Identification of virulence determinants of the human pathogenic fungi Aspergillus fumigatus and Candida albicans by proteomics

International Journal of Medical Microbiology 301 (2011) 368–377

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International Journal of Medical Microbiology journal homepage: www.elsevier.de/ijmm

Mini Review

Identification of virulence determinants of the human pathogenic fungi Aspergillus fumigatus and Candida albicans by proteomics Olaf Kniemeyer a,b,∗ , André D. Schmidt a,b , Martin Vödisch a,b , Dirk Wartenberg a,b , Axel A. Brakhage a,b,∗ a b

Dept. of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology – Hans-Knöll-Institute (HKI), Jena, Germany Dept. of Microbiology and Molecular Biology, Institute for Microbiology, Friedrich Schiller University Jena, Germany

a r t i c l e

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Keywords: Aspergillus fumigatus Candida albicans Proteomics Pathogenicity Two-dimensional gel electrophoresis Mass spectrometry

a b s t r a c t Both fungi Candida albicans and Aspergillus fumigatus can cause a number of life-threatening systemic infections in humans. The commensal yeast C. albicans is one of the main causes of nosocomial fungal infectious diseases, whereas the filamentous fungus A. fumigatus has become one of the most prevalent airborne fungal pathogens. Early diagnosis of these fungal infections is challenging, only a limited number of antifungals for treatment are available, and the molecular details of pathogenicity are hardly understood. The completion of both the A. fumigatus and C. albicans genome sequence provides the opportunity to improve diagnosis, to define new drug targets, to understand the functions of many uncharacterised proteins, and to study protein regulation on a global scale. With the application of proteomic tools, particularly two-dimensional gel electrophoresis and LC/MS-based methods, a comprehensive overview about the proteins of A. fumigatus and C. albicans present or induced during environmental changes and stress conditions has been obtained in the past 5 years. However, for the discovery of further putative virulence determinants, more sensitive and targeted proteomic methods have to be applied. Here, we review the recent proteome data generated for A. fumigatus and C. albicans that are related to factors required for pathogenicity. © 2011 Elsevier GmbH. All rights reserved.

Introduction The release of the genome sequences of Candida albicans and Aspergillus fumigatus has been of great benefit for a more detailed insight into the evolution and pathogenesis of these medically important fungi (Jones et al., 2004; Braun et al., 2005; Nierman et al., 2005). The C. albicans genome consists of 8 chromosomes with 6354 genes (Arnaud et al., 2005; Braun et al., 2005; D’Enfert et al., 2005). A. fumigatus has an about one third greater genome size of 9922 protein-encoding sequences (Mabey et al., 2004; Nierman et al., 2005; Arnaud et al., 2010). The sequence data provide the basis for global studies on the level of proteins, the proteome. The term proteome was coined by the Australian scientist Marc Wilkins. He defined it as the protein complement of the genome of an organism (Wilkins et al., 1996). Proteomic studies were initially represented by quantitative two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by identification of proteins by Edman-sequencing or mass spectrometry (MS) (Klose, 1975;

∗ Corresponding authors at: Leibniz Institute for Natural Product Research and Infection Biology – Hans-Knöll-Institute (HKI), Beutenbergstrasse 11a, 07745 Jena, Germany. Tel.: +49 3641 532 1001; fax: +49 3641 532 0802. E-mail addresses: [email protected] (O. Kniemeyer), [email protected] (A.A. Brakhage). 1438-4221/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2011.04.001

O’Farrell, 1975). Over the years, 2D-PAGE has become a robust, reproducible, and popular technique. One of the major advantages of 2D-PAGE lies in its unique ability to separate complete proteins in high resolution, with all their modifications. It further allows parallel running of many biological replicates and can easily be combined with antibody detection. However, hydrophobic, in particular, membrane proteins and low abundant proteins are extremely underrepresented on two-dimensional gels (Görg et al., 2009; Rabilloud et al., 2009). Since the introduction of the combination of multidimensional chromatography and tandem mass spectrometry (LC–MS/MS) by Yates et al. (1999) and others, a diverse set of gel-free methods was developed (see Fig. 1), allowing relative quantitation of proteins. The application of LC–MS/MSbased techniques enables also the detection of proteins which are hardly amenable to 2D separation: very hydrophobic proteins, membrane proteins, and proteins with extreme pI or molecular mass (Aebersold and Mann, 2003; Speers and Wu, 2007; de Godoy et al., 2008). MS-based proteomics involve digesting the protein extract into peptides first before the resulting peptide mixture is fractionated. Tandem mass spectrometry (MS/MS) is then used to identify the proteins. In MS/MS, selected peptides (precursor ions) are further fragmented and the m/z of their fragment ions is measured. This ‘peptide-centric proteomics’ typically identifies 1000–2000 proteins in a biological sample. However, this approach has intrinsic limitations due to the loss of intact

