Free Access Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary Survival in blood and escape from blood vessels into tissues are essential steps for the yeast Candida albicans to cause systemic infections. To elucidate the influence of blood components on fungal growth, morphology and transcript profile during bloodstream infections, we exposed C. albicans to blood, blood fractions enriched in erythrocytes, polymorphonuclear or mononuclear leukocytes, blood depleted of neutrophils and plasma. C. albicans cells exposed to erythrocytes, mononuclear cells, plasma or blood lacking neutrophils were physiologically active and rapidly switched to filamentous growth. In contrast, the presence of neutrophils arrested C. albicans growth, enhanced the fungal response to overcome nitrogen and carbohydrate starvation, and induced the expression of a large number of genes involved in the oxidative stress response. In particular, SOD5, encoding a glycosylphosphatidylinositol (GPI)-anchored superoxide dismutase localized on the cell surface of C. albicans, was strongly expressed in yeast cells that were associated with neutrophils. Mutants lacking key genes involved in oxidative stress, morphology or virulence had significantly reduced survival rates in blood and the neutrophil fraction, but remained viable for at least 1 h of incubation when exposed to erythrocytes, mononuclear cells, plasma or blood lacking neutrophils. These data suggest that C. albicans genes expressed in blood were predominantly induced in response to neutrophils, and that neutrophils play a key role during C. albicans bloodstream infections. However, C. albicans is equipped with several genes and transcriptional programmes, which may help the fungus to counteract the attack of neutrophils, to escape from the bloodstream and to cause systemic infections. Introduction The majority of nosocomial bloodstream infections are caused by opportunistic pathogens, which are often part of the normal human microbial flora. These microorganisms take advantage of weaknesses of their host, which may have been caused by a depression of the immune system or by intensive medical or surgical treatments, and shift from harmless commensals to become aggressive pathogens. Although bacteria are the predominant microorganisms that cause hospital-acquired infections, the frequency of fungal infections in hospitals has dramatically increased in the past years (Kullberg and Oude Lashof, 2002). Candida species are the agents responsible for almost 80% of nosocomial fungal infections and are the fourth most frequently isolated organisms of all nosocomial disseminated infections. Furthermore, the crude mortality associated with candidaemia is the highest of all bloodstream infections. Despite the emergence of other Candida species as causes of invasive infection, C. albicans remains the leading cause of life-threatening disseminated candidosis (Pfaller et al., 2000). This fungus is polymorphic, with cells developing as yeast, pseudohyphal and true hyphal forms (Berman and Sudbery, 2002). There are two major pathways by which C. albicans may enter the bloodstream: penetration from mucosal surfaces into blood vessels and direct transmission via intravascular catheters (Rex et al., 1994; Velasco et al., 2000; Kullberg and Oude Lashof, 2002). Although several mechanisms and attributes by which C. albicans can colonize and penetrate host tissue have been described (Calderone and Fonzi, 2001), it is not known how this fungus can survive in human blood and escape from blood vessels, both of which are essential steps in the dissemination of infection. Blood is a complex and – to microorganisms – a hostile milieu composed of several types of immunoactive cells and molecules to which C. albicans has to adapt and respond. Furthermore, to cause disseminated infections, the fungal cells need mechanisms to help the cells escape from the bloodstream by invasion of endothelial cells and their surrounding tissues. In a previous report we began to elucidate how C. albicans responds to the challenge of the blood environment (Fradin et al., 2003). To mimic bloodstream infections, human blood was inoculated with C. albicans and fungal gene expression was analysed by transcript profiling with a partial macroarray representing 2002 open reading frames (ORFs) of the fungus. The expression patterns provided evidence that the fungus is able to adapt very quickly to a new environment and that blood cells significantly influence the expression of certain fungal genes. For example, genes involved in the glyoxylate cycle, previously shown to be essential for survival in the phagosome of macrophages (Lorenz and Fink, 2001), were upregulated in blood but not in plasma. Interaction between C. albicans and specific blood cells has been analysed at the cellular level by studies focused on the role of individual host or fungal molecules (Torosantucci et al., 2000; Liu et al., 2001). Only recently have scientists begun to use large-scale transcript profiling methods to investigate how pathogenic microorganisms respond when facing an environment containing phagocytic cells (Lorenz and Fink, 2001; Staudinger et al., 2002; Eriksson et al., 2003). Rubin-Bejerano et al. (2003) have analysed the transcript profile of C. albicans cells that were phagocytosed by isolated neutrophils and monocytes. However, blood cannot be considered as a unicellular compartment and thus one of the major aims of the present study was to determine how different blood components affect the C. albicans gene expression profile. Therefore, we separated the blood into five fractions: enriched in (i) erythrocytes, (ii) polymorphonuclear leukocytes and (iii) mononuclear leukocytes or depleted of (iv) neutrophils or (v) all blood cells (i.e. plasma) and we analysed the impact of these fractions on fungal growth, morphology and virulence. C. albicans microarrays were used to monitor the yeast transcript profile in blood and in the different fractions in order to identify genes that are associated with the infectious process in blood. Furthermore, we investigated the relevance of key regulators of morphology and virulence, virulence genes, a metabolic gene shown to be associated with phagocytosis, and key genes of the oxidative stress response during experimental blood infections. Our observations not only point to fundamental mechanisms by which the innate immune system fights fungal pathogens, but also provide insights into fungal strategies used to counteract host attacks, to escape from the bloodstream and to cause systemic infections. A previous study in our laboratory has demonstrated that blood cells significantly influence the gene expression pattern of C. albicans in human blood (Fradin et al., 2003). To dissect the impact of different blood cells and components on C. albicans transcript profiles in human blood, we isolated three different cellular fractions from fresh human blood: erythrocytes (RC), polymorphonuclear (PMN) cells consisting of neutrophil, eosinophil and basophil granulocytes, and mononuclear cells (MNC) composed of lymphocytes and monocytes. The respective percentage of each type of leukocyte in the blood and in the enriched fractions is listed in Table 1. The different cell fractions were resuspended in plasma purified from the same blood to reconstitute the original concentration of the particular cell type in blood. To determine which blood fractions induced the expression of which C. albicans genes, the cellular fractions and fresh blood were inoculated with C. albicans at a density of 5 × 106 fungal cells per millilitre, which corresponded to a C. albicans : leukocyte ratio of 1 : 1 in blood. As host cells had the strongest influence on fungal gene expression in blood after 30 min of incubation (Fradin et al., 2003), all enriched fractions containing C. albicans were incubated for 30 min. To compare the global influence of blood and its cellular fractions on the C. albicans transcript profile in the absence of host cells, fungal cells were also incubated in plasma. Cells were harvested and shock frozen. The RNA was isolated, amplified and labelled for transcript profiling with cDNA microarrays representing almost the complete genome of C. albicans. The differential regulation of selected genes was confirmed by Northern analysis (Fig. 1). For all experiments, we determined microscopically the morphology of cells in all fractions and monitored whether cells were attached to or phagocytosed by blood cells. Table 1. Proportion of the different leukocytes in blood, blood depleted of CD15+ cells, polymorphonuclear cells (PMN) and mononuclear cells (MNC) fractions in %± standard deviations. The erythrocyte fraction contained   0.5% of the concentration of leukocytes found in blood. Northern blot analysis of selected differentially expressed genes in blood and different blood fractions confirms the expression pattern obtained in the microarray experiments. Approximately equal amounts  of  total  RNA  (5 µg)  from  blood,  RC,  PMN,  MNC,  plasma and blood depleted of CD15+ (blood –) or not depleted of CD15+ (blood +) (see Experimental procedure) incubated with C. albicans for 30 min were electrophoresed, blotted and probed with DIG-labelled PCR-amplified gene fragments for ACT1, RPS10, HYR1, CDR1, ALS1, TRR1, CTA1/CAT1 and ICL1. As observed previously (Fu et al., 2002), ALS1 was transcribed as two mRNA species of different lengths. The quantity of each transcript species seems to vary depending on the conditions investigated. Note that CTA1/CAT1 was more highly expressed in the RC fraction, while TRR1 was more highly expressed in plasma which is in agreement with the array data for these genes (see Table 3). Lane (6) contained RNA from blood prepared using the depletion protocol, but without CD15+ beads to show that the modified expression pattern resulted from the lack of neutrophils, and was not caused by the experimental depletion procedure (see Experimental procedures). Set 2, set 3 and set 5 correspond to the sets shown in Fig. 3. When C. albicans was incubated in blood, cells interacted rapidly with leukocytes. After 30 min of incubation, more than 90% of C. albicans cells were either bound to or ingested by leukocytes (Fradin et al., 2003). At this