in the soil can cause meningoencephalitis and cryptococcosis in immunocompromised patients. Missall et al. [35] investigated the impact of reactive nitrogen species (RNS) on the transcriptome and proteome by treating C. neoformans cells with sodium nitrite at pH 4.0, which decomposes spontaneously to form nitric oxide (NO) at acidic pH. The exposure of C. neoformans to NO resulted in changes of the level of many proteins involved in the general stress response, e.g. proteins with antioxidative activity. Among others, the thioredoxin-dependent peroxide reductase, Tsa1, was induced upon treatment with NO. In a previous study, Tsa1, which is also a known antioxidant enzyme, was shown to exhibit a protective function under nitrosative and oxidative stress conditions and at high temperatures. The important contribution of Tsa1 to the stress response of C. neoformans was further corroborated by proof that it is important for virulence in murine infection models of cryptococcosis [36]. Besides Tsa1, the proteomic analysis also revealed that the glutathione-disulphide reductase, Glr1, a member of the glutathione antioxidant system, was induced upon NO treatment. Glutathione reductase regenerates oxidized glutathione to its reduced form thereby maintaining cellular redox balance. The lack of the glutathione reductase activity in a knock-down mutant led to an enhanced sensitivity to nitric oxide stress, while the sensitivity to peroxides remained similar to the wild-type. Interestingly, Glr1 seems to be crucial for the pathogenicity of C. neoformans, since a Δglr1 deletion mutant was shown to be avirulent in a mouse infection model [35]. In addition, the oxidoreductases, Oye3 and Oxr3, which potentially play a role in the oxidative stress and high temperature response, were also increased significantly along with changes in the level of metabolic enzymes. Some of these metabolic enzymes were also posttranslationally modified upon NO stress, which suggests that nitrosative stress conditions induce protein modifications such as nitrosylation or phosphorylation. The contribution of ROS detoxification in the virulence of pathogenic fungi was also investigated in the polymorphic yeast, C. albicans. It is usually a harmless commensal organism found in the gastrointestinal tract of most people. However, C. albicans can also cause life-threatening systemic infections in immunocompromised patients [37,38]. Several studies showed that an elaborate stress response contributes to the survival and virulence of C. albicans in the host [39]. The oxidative stress response of C. albicans was analysed via a large scale 2-DE proteomic approach. Yeast cells were treated with 1 mM hydrogen peroxide and 3 mM diamide, respectively [40]. A pulse labelling of C. albicans cells with radioactive L-[35S]-methionine at

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Most eukaryotes require molecular oxygen for growth. In general, oxygen is the terminal electron acceptor of the respiratory chain and represents an important substrate for the biosynthesis of cellular compounds such as haem, unsaturated fatty acids or sterols. In yeast and fungi the sterol ergosterol is an essential component of the cell membrane maintaining its structure and fluidity. In fungi such as S. pombe, C. neoformans and A. fumigatus the SREBP (sterol regulatory element binding protein) pathway represents an essential adaptation mechanism for sterol homeostasis when oxygen availability is limited [57–60]. This mechanism was first characterized in S. pombe [57]: Under normoxic conditions the SREBP transcription factor, Sre1, is bound to the membrane of the endoplasmatic reticulum. During hypoxia the oxygendependent synthesis of ergosterol is reduced. Under these conditions Sre1 is proteolytically cleaved and transported into the nucleus where it activates the expression of genes involved in sterol biosynthesis and the adaptation to hypoxia. In the human pathogenic fungi C. neoformans and A. fumigatus a deletion of the SREBP transcription factor resulted in impaired growth under hypoxia and attenuated virulence in a murine infection model [58–60]. To examine the impact of oxygen limitation on protein expression, several groups performed proteome studies on A. fumigatus, A. nidulans and Pichia pastoris [61–64]. It is generally believed that the pathogenic mould A. fumigatus is confronted with less than 1% of normal oxygen levels in inflammatory and necrotic tissue at the site of infection [65]. To investigate the adaptation process of A. fumigatus towards hypoxia (0.2% oxygen) the changes of the protein level were studied for both long and short periods of hypoxia using the 2D-DIGE (differential gel electrophoresis) technique [61,62]. To analyse the long-term adaptation to hypoxia, A. fumigatus was cultivated in an oxygen-controlled and glucose-limited chemostat for 10 days [61]. Proteome analysis of the intracellular protein fraction revealed an upregulation of primary metabolic pathways such as glycolysis, the TCA cycle and pentose phosphate pathway. Surprisingly, the abundance of