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Fig. 1. General procedure in 2D-PAGE- and MS-based proteomics workflows. After separation of proteins by isoelectric focusing and SDS–PAGE, gel images are acquired using a scanner, and gel images are analysed by a 2D gel analysis software. Protein spots of interest are excised, tryptically digested, and analysed by mass spectrometry. The 2D-DIGE method (2D-fluorescence difference gel electrophoresis) is a multiplexing technology, where protein samples are covalently labelled with fluorescent dyes with different excitation wavelengths. This allows the separation of up to 3 samples in one 2D gel. Gel-free proteomic approaches are usually based on the separation of peptides by nano-LC and their subsequent identification by mass spectrometry. For the quantification of proteins, methods for non-radioactive isotopic labelling are often applied. Here, the isotopic label can be introduced during the cultivation (metabolic labelling), after protein extraction at the protein level, and during or after the tryptic digest on the peptide level. With the introduction of mass analysers with high resolution and sensitivity and reproducible LC–MS systems, the application of label-free quantitative methods has increased. The method uses either changes in chromatographic ion sensitivity or spectral countings for the quantification of protein expression. Abbreviations: IEF (isoelectric focusing), ICAT (isotope-coded affinity tags), ICPL (isotope-coded protein labelling), SILAC (stable isotope labelling with amino acids in cell culture), iTRAQ (isobaric tag for relative and absolute quantitation), and TMT (tandem mass tags).

protein information and the high complexity and large dynamic range in peptide concentrations of complex protein samples, which cannot be resolved and completely analysed by even the fastest mass spectrometers. This limitation often results in a significant variation of the data obtained (Duncan et al., 2010). The average overlap of identified protein species between technical replicates

of the same protein extract is typically only 60% and varies even more between different MS-platforms (Elias et al., 2005). Today, directed and targeted proteomic approaches are being explored to overcome some of the aforementioned limitations of LC–MS/MSbased proteomics. Here, mass spectrometers are employed to select and analyse non-redundantly a specific set of proteins and pep-

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Fig. 2. First level functional catalogue (MIPS FunCat; http://mips.helmholtz-muenchen.de/proj/funcatDB/search main frame.html) annotation for all A. fumigatus proteins (631) detected in the most recent 2D-PAGE studies published since 2006 (Albrecht et al., 2010a; Lessing et al., 2007; Vödisch et al., 2009; Teutschbein et al., 2010; Kim et al., 2007; Zhang et al., 2009; Schmaler-Ripcke et al., 2009; Grosse et al., 2008; Mouyna et al., 2010; Gautam et al., 2008; Asif et al., 2006; Carberry et al., 2006; Kniemeyer et al., 2006). Many proteome data are also accessible at www.omnifung.hki-jena.de.

tides that avoids oversampling of high intensity precursor ions (reviewed in Gstaiger and Aebersold, 2009; Schmidt et al., 2009). Such a targeted approach allows to detect proteins in concentrations as low as 50 protein copies in a baker’s yeast cell (Picotti et al., 2009). The enormous progress made in optimisation of proteomic techniques has also the potential to help elucidating the complex steps in the infection process of the fungal pathogens A. fumigatus and C. albicans. Efforts towards post-genomic proteome studies in these pathogenic fungi have been intensified in the last years, and the level of knowledge has already considerably increased (Rupp, 2004; Diez-Orejas and Fernandez-Arenas, 2008; Kim et al., 2008; Kniemeyer and Brakhage, 2008; Rupp, 2008; Andersen and Nielsen, 2009; Kniemeyer et al., 2009; Vialas et al., 2009). The aim of this review is to provide an overview about proteome-related studies with an emphasis on pathogenicity. Proteome maps Proteome maps are helpful tools for comparative proteome studies. They give an impression of the kind and quantity of proteins which can be separated and visualised by a 2D gel approach. For both pathogenic fungi A. fumigatus and C. albicans, 2D gel proteome reference maps are available. For filamentous fungi such as A. fumigatus, a clean-up of the protein extract is often crucial prior to the 2D gel separation (Kniemeyer et al., 2006). Vödisch et al. (2009) identified 334 different mycelial proteins via 2D gel electrophoresis of A. fumigatus cells, which had been cultivated in minimal medium with glucose in shake flasks. The molecular mass and the isoelectric points (pI) of separated proteins varied between 10 and 142 kDa and 4.1 and 11.5, respectively. In the same study, A. fumigatus mitochondria were isolated and a mitochondrial proteome map was established (Vödisch et al., 2009). This subproteomic approach led to identification of 55 proteins, which had not been found in the mycelial proteome map before. This result clearly illustrates the