stage, the fungal cells in blood were mainly found in the yeast form, with only 42.3 ± 2.4% of cells producing germ tubes. Incubation of C. albicans with either erythrocytes, monocytes or plasma induced germ tube formation in 82.6 ± 1.9%, 79.9 ± 1.7% and 85.3 ± 1.9% of the yeasts respectively. In sharp contrast, 96.5 ± 0.7% of C. albicans cells remained in the yeast form in the PMN fraction, in which 38% of fungal cells were phagocytosed by and 57.5% attached to neutrophils after 30 min (Fig. 2A). Germ tubes produced in the blood fractions lacking neutrophils were noticeably longer compared with germ tubes produced in whole blood (5.76 ± 0.57 µm and 3.18 ± 1.1 µm respectively). Survival assays confirmed that the majority of C. albicans cells were still alive after 30 min of incubation in blood and all the blood fractions. A. C. albicans was incubated for 10, 20 and 30 min with neutrophils and the percentage of fungal cells unbound, bound to or phagocytosed by neutrophils was determined.B. The effect of cytochalasin D on the interaction of C. albicans with neutrophils after 30 min of incubation was analysed after pretreatment of neutrophils with 0.5% DMSO alone (–) or 0.5% DMSO containing cytochalasin D (+).C. C. albicans transformed with pACT1-GFP and pSOD5-GFP were incubated for 10, 20 and 30 min with neutrophils and the percentage of fluorescent cells was monitored. D. Neutrophils were pre-incubated for 15 min with (+) or without (–) cytochalasin D and incubated for further 30 min with pACT1-GFP and pSOD5-GFP transformants. In order to study the impact of the different blood components on fungal gene expression, C. albicans microarrays were hybridized with the RNA isolated from whole blood and the blood fraction experiments. To allow a direct comparison of data obtained from each experiment, RNA samples were co-hybridized with RNA isolated from C. albicans cells incubated with erythrocytes. From the 6039 ORFs represented on the microarray, expression of 1518 was shown to be statistically different under at least one of the five conditions tested. Cluster analysis revealed that the transcript profile of C. albicans cells in whole blood was clearly different from cells incubated in plasma, confirming the strong influence of leukocytes on the gene expression of the fungus in blood (Fig. 3A; Fradin et al., 2003). Blood, plasma and blood fractions were isolated from a single healthy donor. In order to show that the major observations made are independent of the blood donor, we compared the transcript profiles of C. albicans cells exposed to blood and plasma from the single donor and pooled donors. Very few differences were observed between profiles of cells exposed to blood or plasma from single and pooled donors (correlation coefficient 0.953 for blood and 0.946 for plasma). However, significant differences were  detected  between  profiles  of  cells  exposed to blood versus cells exposed to plasma [correlation coefficient  0.809  for  blood  (single  donor)  versus  plasma (pooled donors) and 0.827 for blood (single donor) versus plasma (single donor)] (see supplementary material at http://www.pasteur.fr/recherche/unites/Galar_Fungail/data2.html). The five genes found differentially expressed when the two blood samples (single donor versus pooled donors) were compared (ALS3, HXT62, PUT2, STL1 and SUR1) did not affect the interpretation of the further experiments. Cluster analysis of differentially expressed genes in blood and different blood fractions. A. A hierarchical clustering was performed for the 1518 C. albicans genes which showed a statistically different expression in blood, the PMN fraction, the MNC fraction, plasma and/or the RC fraction. The similarity between gene expression is represented by the vertical dendrogram and the similarity between experiments by the horizontal tree. The transcript profile of the same 1518 C. albicans genes expressed in whole blood (CD15+) as compared with genes expressed in blood depleted of CD15+ cells (CD15–) is juxtaposed to the clusters of blood and the cell fractions to illustrate the response of C. albicans to neutrophils. Note that the profile resembles the pattern of genes expressed in blood and the PMN fraction. B. Genes with expression levels statistically different in at least one of the five conditions tested were selected and separated into five sets by k-means clustering of the global transcriptional profile of all fractions shown in (A). The location of these sets within the global pattern were marked by colours and by numbers. For each set, the proportion of regulated genes that are known or predicted to belong to a functional category is shown in a pie chart. The histograms represent the percentage of regulated ORFs that are predicted to belong to subcategories of the functional categories metabolism, energy and cell rescue. When C. albicans was incubated with the red cell fraction, the transcript profile was similar to that of cells in plasma, suggesting that erythrocytes have only a minor influence on C. albicans gene expression in whole blood. Surprisingly, the general expression pattern of fungal cells in