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abundance under ROS. Contradictory to the view that heat shock proteins, which ensure proper protein folding, increased under oxidative stress, members of this functional class were repressed under long-term MSB treatment, while the strong induction of proteasome components (Prn8 and Pre6) indicate an increased degradation of improperly-folded proteins in A. nidulans. In conclusion, proteomic studies could contribute in helping to gain insights into the global fungal response upon exposure to oxidative and nitrosative stress conditions (Fig. 1). Interestingly, RNS and ROS provoke a relatively similar protein signature, as proteins with antioxidant function, like oxidoreductases and enzymes involved in the antioxidant thioredoxin and glutathione system, and heat shock proteins are highly increased under both stress conditions. The reason may be that both ROS and RNS interfere with cellular respiration, induce protein modifications and NADPH-dependent detoxifying reactions.

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sequence similarities with the aforementioned C. albicans peroxidase, Ahp1p. Besides AspF3, other antioxidative proteins were induced, like a Cu/Zn-superoxide dismutase, and the mitochondrial peroxiredoxin Prx1. The significant upregulation of proteins of the thioredoxin system in response to increased levels of H2O2 indicates its importance for the oxidative stress response of A. fumigatus. Furthermore, proteins of primary metabolic pathways were influenced by ROS treatment as well. These include enzymes from glycolysis, the TCA cycle and the pentose phosphate shunt. Also phytopathogenic fungi are confronted with even larger quantities of ROS, which are produced by the plant in response to pathogen infection [49–51]. Penicillium expansum, a widespread phytopathogenic fungus, is one of the most common foodborne fungi on fruits and a producer of several mycotoxins [52]. In order to investigate the mechanistic process that leads to the death of P. expansum after ROS exposure, Qin et al. [53] stressed this fungus for 60 min with 30 mM H2O2 [53]. In total, the intensity of 28 protein spots, many with metabolic function, changed significantly. Ten of which were identified as proteins of mitochondrial origin. The observation that many mitochondrial proteins were influenced upon H2O2 exposure pointed to an important role of the mitochondria in the fungal oxidative stress response and induction of death. Consequently, a mitochondrial sub-proteome approach was performed to get deeper insights in the mitochondrial proteome response to H2O2. The identified proteins included proteins of the respiratory chain complexes I and III, the F1F0 ATP synthase complex, and several mitochondrial membrane carrier proteins. Except for the 12-kDa subunit of complex I, all identified members of the electron transport chain decreased in their abundance. Further investigations revealed that specifically respiratory complex III contributes to the accumulation of ROS under H2O2 stress. As a result, the oxidative damage of mitochondrial proteins and the loss of the mitochondrial membrane may finally lead to cell death [53]. In general, clear similarities of the oxidative stress response among fungi exist, but the specific proteome signatures after induction of oxidative stress are particularly influenced by the type of oxidative stress-inducing agent and the time of exposure. For example, Pusztahelyi et al. [54] investigated the genome-wide transcriptional and proteome-wide translational changes after long-term (6 h) exposure to oxidative stress caused by menadione sodium bisulphite (MSB) [54]. Their gel-based proteomic approach revealed, that 82 stressrelated intracellular proteins changed significantly in their abundance compared to unstressed A. nidulans cells. Among them, 17 metabolic enzymes catalysing primary metabolic processes were affected upon MSB-treatment. As a consequence of the long-term oxidative stress, peroxide-detoxifying peroxiredoxins and the enzyme cytochrome c peroxidase of the early antioxidative response were replaced by other typical stress responsive proteins such as thioredoxin reductase TrxR, the two glutathione S-transferases GstB and Gst3, a nitroreductase and a flavohemoprotein. Interestingly, the induction of the NO-detoxifying flavohemoprotein coincides with the observed induction of the nitrosative stress response upon H2O2 and MSB exposure in S. cerevisiae [55,56]. Another interesting observation was that a large set of stress-response proteins containing oxidoreductase domains increased their