advantage of subcellular fractionation that can give access to lower abundant proteins by depletion of high abundant cytosolic proteins. The asexually produced spores of A. fumigatus, designated as conidia, represent the infectious agent in most cases. Hence, they have the initial contact with the host. For this reason, Teutschbein et al. (2010) characterised the proteome of dormant conidia of A. fumigatus. Dormant conidia contain in particular proteins in high abundance, which are required for stress tolerance and rapid reactivation of metabolic processes and account therefore for the enormous stress resistance of conidia and their ability to start germinating within a couple of hours. In summary, the generation of 2D gel reference maps for A. fumigatus illustrated that especially proteins involved in biological processes such as translation, primary metabolism, respiration, and stress response are easily accessible to gel-based proteomic approaches (see also Fig. 2 for a summary of all detected proteins in recent 2D-PAGE-based studies). Comparable results were also found for C. albicans by Kusch et al. (2008). The authors created a 2D reference map of cytoplasmic proteins present at both the exponential and stationary growth phase of C. albicans. A total of 746 protein spots was identified representing 360 different proteins. Altogether, these studies revealed that around 6% of all predicted proteins of A. fumigatus and C. albicans can be easily analysed by 2D-PAGE across a broad pH range. To achieve significant higher proteome coverages in 2D-PAGE, narrower pH ranges during IEF or prefractionation techniques have to be applied (Righetti et al., 2005). Some of the proteome reference maps are also accessible from data repositories, namely the data warehouse for integrating fungal omics data ‘Omnifung’ (http://www.omnifung.hki-jena.de) and specific C. albicans databases such as Proteopathogen database for studying host–pathogen interaction (http://proteopathogen.dacya.ucm.es) and Compluyeast (http://compluyeast2dpage.dacya.ucm.es/cgibin/2d/2d.cgi).

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Proteome of the cell wall and membrane proteomics The cell wall of fungi is responsible for maintaining the integrity of the cell against physical and chemical damage. It is also essential for the interaction with the environment and represents a known target for antifungals. The fungal cell wall is mainly composed of polysaccharides, while a smaller part is represented by proteins (reviewed in Latge, 2007; Chaffin, 2008; Gastebois et al., 2009). These cell wall proteins are known to play specific roles as adhesins, enzymes, allergens, or immunomodulators (Castillo et al., 2008) and may be targeted during recognition of the pathogen by the host’s innate and adaptive immune system. The cell wall proteome has been studied intensively in C. albicans, and enrichment strategies were established by several groups (Pitarch et al., 2002; de Groot et al., 2004). Castillo et al. (2008) applied different methods to extract glycosylphosphatidylinositol (GPI)-anchored, disulphide-linked, and alkali-released proteins from purified cell walls. After tryptic digestion, the peptide mixture was analysed via LC–MS/MS. The authors identified 21 proteins predicted to carry a signal peptide, having potential O- and Nglycosylation and occasionally GPI-modification sites. By contrast, in silico analyses revealed more than 100 ORFs encoding putative GPI-anchored proteins in the C. albicans genome (De Groot et al., 2003). Castillo et al. (2008) discussed several reasons for this discrepancy: (1) the identified proteins are the most abundant ones; (2) hindered tryptical protein hydrolysis or peptide analysis due to high amounts of sugar residues, or (3) developmental stage or stress-dependent protein synthesis. Interestingly, Castillo et al. (2008) found several ‘atypical’ cell wall proteins which had previously been detected in cytosolic fractions or other cellular compartments. Whether these proteins are trapped in the cell wall or whether they are strongly associated to certain cell wall polymers has not been elucidated yet. However, contaminations of the cell wall fractions during isolation by cytosolic proteins cannot be ruled out despite extensive washing. To reduce possible contaminations to a minimum another relatively easy technique to study cell surface proteins of intact C. albicans yeast cells was introduced by Hernaez et al. (2010). Cells were directly treated with trypsin followed by peptide separation and identification using LC–MS/MS. To prevent cell lysis and subsequent contamination with intracellular proteins, the incubation time with trypsin was limited to 5 min, and the viability of the treated cells was determined by flowcytometry. The authors were able to identify 17 proteins, 40% of which were functionally classified to cell wall organisation and biosynthesis. The comparative proteome analysis of cell wall-enriched fractions derived from different growth forms of C. albicans, namely yeast cells, hyphae and biofilms, revealed typical differences in the protein pattern, indicating a morphotype-dependent synthesis of cell wall proteins (Ebanks et al., 2006; Maddi et al., 2009; Martinez-Gomariz et al., 2009). Proteome studies detected also a new regulation mechanism for enzymes involved in the biosynthesis of cell wall compounds. In a global analysis of the C. albicans phosphoproteome, the chitin synthase 3 (Chs3) was shown to be phosphorylated. Mutation of the phosphorylation site at Ser139 revealed that phosphorylation and dephosphorylation are required to target Chs3 to the sites of polarised growth of C. albicans (Lenardon et al., 2010). In comparison to the cell wall composition of the yeasts C. albicans and Saccharomyces cerevisiae, the mycelial cell wall of A. fumigatus is very different (Latge et al., 2005). Hence, extraction protocols, which have been established for yeasts, are not easily transferable to A. fumigatus. This could be one of the reasons why little information is available about the cell wall proteome of A. fumigatus. In the pioneering study of Bruneau et al. (2001), several

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GPI-anchored proteins were identified, which had been released from isolated membranes by the activity of an endogenous GPIphospholipase. Asif et al. (2006) characterised the conidial surface proteome and found 26 proteins, which were released upon ␤-1,3glucanase treatment. Among these were the conidial surface layer protein RodA, the aspartic proteinase PEP2, a lipase, and surprisingly, many proteins without a signal for secretion. The authors speculated that some of the conidial proteins may be important for the interaction with the host by, e.g., mediating cell damage or adhesion. Besides the cell wall, the cytoplasmic membrane is another crucial structure, which enables a cell to respond and interact with the environment, including the human immune system. However, analysis of membrane proteins is still a challenging task due to their low water solubility (Rabilloud, 2009). Two recent publications focused on the cytoplasmic membrane proteome of A. fumigatus (Ouyang et al., 2010) and C. albicans (Cabezon et al., 2009). Ouyang et al. (2010) identified 530 proteins from a membrane-enriched fraction of A. fumigatus using a combined 1D-PAGE–LC–MS/MS strategy. They found 17 integral membrane proteins involved in N-, O-glycosylation or GPI-anchor biosynthesis. On the basis of this information, glycosylation pathways were predicted in A. fumigatus. To obtain a global picture about the protein arrangement of the C. albicans cytoplasmic membrane, Cabezon et al. (2009) purified membranes via generation of protoplasts, mechanical disruption, ultracentrifugation, and Na2 CO3 treatment. To isolate GPI-anchored proteins, 2 additional steps were included: two-phase separation using Triton X-114 and phosphatidylinositol-phospholipase C treatment. LC–MS/MSbased peptide analysis revealed 214 different membrane proteins. Among them, proteins involved in transport processes, endocytosis, and biopolymer biosynthesis were enriched, e.g., enzymes important for ergosterol (Erg1, Erg3, Erg6) and cell wall biosynthesis (Utr2, Exg2). In conclusion, most recent studies applied MS-based, gel-free approaches for the functional characterisation of the A. fumigatus or C. albicans membrane proteomes, thus overcoming the limitations of gel-based proteomics. Nevertheless, the information about membrane proteins in both pathogenic fungi remains rather limited.

Oxidative stress During the infection process, the release of reactive oxygen species (ROS) by immune effector cells was suggested to play an important role in killing microbes. Recent data suggest that it is unlikely that ROS are one of the main factors, which are directly responsible for killing microbes (Segal, 2005). Oxidative stress response was analysed in both pathogenic fungi C. albicans and A. fumigatus by 2D gel electrophoresis. Kusch et al. (2007) showed that the synthesis of many proteins with antioxidant functions, i.e. catalases, enzymes of the thioredoxin and glutathione system, a set of oxidoreductases, and heat shock proteins increased significantly upon treatment of cells with hydrogen peroxide or diamide in C. albicans. Yin et al. (2009) confirmed the increased production of enzymes involved in redox regulation. Further proteins up-regulated in response to peroxide stress play roles in protein folding and protein degradation. In addition, the authors postulated the existence of a core stress response in C. albicans, consisting of a set of proteins, which always display a higher level of expression under different conditions of stress (osmotic, heavy metal, and oxidative stress). This group of proteins included alcohol dehydrogenase Adh1, ATPase complex proteins, cadmium-induced protein Cip1, elongation factor 2 as well as the heat shock proteins Ssa2 and Ssb1. In A. fumigatus, the oxidative stress response resembled the one described for C. albicans: the level of many proteins with

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antioxidant functions, heat shock proteins, and proteins involved in the biosynthesis of the stress protectant sugar trehalose increased. In addition, the primary metabolic pathways glycolysis, TCA cycle, and pentose phosphate shunt were affected by oxidative stress. In contrast to C. albicans, also the expression of cytoskeletal proteins was influenced (Lessing et al., 2007).