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proteins involved in aerobic respiration also increased during hypoxia. Therefore, the changes in the mitochondrial proteome during hypoxia were additionally analysed by 2-DE blue-native/ SDS-PAGE. Since the strategy of adapting to short periods of low oxygen levels may differ from the long-term hypoxic response, A. fumigatus was cultivated in an oxygen-controlled batch fermenter using glucose as the carbon source [62]. Samples were taken over a period of 24 h and proteins were subsequently separated and analysed by the DIGE technique. When comparing both the long- and short-term responses of A. fumigatus during hypoxic growth conditions, only one third of differentially regulated proteins were identical in both studies. Independent of the time period of oxygen limitation, the glycolytic efflux was enhanced. Glycolysis provides precursor molecules for cell wall biosynthesis as well as pyruvate and ATP. This suggests that the cell wall composition changes under hypoxia and that the depletion of the terminal electron acceptor, O2, is compensated by the upregulation of glycolysis to generate ATP. Indeed, Shepardson et al. [10] showed that hypoxic growth conditions alter the composition of the fungal cell wall [66]: The cell wall thickness and the β-glucan level increased, which led to an increased activation of the immune response. In this context it is worth mentioning that also in C. albicans the fungal cell wall is modified under hypoxia, Here, a rising level of iron-acquisition proteins localised in the cell wall was observed [67]. Proteome analysis of the long-term response revealed also an upregulation in the pseurotin A secondary metabolite biosynthetic gene cluster. This is noteworthy because the biosynthesis of pseurotin A consumes atmospheric oxygen, but it still remains unclear whether the activation of the

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Fig. 1 – Induction of oxidative stress associated enzymes upon different stress conditions. A. Interestingly, diverse stress conditions, including nutrient limitation, hypoxia, and nitrosative stress provoke the oxidative stress response as a general reaction. B. The comparison of a large subset of proteomic studies revealed, that specific antioxidative proteins, such as peroxiredoxins, superoxide dismutases, and catalases are part of the general stress response. (−) means limitation of oxygen (O2), glucose (C6H12O6), nitrogen (N) or amino acids (aa). (+) means addition of a stress provoking compound such as hydrogen peroxide (H2O2) or nitrite at pH 4 (NO2−).

pseurotin A cluster is induced by long-term exposure to hypoxia, glucose limitation, a combination of both or some other stress conditions. A further remarkable result was the upregulation of proteins involved in respiration and oxidative phosphorylation under both tested hypoxic conditions. This was accompanied with an increased number of mitochondria within a cell and a higher respiratory capacity. This suggests that A. fumigatus compensates a drastic drop in oxygen levels by enhancing the formation of mitochondrial respiratory complexes. A recent genetic study confirmed that a functional mitochondrial respiration chain is essential for the adaptation of A. fumigatus to hypoxia [68]. The proteomic response to hypoxia was also investigated in the Aspergillus species, A. nidulans. A. nidulans is able to perform ammonia fermentation to support growth under hypoxia [69]. Here, nitrate is reduced to ammonium while ethanol is oxidized to acetate. For analysing the proteomic changes upon hypoxia by 2D-gel electrophoresis, A. nidulans was cultivated for 6, 12 and 24 h under normoxic and hypoxic conditions using ethanol as sole carbon source [63]. In contrast to A. fumigatus [61,62], the proteome analysis of A. nidulans revealed a downregulation of proteins involved in glycolysis and the TCA cycle during oxygen depletion. Whereas proteins involved in the biosynthesis of thiamine pyrophosphate (TPP) showed an increased abundance during hypoxia. TPP is an essential cofactor for many metabolic enzymes such as transketolase, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Consistent with this, proteins of the pentose phosphate pathway (PPP) were upregulated during hypoxia. One function of the PPP is the generation of NADPH for reductive biosynthesis. And in fact,

Please cite this article as: Kroll K, et al, Elucidating the fungal stress response by proteomics, J Prot (2013), http://dx.doi.org/10.1016/ j.jprot.2013.06.001