Response to iron limitation For a pathogen, its potential to deal with the limited availability of nutrients in the host is of immense importance. In particular, the acquisition of iron is one of the crucial steps to initiate and establish an infection (Miethke and Marahiel, 2007). A. fumigatus requires a functional siderophore system for pathogenicity under such limited conditions to chelate iron (Schrettl et al., 2004, 2007). To examine the global impact of iron limitation on A. fumigatus and to investigate how fungi cope with this metal shortage, comparative proteome studies were performed by 2D gel electrophoresis analyses. Among the down-regulated proteins under iron deficiency, the major group was involved in primary metabolism, which includes processes like glycolysis (3%), citrate cycle (6%), and oxidative phosphorylation (16%). The reason for this is the high proportion of enzymes in these pathways, which contain iron as a cofactor. Moreover, proteins of the amino acid metabolism (21%) represented the largest fraction of up-regulated proteins probably to provide efficient amount of precursors for siderophore biosynthesis. In addition, the induction of members of the proteasome machinery and enzymes involved in the detoxification of ROS emphasised the exposure of A. fumigatus to nutrient shortage and oxidative stress as a result of iron limitation (A. Schmidt, unpubl. results). Beside this general approach to study the response on iron depletion, the regulatory mechanisms of iron homoeostasis were examined in more detail in the related fungus A. nidulans, which shares a high level of similarity in the mode of iron regulation with A. fumigatus. The central component of the regulation is the transcriptional regulator HapX (Hortschansky et al., 2007). HapX represses iron-dependent pathways under –Fe conditions by interaction with the heterotrimeric CCAAT-binding core complex (CBC) that is involved in redox regulation (Brakhage et al., 1999; Thön et al., 2010). The proteome analysis, performed on mycelia grown under iron limitation, revealed 30 different regulated proteins in the hapX mutant strain. Among these, haem-containing enzymes as well as the iron–sulphur cluster containing proteins such as the 5-aminolevulinic acid synthase were overrepresented. The latter enzyme catalyses the first step in haem biosynthesis and its induction under –Fe conditions underlined the enormous deregulation in iron homoeostasis in the hapX strain. In contrast to A. fumigatus, the genome of C. albicans does not contain any genes encoding enzymes for the biosynthesis of siderophores. However, C. albicans is able to use siderophores produced by other members of the microbial flora and host proteins such as haemoglobin, transferrin, and ferritin as iron source. The fungus possesses 3 independent high-affinity uptake systems for the utilization of iron from the environment (Almeida et al., 2009). Very few proteomic studies have been carried out until now, targeting the global response of C. albicans to iron restriction. Sosinska et al. (2008) investigated the variability of the cell wall proteome as a function of the ambient oxygen or iron concentration. The addition of the iron chelator ferrozine resulted in higher levels of the cell wall proteins Hwp1 and Rbt5. Rbt5 binds haemin and haemoglobin and is thus directly involved in iron metabolism. Nevertheless, iron limitation also seems to affect other processes such as adhesion: the hyphal-specific cell wall protein Hwp1 is known to mediate adherence to epithelial cells.

Temperature adaptation in A. fumigatus and C. albicans Fungal survival at the elevated temperature of a human host is essential for virulence. A. fumigatus is able to grow at temperatures as high as 55 ◦ C and can even survive temperatures of around 70 ◦ C. This characteristic distinguishes A. fumigatus from other Aspergilli and may contribute to its virulence (Bhabhra and Askew, 2005). To extend the knowledge about the heat shock response in A. fumigatus, which was previously based solely on transcriptome and molecular biology data (Nierman et al., 2005; Angiolella et al., 2009; Do et al., 2009), Albrecht et al. (2010a) analysed the change of the A. fumigatus proteome during a temperature shift from 30 to 48 ◦ C by applying proteome analysis based on the DIGE (differential gel electrophoresis) technique. As a result, 91 spots representing 64 different proteins were differently regulated. In general, most changes in protein spots were observed within 120 min after the induced temperature shift, but enormous changes were already seen after 30 min, like the increase of heat shock proteins (HSPs) of the cytoplasm, mitochondria, and ER. During the heat shock response, the proteins HSP30/HSP42 and HSP90 showed the highest increase in abundance. In addition, enzymes of the oxidative stress response were induced, presumably because of a higher respiration rate which leads to an elevated production of ROS (Sugiyama et al., 2000). In particular, the upregulation of both the thioredoxin peroxidase AspF3 and the cytochrome c peroxidase Ccp1 appeared to be heat shock-dependent. Moreover, enzymes of the NADPH-generating pentose phosphate pathway were upregulated and could thereby maintain the pool of the required reducing equivalents for the reduction of ROS, oxidized glutathione or thioredoxin. Finally, besides proteins involved in cellular processes like cytoskeleton organisation, transcription and translation, enzymes of the biosynthesis of amino acids and fatty acids were up-regulated, probably due to a higher turnover of proteins and increased lipid damage under heat shock. In C. albicans, the temperature plays a pivotal role for dimorphism. During infection and in culture medium supplemented with blood serum at 37 ◦ C, C. albicans undergoes a morphological change from yeast to branching hyphal cells. Recently, some details were elucidated how temperature orchestrates fungal morphogenesis. Shapiro et al. (2009) showed that at the lower temperature of 30 ◦ C, the heat shock protein HSP90 negatively regulates hyphae formation by interacting with positive regulators of the filamentation pathway, i.e. components of the Ras-protein kinase pathway. Binding of Hsp90 probably maintains these proteins in an inactive form, whereas at higher temperatures of 37 ◦ C the block is relieved. Moreover, global analysis of the regulation of genes by the heat shock transcription factor Hsf1 has been intensively studied in C. albicans (Nicholls et al., 2009), but little information is available about the regulation of the heat shock response on the protein level. Zeuthen and Howard (1989) investigated the induction of stress proteins by thermal and other stresses and provided evidence for the biosynthesis of new HSPs at temperature shifts from 23 to 27 ◦ C and from 37 to 45 ◦ C. De Backer et al. (2000) explored the unexpected resistance of a C. albicans strain containing one disrupted allele of the gene CGT1 by 2D gel electrophoresis. CGT1 encodes the mRNA capping enzyme 5 -guanyltransferase. The proteome analysis revealed a significant overexpression of the elongation factor subunit Ef1-␣ and the cell wall heat shock protein Ssa2p, which may explain the strain’s increased resistance to heat stress.