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Nitrogen limitation appears to be an essential stimulus for the activation of virulence functions in plant pathogenic fungi. Many phytopathogenic fungi penetrate the host tissue by forming a highly specialized infection cell called an appressorium, which is then followed by invasive growth. Interestingly, it was shown for the rice blast fungus, Magnaporthe grisea, that genes expressed during the appressorium stage were induced by nitrogen limitation [72]. The importance of global nitrogen regulators for the development of pathogenicity was shown for M. grisea and many other fungal plant pathogens, e.g. Colletotrichum lindemuthianum, Colletotrichum acutatum and Fusarium oxysporum [73–75]. So far only a few studies investigated the impact of nitrogen starvation on the protein expression of fungal plant pathogens. One notable example is Fusarium graminearum which causes crop disease by the production of the secondary metabolite deoxynivalenol (DON) [76], which was also identified as a virulence factor [77]. Nitrogen starvation activates the trichothecene pathway and induces the biosynthesis of DON. In the presence of the kinase inhibitor staurosporine a reduced production of DON was observed [78]. The authors hypothesized that phosphorylation events may play a role in the biosynthesis of DON. Thus, the phosphoproteome of F. graminearum during nitrogen depletion was analysed and both gel-based and gel-free approaches were applied [78]. Using 2D-gel electrophoresis and ProQ Diamond staining, 20 phosphoproteins were analysed by MALDI-MS. About half of them showed a change in their phosphorylation level over time. Since phosphorylated lowcopy proteins are less tractable in 2D-gels the authors also applied LC–MS based approaches. Indeed the number of identified phosphopeptides (in total 298) increased significantly by SDS-PAGE coupled with LC–MS separation and gel-free LC–MS using anion exchange chromatography. The phosphorylated peptides were enriched by immobilized metal ion affinity chromatography (IMAC) and TiO2 columns. Combining the results of the different methods a total of 348 phosphorylation sites from 241 proteins were identified. A large group of peptides belonged to proteins of unknown function, which, however, contained known consensus kinase substrate sequences.

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However, the overall conclusion can be drawn that hypoxia is 509 linked to a strong regulation of metabolic enzymes (Table 1). 510

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dehydrogenases using NADPH as the co-substrate were upregulated during hypoxia including sulphite reductase, nitroreductase, glutamate-DH and succinic-semialdehydeDH. The latter two enzymes are components of the GABA shunt, a metabolic pathway that bypasses two steps of the TCA cycle. It is believed that it regulates the NAD+/NADH balance of the cell in the absence of oxygen. The methylotrophic yeast P. pastoris is widely used for heterologous protein production in academia and the biotechnical industry. Stress conditions like nutrient and oxygen depletion have some impact on the product stability during the large-scale fermentation process [70]. Baumann et al. [64] analysed the impact of oxygen depletion on recombinant protein production in P. pastoris by combining proteomics, transcriptomics and metabolic flux studies [64]. For this study a production (secretion of Fab antibody fragment) and a non-production strain of P. pastoris were cultivated under glucose limitation in a chemostat using three different oxygen concentrations: 21% (normoxia), 11% (oxygen limitation) and 8% (hypoxia) oxygen (v/v). Similar to A. fumigatus [61,62], proteins involved in glycolysis, amino acid metabolism and stress response were upregulated during hypoxia. However, in contrast to A. fumigatus cultivated in a chemostat [61], the TCA cycle and vitamin metabolism was downregulated in P. pastoris. Furthermore, when oxygen availability was limited to 8% of oxygen (v/v), ethanol was detectable in the supernatant. This suggests that the downregulation of the TCA cycle goes along with a reduction of the respiratory activity and an induction of ethanol fermentation during hypoxia. Besides ethanol, arabitol was also found in the culture supernatant. For the pathogenic yeast C. albicans, elevated levels of arabitol were reported to indicate oxidative and temperature stress [71]. Consistent with this observation, P. pastoris proteins involved in the oxidative stress response, e.g. the thioredoxin peroxidase TSA1 or cytochrome-c peroxidase, were upregulated during oxygen depletion. Surprisingly, under hypoxic conditions the production of the recombinant protein was increased. Transcriptome data revealed an increased expression of genes involved in the biosynthesis of ergosterol and sphingolipids. Thus, allowing the authors to speculate that an altered composition of the cell membrane may facilitate the secretion of recombinant proteins under hypoxic conditions. Due to the different experimental set ups, it is difficult to compare the above described proteomic studies in great detail.

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Table 1 – Regulation of metabolism on the proteome level in response to hypoxia. ↑ indicates an upregulation and ↓ indicates a downregulation of the shown pathways during hypoxia. The bar (–) means there was no corresponding regulation found on the proteome.

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Amino acids are the essential building blocks for the biosynthesis of proteins and nucleotides. Thus, their biosynthesis is strictly regulated. In S. cerevisiae, depletion of a single amino acid induces the expression of all amino acid biosynthetic

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pathway genes in a mechanism that is called general amino acid control (GCN response) [85]. This is regulated by the bZIP transcription factor, ScGcn4p [86]. Also in C. albicans the functional homologue, CaGcn4p was identified [87]. The degree of conservation between the GCN response of the budding yeast S. cerevisiae and the fungal pathogen C. albicans was analysed on the proteome level by Yin et al. [88,88]. To identify Gcn4p-dependent changes the protein levels of the wild type and the corresponding homozygous Gcn4p-deletion strain were compared. To induce amino acid limitation the histidine analogue, 3-aminotriazole (3AT) was used. Overall, there was a high similarity of the Gcn4p-dependent proteomic response of C. albicans and S. cerevisiae to amino acid starvation. Proteins of the amino acid biosynthetic pathways showed an increased abundance during amino acid depletion conditions. Furthermore, carbon metabolism and the general stress response were induced. Hence, these data confirmed the conservation of the GCN response in C. albicans and S. cerevisiae. However, enzymes involved in the biosynthesis of adenine were only induced in a Gcn4p-dependent manner in the budding yeast S. cerevisiae, but not in the fungal pathogen C. albicans. In a previous study, it was shown that the biosynthesis of purines and histidine is co-regulated in S. cerevisiae, which may explain the observed differences [89]. Interesting in this connection is also a proteome study of C. albicans analysing the effect of the carbon source N-acetylglucosamine (GlcNAc) on morphogenesis. GlcNAc was shown to induce the GCN response [90]. It was further demonstrated for C. albicans that under amino acid-limiting conditions GCN4 promotes a morphogenetic switch to pseudohyphae, which was not observed in S. cerevisiae [87]. Although the GCN response is conserved in yeast, divergences in the functional role of ScGcn4 and CaGcn4 were identified. Another aspect of amino acid starvation in S. cerevisiae is that it triggers adhesive growth in haploid and diploid cells accompanied by the expression of the adhesin Flo11, a cell surface flocculin [91]. The influence of amino acid starvation during adhesive growth of S. cerevisiae was studied in a time-dependent manner on the protein level using the 2D-DIGE technique [92]. A diploid wild type strain was incubated in the presence and absence of 3AT. Only seven proteins were highly upregulated under these conditions, including, e.g. the receptor of activated C kinase (RACK1) orthologue Cpc2p, the translation elongation factor Efb1p, the chaperone Hsp60p and the ROS detoxifying Sod1p. Interestingly, the increased abundance of these proteins during amino acid depletion could not be confirmed by the corresponding transcriptome data [93]. Thus, a posttranscriptional regulation, e.g. by phosphorylation or acetylation was postulated by the authors [92]. In the presence of amino acids the transcription factor, Cpc2p, is known to repress Gcn4p [94]. Based on the observed upregulation of Cpc2p on the proteome during depletion of amino acids the functional role of Cpc2p was studied in more detail: Under amino acid starvation conditions the expression of FLO11 and the induction of adhesive growth were Cpc2pdependent [92]. Interestingly, in the presence of amino acids the 2D-phosphoproteome of the Cpc2p-deletion strain revealed an increased phosphorylation of proteins, e.g. of the