Antifungal substances Because of the rising number of severe fungal infections and the development of drug resistance (Verweij et al., 2009; Lass-Flörl et al., 2010; Leventakos et al., 2010; Morio et al., 2010), novel anti-

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fungal drugs are urgently needed. To identify new targets and to elucidate the molecular interactions of antifungal molecules with the fungal cell, proteomic approaches are helpful tools. Dabur et al. (2007) and Chhillar et al. (2009) studied the molecular mechanism of the antifungal compounds 2-(3,4-dimethyl-2,5-dihydro-1Hpyrrol-2-yl)-1-methylethyl pentanoate (DHP), a pyrrole derivative from Dature leaves, and diethyl 4-(4-methoxyphenyl)-2,6dimethyl-1,4-dihydropyridin-3,5-dicarboxylate (2e), a chemically synthesized dihydropyridine derivative, by proteomic methods. The authors found that both DHP and 2e inhibit the expression of secreted proteins and low molecular mass antigens in A. fumigatus. Furthermore, the antifungal action of the drug Amphotericin B (AMB) was studied by a proteomic approach to get more detailed information about its molecular targets. Using 2D-PAGE, Gautam et al. (2008) identified 48 proteins affected by continuous exposure to AMB (44 up-regulated and 4 down-regulated), which are involved in various metabolic processes like the ergosterol pathway (upregulation of Erg13, Heme13), the cell wall maintenance (downregulation of RodB), oxidative stress (upregulation of MnSOD, Cat1, LsfA), transporter proteins, carbohydrate, amino acidand energy metabolism. In C. albicans, several recent proteomic studies elucidated drug resistance (Kusch et al., 2004; Yan et al., 2007; Lis and Bobek, 2008; Xu et al., 2009), susceptibility to (Marinach et al., 2009) and the mode of action of antifungals (Angiolella et al., 2009). For a deeper understanding of resistance mechanisms to antifungal substances, Yan et al. (2007) employed proteomic analysis for a comparison of a fluconazole-resistant strain of C. albicans with a susceptible strain. Proteins involved in energy metabolism, stress response, macromolecule biosynthesis, and others were found to be differently synthesized. The fluconazole-resistant strain showed a decreased intracellular ATP content and a lower mitochondrial membrane potential. The concurrent upregulation of glycolytic proteins was interpreted as a hint for an additional source of ATP generation presumably via substrate level phosphorylation. The authors speculated that the impaired mitochondrial respiration leads to a decrease of the endogenous ROS level and as a result, to higher drug resistance. Comparable changes in the primary metabolism were also observed in a C. albicans mutant resistance to the human antimicrobial peptide MUC7 12-mer (Lis and Bobek, 2008). The application of fluconazole combined with the alkaloid berberine exerted a synergistic action against clinical, fluconazole-resistant C. albicans strains (Xu et al., 2009). The combined use of these 2 antifungal compounds led to a decreased intracellular ATP content, inhibition of ATP synthase, and an increase in endogenous ROS. The described studies nicely demonstrate how the combination of proteomic and functional analyses helps to elucidate complex molecular processes.

Biofilm Biofilms consist of cells that enable the attachment to artificial or biotic surfaces leading to a robust and often non-eradicable biolayer by the production of an extracellular matrix. Increasing attention has been paid to biofilms of human pathogenic fungi since the incidence of C. albicans infections is strongly correlated with its adherence on central venous catheters and other medical devices. This results in severe and often lethal blood stream infections. To get a better insight into the C. albicans biofilm structure and its formation, several proteomic investigations were performed. Thomas et al. (2006) characterised the cell surface proteome and the secretome of early C. albicans biofilms in comparison with planktonic-grown cells. The authors identified 8 cell wall-associated proteins being higher abundant in biofilm cells. Among them were glycolytic enzymes like Adh1p and stress pro-