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The response of the rice blast fungus, Magnaporthe oryzae, to nitrogen starvation was studied in a comparative secretome analysis using 2D-gel electrophoresis [79]. In this study 85 differentially regulated proteins were identified. About 82% of the identified proteins were classified as proteins with signal peptides, leaderless proteins (non-classical secretion pathway) or proteins with localisation out of the membrane. For the classical secretion pathway signal peptides were predicted, e.g. for proteins involved in cell wall modifications including enzymes like β-1,3-glucosidase or chitinase. They all increased in abundance during nitrogen starvation. Presumably, components of the plant cell wall can be used as substrates and their degradation facilitates the penetration of the appressorium into the host plant. Besides, an increased abundance of the ROS detoxifying enzymes such as catalase and Cu/Zn-superoxide dismutase was detected when nitrogen was limited. It is interesting to note that in contrast to the Cu/Zn-superoxide dismutase no signal peptide for secretion was predicted for the catalase. Altogether, the increased formation of extracellular enzymes with antioxidative activity may help M. oryzae to tolerate ROS produced by plants as a defence mechanism against pathogen attack. Tallying this with the antioxidative enzymes of M. grisea, they were shown to be associated with virulence [80,81]. Furthermore, the strawberry fungal pathogen C. acutatum also showed an increased production of ROS detoxifying enzymes in response to nitrogen starvation at the proteome level [82]. An increased abundance and activity of the Cu/Zn-superoxide dismutase was found during nitrogen limitation and appressorium formation. Hence, all proteomic studies confirmed that not only increased levels of ROS, but also nitrogen limitation induced antioxidative enzymes during the initial phase of infection to protect the fungal pathogen from ROS generated by the plant host. During plant infection an increased accumulation of glycerol is required to generate the substantial turgor pressure of the appressorium. Under nutrient limiting conditions glycerol is produced from storage products of the fungal pathogen. In M. grisea glycogen, trehalose and lipids serve as sources for glycerol production [83,84]. Concordantly, the proteome study of C. acutatum revealed an induction of the glyoxylate cycle and fatty acid metabolism during the appressorium stage [82]. Likewise, M. oryzae showed an increased abundance of lipases in the secretome during nitrogen starvation [79]. This means that fatty acid degradation supplies the fungal pathogen with energy and acetyl-CoA as building block for biosynthetic processes under nutrient limiting conditions including nitrogen depletion. In short summary, studying the proteome of plant pathogenic fungi during nitrogen starvation confirmed that nitrogen starvation plays an essential role as a signal for the induction of virulence determinants, e.g. antioxidative and cell-wall degrading enzymes.

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In contrast to standard laboratory conditions, glucose availability is limited in the natural habitat of microorganisms. Many transcriptome data are available about the gene expression of S. cerevisiae under glucose limitation [95–97]. However, only a few data have been published on the changes of the proteome during glucose limitation. Recently, the metabolic adaptation of S. cerevisiae on different glucose concentrations (0.5, 2 and 20% glucose) was analysed by proteome analysis using 2D-gel electrophoresis [98]. The standard cultivation condition, containing 2% glucose in the media, was used as control. When comparing the proteome of cells grown on 0.5, 2 and 20% glucose, 21 proteins were differentially regulated. At high glucose concentrations an increased abundance of proteins involved in gluconeogenesis, ethanol fermentation and glycerol biosynthesis were observed. The higher production of glycerol presumably protects the yeast cell from osmotic stress caused by high glucose concentrations [99]. At low glucose levels proteins involved in amino acid metabolism and respiration were more abundant. Surprisingly, the abundance of the antioxidative protein TSA1, a peroxiredoxin, was increased under both conditions, although excess glucose was reported to induce higher levels of oxidative stress [98]. This may be explained by the following observations: Increased levels of oxidative stress as well as elevated temperatures induce the formation of a TSA1 complex exhibiting a chaperone function [100]. In contrast, when the oxidative stress level is low, TSA1 is mainly associated with actively translating ribosomes protecting the translation machinery against endogenous ROS [46]. To put it in a nutshell, the study from Guidi et al. [98] showed that excess glucose induces osmotic and oxidative stress, whereas glucose restriction shows more of a beneficial effect on the growth and metabolism of S. cerevisiae [98]. Another study analysed the effect of carbon source limitation on the yeast proteome by 2D-gel electrophoresis [101]. For this study, S. cerevisiae was cultivated in an aerobic chemostat under glucose- or ethanol-limiting conditions. Among the 44 differentially regulated proteins, most proteins were involved in central carbon metabolism, such as glycolysis, the TCA cycle and gluconeogenesis. For example, the hexokinase, Hxk1p, showed an increased abundance under glucose limiting conditions. Hxk1p catalyses the first step of glycolysis and is involved in glucose sensing [102–104]. However, the corresponding gene HXK1 is known to be repressed when yeast cells were grown on fermentable medium, but were rapidly de-repressed after shifting the cells to non-fermentable carbon sources. This may also explain the induction under glucose limitation [105]. Further studies by the same authors, Rodriguez et al. [105], have analysed the response of yeast to glucose limitation by a

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Until now, surveys of the fungal proteome have mainly been based on classical 2D-gel electrophoresis techniques with certain exceptions for eukaryotic model organisms like baker's or fission yeast. Despite the advantages of this technique (robustness, separation of intact proteins, high resolution), hydrophobic and low abundant proteins are poorly represented on 2D-gels. While 2D-gel electrophoresis allows conveying information about enzymes and stress proteins of high abundance, which mediate stress tolerance, it fails to detect the whole set of proteins which are involved in a specific metabolic or signal transduction pathway.

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shotgun proteomics approach [106]. S. cerevisiae proteins were metabolically labelled with either 14N or 15N isotopes. Protein extracts were separated by 1D-gel electrophoresis, tryptically digested and further separated and identified by nanoflowLC–MS/MS. When glucose was limited, proteins involved in glycolysis, the TCA cycle and oxidative phosphorylation were upregulated. Furthermore, PEX11, a protein involved in the biogenesis of peroxisomes, showed an increased abundance under glucose-limiting conditions. This pointed to an increased β-oxidation of fatty acids in peroxisomes under glucose-limitations, which is further supported by the fact that both the degradation of fatty acids and the biogenesis of peroxisomes are regulated via the glucose repressible activator Ard1p [107]. Indeed, the proteome data showed an upregulation of proteins that catalyse critical steps in the degradation of fatty acids under glucose limiting conditions [101]. Using 2D-gel electrophoresis, the influence of glucose starvation and its impact on autophagy was also analysed in the mould A. nidulans [108]. A. nidulans was cultured in the presence and absence of glucose and the autophagy inducer rapamycin. Using the glucose culture as the control, 20 differentially regulated proteins were identified by LC–ESI–MS/MS. However, most of the identified proteins were annotated as putative uncharacterised proteins. In addition, the aldehyde dehydrogenase, AldA, showed an increased abundance during glucose starvation indicating an activation of the ethanol utilisation pathway (alc). The alc pathway is induced by the transcription factor AlcR, which is tightly regulated by the catabolite repressor CreA [109,110]. In the presence of glucose CreA represses AlcR. Thus, when glucose is depleted, ethanol catabolism gets activated. Furthermore, the vacuolar alkaline phosphatase, Pho8, showed an increased abundance during glucose starvation. In yeast Pho8 is typically used as a marker for autophagy [111], which suggests that glucose starvation induces autophagy. This was further evidenced by comparing the proteome during glucose starvation and induction of autophagy via rapamycin. In both cases the abundance of Cdc37 decreased. In yeast the chaperone complex of Cdc37 and Hsp90 interacts with various kinases. It was shown that Cdc37 is stabilizing the catalytic subunit of protein kinase A (PKA) [112], which is an important regulator/ repressor of autophagy [113]. Thus, a downregulation of Cdc37 results in a reduced activity of PKA and an induction of autophagy.

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Gcn2-dependent elongation factor eIF2α [92]. Hence, independent of the availability of amino acids Cpc2p may have different functions. So the authors suggested that in the presence of amino acids, Cpc2p represses the GCN response by preventing phosphorylation, whereas under amino acid limiting conditions Cpc2p activates adhesive growth by induced expression of FLO11.