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teins (Ssa1p). Also, the mannoprotein Mp58p/Pra1 was found to be secreted in higher amounts during biofilm growth. Interestingly, this protein has been reported to be able to bind human complement regulators for immune evasion (Luo et al., 2009). Overall, this study showed only little difference between biofilm and planktonic-grown cells. By contrast, Seneviratne et al. (2008) analysed cytoplasmic and membrane fractions of mature C. albicans biofilms. The authors identified 20 differentially expressed proteins in biofilm in comparison to planktonic-grown cells. Particularly, proteins involved in yeast-hyphal transition (Sti1p, Ald5p, Cit1p) showed higher abundance in biofilm-grown cells which underlined the importance of hyphae formation during biofilm development (Ramage et al., 2002; Paramonova et al., 2009). Interestingly, many antioxidant proteins like alkyl hydroxyl peroxidase (Ahp1p) and thioredoxin peroxidase (Tsa1p) were up-regulated in the biofilm phase. It was concluded that C. albicans biofilms contain less ROS and exhibit a higher activity of antioxidants and consequently a higher resistance to antifungals. Martinez-Gomariz et al. (2009) characterised cytoplasmic and non-covalently bound surface proteins of mature biofilms, planktonic-grown yeast cells, and hyphae by 2D-DIGE. The authors reported about an increased use of the glyoxylate cycle and confirmed the high abundance of antioxidant enzymes in biofilms. On both the surface and in the cytoplasm, the abundance of the alcohol dehydrogenase Adh1 was greatest in the biofilm, which confirmed the previous observation that this class of enzymes contributes to the regulation of C. albicans biofilm formation (Mukherjee et al., 2006; Nobile et al., 2009). Taken together, all proteome analyses revealed high similarities between the set of formed proteins in C. albicans biofilms and their planktonic equivalents, whereas the quantities of various proteins differ in a morphological stage-dependent manner. Also A. fumigatus has the ability to develop a complex growth form of dense, intertwined mycelium enclosed in an extracellular matrix, which is able to adhere to surfaces. This morphotype thus resembles biofilms known from Candida spp. and other microorganisms (Ramage et al., 2009). A combined transcriptomic and proteomic study about the A. fumigatus biofilm lifestyle revealed an enhanced production of the mycotoxin gliotoxin during ‘multicellular’ growth. The authors speculated that this finding could also play a role in chronic A. fumigatus infections, e.g., in cystic fibrosis patients, where an increased synthesis of gliotoxin may confer protection from the host innate immune system (Bruns et al., 2010).

Immunoproteomics The identification and characterisation of antigenic proteins is a requirement for the development of antifungal vaccines which have retained increased interest as an alternative therapeutic treatment of fungal infections. For investigations of the immunoreactivity of fungal proteins with human or murine antibodies, the combination of 2D-PAGE and immunoblotting represents the method of choice. Pitarch et al. (1999) extracted proteins from 3 different subcellular compartments of C. albicans and performed immunoblotting with sera of humans with systemic candidiasis. One of the most reactive proteins found in each subfraction was the glycolytic enzyme enolase. Among other studies (Manning and Mitchell, 1980; Barea et al., 1999; Pardo et al., 2000), a more recent investigation was carried out by Hernando et al. (2007). The analysis of blastoconidia and germ tube extracts of C. albicans revealed several manno proteins showing antigenic reactivity against serum of a patient with systemic candidiasis, but not against control serum, i.e., kexin precursor and a mitochondrial complex I chaperone. In a different approach, low-virulent C. albicans mutant strains were used for vaccination of mice which conferred

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a protection against subsequent infections with a virulent C. albicans wild-type strain (Fernandez-Arenas et al., 2004a, 2004b). The same group immunized BALB/c mice with an ecm33 deletion mutant and gained protection for over 90% of the vaccinated mice against a subsequent infection with a C. albicans wild-type strain (Martinez-Lopez et al., 2008). In the same study, from 29 detected immunoreactive proteins, 6 novel antigens were identified: 6phosphogluconate dehydrogenase (Gnd1p), translation elongation factor eEF1beta (Efb1p), citrate synthase (Cit1p), cystathionine beta-synthase (Cys4p), and the ribosomal proteins Rpl10Ep, and Yst1p. Similarly, several immunoproteomic studies have been carried out for A. fumigatus. Most of these analyses screened for immunoreactive anti-Aspergillus IgE antibodies, since A. fumigatus is the causative agent of allergic diseases such as allergic bronchopulmonary aspergillosis (ABPA) and allergic asthma. In this context, Gautam et al. (2007) characterised the secreted antigens from a static-grown A. fumigatus culture. The authors used sera of A. fumigatus-sensitised asthmatics and identified 11 novel antigens. Among them, an extracellular arabinase and a non-characterised chitosanase were supposed to be major allergens. A putative cell wall protein (PhiA) only showed little immunoreactivity with the human sera. By contrast, Glaser et al. (2009) detected specific IgE antibodies against PhiA in the sera of 94% of all investigated ABPA patients. In another study, a rabbit model of invasive aspergillosis was used to screen for A. fumigatus antigens with an ability to elicit a protective immune response. Fifty-nine different proteins were found which included proteins related to oxidative stress response and primary metabolism (Asif et al., 2010).