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modifications in a subset of 41 different proteins, thereby affecting a variety of different pathways. The study of Garcia-Santamarina et al. (2008) is another example for the identification of oxidative stress-induced modifications on proteins. The authors conducted one of the first in-depth analysis of the thiol proteome of the yeast S. pombe by applying an optimised protocol of the abovedescribed OxICAT technology: They froze the redox state of the cells prior to cell breakage by strong acid treatment and additionally employed a stable-isotope dimethyl-labelling step for improved quantification of relative changes in protein abundances. The main result of the study was that the peroxiredoxin Tpx1 and the AP-1 like transcription factor Pap1 were the major targets of H2O2-dependent oxidations of thiol [123]. The above-described impressive technological and methodological advances, which have so far only been applied on the yeast proteome, have demonstrated that these technologies can accelerate the gain of knowledge in the biology of the fungal stress response. Without a doubt, the future success also depends on the spread of new technology among the fungal research laboratories.

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Significant technological advances have been developed in the last 5 years in mass spectrometry instruments and LC– MS/MS based techniques. So it is safe to say that proteomics will provide a far more complete picture of the fungal stress response in the future. The recent reports about the characterisation of the S. cerevisiae proteome are already quite impressive. In a study from Thakur et al. [114] the authors were able to identify 2990 yeast proteins in a single shotgun-proteomics experiment by employing long liquid chromatography runs (140 and 480 min) coupled to an Orbitrap-MS instrument with fast sequencing speed [114]. Another strategy is the so-called targeted MS-approach by Selected Reaction Monitoring (SRM). It allows the selective detection and quantification of predetermined peptide ions and excels by high detection sensitivity. By applying this approach, Picotti et al. [115] were able to detect yeast proteins of very low abundance below 50 copies/cell [115]. This method, however, is limited in its ability to quantify large fractions of the proteome. So the same group developed a data independent LC–MS/MS acquisition method termed SWATH technology on a TripleTOF instrument. It promises to provide quantitation on every peptide in a single proteomics sample analysis [116]. In a new study, from the same group, the power of the SRM technology could be impressively illustrated [117]. By creating a whole chemically synthesised peptide library of the yeast proteome, the authors were able to generate an almost complete reference map of the S. cerevisiae proteome (97% of the genome-predicted proteins). Apart from the quantitative analysis of the proteome, it is also important for the characterisation of the stress proteome to determine the post-translational modifications (PTMs) of proteins which influence and regulate the response to adverse conditions. The arsenal of methods for the determination of PTMs is diverse and their detailed description is going beyond the purpose of this review. For the determination of PTMs, in particular redox-induced alterations of amino acids and phosphorylations, the reader is referred to some excellent recently published reviews [118–121]. Here only two studies will be specified as examples. The exposure of fungi to oxidative and nitrosative stress may lead to posttranslational modifications of proteins, for instance, the oxidation of methionine and cysteine residues or the nitrosylation of tyrosine. A growing body of evidence indicates that such modifications may be of physiological significance and may be involved in cellular signalling. Brandes et al. [122] applied the OxICAT technology for the characterisation of the yeast redoxome [122]. The ICAT (isotope-coded affinity tags) reagent consists of an iodoacetamide-moiety that reacts with the protein's cysteine residues, a cleavable biotin affinity tag and a 9-carbon linker, which exists in an isotopically light 12C- and heavy 13C-form. The gel-free OxICAT technology is based on the sequential labelling of all reduced cysteines of a sample with the light version of the ICAT mass tag and the subsequent reduction of all reversible oxidized cysteines using a strong thiol reductant. These reduced cysteines are then subsequently labelled with the heavy ICAT version. The main findings of the study from Brandes et al. [122] was that a substantial number of cytosolic and mitochondrial proteins are partially oxidized during exponential growth. Treatment with sublethal H2O2 concentrations did only cause thiol

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The author's work is currently supported by funding from the German–Israeli Foundation for Scientific Research and Development (GIF No. 996-47.12/2008), the International Leibniz Research School for Microbial and Biomolecular Interactions Jena (ILRS) as part of the excellence graduate school Jena School for Microbial Communication (JSMC) and the Federal Ministry of Education and Research (BMBF), Germany, FKZ: 01EO1002. All authors thank Derek Mattern for critically reading the manuscript.

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