Host–pathogen interactions The interplay of human effector cells with fungal pathogens is of particular interest since the innate immune system represents the most important defence system against infections with C. albicans and A. fumigatus (Netea et al., 2008; Brakhage et al., 2010). However, there is only little knowledge available about the mechanisms how fungal pathogens are obliterated. In addition, the strategies of fungi to bypass phagocytosis are hardly known. Two studies characterised the proteome of murine macrophages which had been exposed to living C. albicans yeast cells for either 45 min or 3 h (Shin et al., 2005; Martinez-Solano et al., 2006). At the later time point, yeast cells formed hyphae. Under these conditions, a significantly lower abundance of key glucose metabolic enzymes was observed. Thus, energy depletion might be responsible for C. albicans-induced death of infected macrophages. Furthermore, the synthesis of proteins involved in NO production and cytoskeletal stability was altered which might affect the microbicidal potential and cellular integrity of the macrophages. Likewise, Martinez-Gomariz et al. (2009) identified proteins as differentially regulated which are involved in a variety of cellular processes including cytoskeletal organisation and oxidative response. A recent study used heat-inactivated C. albicans cells for stimulation of murine macrophages (MartinezSolano et al., 2009). Changes in macrophage proteome revealed 24 differentially regulated proteins involved in cytoskeletal organisation, signal transduction (Annexin I, Ran GTPase), metabolism (protein disulphide isomerase), protein fate (proteasome protein Psma1p), and stress response. The authors speculated that the altered abundance of the proteins listed in brackets might contribute to an anti-inflammatory response against heat-inactivated C. albicans cells, whereas live cells provoke an inflammatory response. Complementary, the proteomic changes of C. albicans yeast cells due to macrophage confrontation were analysed by

Fernandez-Arenas et al. (2007). They showed that the abundance of chaperones and detoxifying proteins related to stress response was increased upon interaction with macrophages. Furthermore, a huge amount of metabolic enzymes was affected indicating that starvation conditions were induced in C. albicans cells. In contrast to C. albicans, no proteomic studies on the host–pathogen interaction of A. fumigatus have been reported, yet.

Conclusions Until now, proteomic surveys are underrepresented in the area of medical mycology. However, a lot of progress has been made, and exciting insights into the stress response, host–pathogen interaction, and antigenic activity have been gained, e.g., new putative targets of global stress regulators were found, new responsive pathways upon phagocytic cell interaction were identified, and new protein antigens with immunoprotective capacity were discovered. Proteome analysis represents a powerful tool for the investigation of fungal metabolism and the adaptation of fungi to environmental changes. The application of new proteomic technologies and the reduction of costs for instrumentation will accelerate the gain of knowledge by proteome analysis on the infection biology of A. fumigatus and C. albicans. To scrutinise the intimate interplay of pathogenic fungi and their hosts, difference gel electrophoresis (DIGE) is definitely the method of choice for gel-based studies. In DIGE-based proteomics, the experimental and control samples are labelled with different fluorophores and are run in the same gel, thereby minimising gel-to-gel variations and most importantly, providing high sensitivity (see Fig. 1). Even small quantities of protein in the range of 5 ␮g are sufficient to be visualised (Minden et al., 2009). For MS-based studies, directed and targeted MS methods with high performance instruments in which prior information is used to define sets of peptides to be analysed, will probably become increasingly important. These approaches avoid the identification of proteins with no relevance to the experiment and produce less noisy and more reproducible data as, e.g., reviewed in Domon and Aebersold (2010). Furthermore, appropriate cell separation and protein extraction methods have to be developed and optimised for host–pathogen interaction studies at the proteome level. Nevertheless, the complete characterisation of the proteome remains a tremendous challenge, and the dynamic range needed to obtain a complete proteome has not been achieved for eukaryotic cells, yet. There is also a long road ahead to improve the methods for research on fungal cell wall and membrane proteomes in addition to tools for posttranslational modification (PTM) mapping. The established methods are far from being easy, reproducible, and comprehensive, and standardised protocols are missing. Future progress in the development of more sensitive and refined MS instrumentations with higher speed for collecting fragmentation spectra can be expected. Such improvements will probably lead to an increase of the dynamic range for peptide detection beyond the currently achieved range in protein copy numbers of 103 –104 and would therefore be a big step forward to the complete depiction of a eukaryotic proteome, which has an estimated range of protein copies per cell from 1 to 106 (Mann and Kelleher, 2008). Besides, it will be equally important to improve the software for data acquisition and processing, to complete the proteome databases, and to make the new proteomics technologies more robust and reproducible so that it becomes easier to be used by non-experts. Or to put it in John Yate’s words: “. . . the cutting-edge technological developments in proteomics labs have to disseminate to all levels of the research community. What we need, in short, is the democratisation of mass spectrometry” (Yates, 2005). Moreover, one of the major challenges for the future will be to make use of

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the mass of information that is being collected by transcriptome and proteome studies. Thus, integrative data analyses, computational modelling, and the further development of data warehouses are strongly needed (Albrecht et al., 2010b).

Acknowledgements The proteome analysis in the authors’ laboratory was supported by the Deutsche Forschungsgemeinschaft (Priority Programme 1160), the LSH project MANASP of the European Union, the International Leibniz Research School for Microbial and Biomolecular Interactions Jena as part of the excellence graduate school Jena School for Microbial Communication, and the German-Israeli Foundation for Scientific Research and Development. We thank Daniela Albrecht for the FunCat analysis and the generation of pie charts.

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