the MNC fraction was also very similar to that of C. albicans in the red cell fraction, although the pattern still clustered with the expression pattern of cells exposed to whole blood and the PMN fraction (Fig. 3A). Therefore, the transcript profile of C. albicans in blood seemed to be influenced only to a minor extent by red cells and mononuclear cells after 30 min. In sharp contrast, the gene expression profile of C. albicans in whole blood was very similar to the gene profile of cells exposed to the PMN fraction. As neutrophils represent more than 90% of the PMN fraction, we concluded that neutrophils dominate the transcript profile of C. albicans cells when exposed to whole blood. A k-mean clustering was used to divide the differentially expressed genes into five groups (Fig. 3B) and to identify groups of genes whose expression was associated with the influence of certain blood fractions. Set 1 consists of C. albicans genes predominantly upregulated in whole blood and the RC fraction. Sets 2 and 3 correspond to genes most clearly induced in whole blood due to the influence of neutrophils. Some of these C. albicans genes were also up- (set 3) or downregulated (set 2) in the MNC fraction or plasma, while sets 4 and 5 represent C. albicans genes almost exclusively repressed by neutrophils but differentially expressed in the MNC and plasma fraction. Neutrophils seem to have a dramatic effect on fungal proliferation in blood because these cells strongly inhibit germ tube formation, which is the dominant growth form of C. albicans in plasma. This is consistent with the observation that the transcript level of genes involved in protein synthesis, including genes coding for ribosomal proteins (RPS10, RPL12), translation elongation factors (EFB1, EFT3), or translation initiators (GCD7, GCN3) was dramatically reduced when C. albicans was incubated in the PMN fraction or whole blood as compared with the other blood fractions (Fig. 3; Table 2). We concluded that C. albicans cells exposed to neutrophils downregulate protein synthesis, but not when exposed to plasma, or the erythrocyte or MNC fractions. Expression levels of genes involved in protein synthesis were even more reduced in the PMN fraction as compared with cells in blood. Table 2. Normalized data of selected genes differentially expressed in at least one of the conditions tested. . For blood, PMN, MNC and plasma samples, normalized median signals were compared with signals from the RC fraction. For CD15+/–, fold-regulation corresponds to the normalized median signals of blood samples compared with signals obtained from depleted blood samples. One reason for the repression of proliferation could be a lack of nutrients, such as nitrogen sources and carbohydrates, in the environment surrounding fungal cells. Such starvation would probably be reflected in the expression levels of genes involved in amino acid or carbohydrate metabolism. Numerous genes associated with arginine, leucine, lysine and methionine biosynthesis pathways and GCN4, encoding a transcriptional activator of amino acid biosynthetic genes (Tripathi et al., 2002), were all expressed by C. albicans at higher levels in whole blood and the PMN fraction (Fig. 3B; Table 2). In addition, several genes encoding amino acid transporters, genes involved in general nitrogen metabolism and genes encoding the ammonium permeases Mep2 and Mep3 (known to be induced in nitrogen-poor media; Marini et al., 1997) were induced by neutrophils in whole blood and the PMN fraction. Finally, genes encoding the vacuolar proteases Prb1, Prb2 and Apr1 or the carboxypeptidases Prc1/Cpy1 and Prc2 were also expressed at higher levels in the presence of neutrophils as compared with blood fractions lacking neutrophils. Prb1 and Prb2 and the carboxypeptidases are known to be involved in the recruitment of internal nitrogen sources in Saccharomyces cerevisiae (Zubenko and Jones, 1981). These data suggest that C. albicans cells exposed to neutrophils do not have sufficient access to a nitrogen source. The availability of carbohydrates to C. albicans also seemed to be reduced in the presence of neutrophils, as the genes encoding the key enzymes of the glyoxylate cycle (MLS1, ICL1, ACS1) were strongly upregulated (Fig. 3; Table 2). This was also true for members of the so-far-undescribed FRP gene family of C. albicans. These genes are related to GPR1 of Yarrowia lipolytica which is required for growth on acetate as carbon source (Augstein et al., 2003). At the same time, genes involved in glycolysis were downregulated when exposed to PMN cells. Reactive oxygen species are potential anti-microbial agents produced by phagocytic cells to kill microbes. H2O2 is the key oxidizing agent produced by immunoactive cells which generates other extremely toxic radicals that are active against microorganisms. To detoxify these components, pathogenic microorganisms need a number of enzymes that catalyse the transformation from reactive oxygen species via H2O2 to water. We observed that C. albicans cells confronted with neutrophils showed up to a sevenfold upregulation of a large number of genes (summarized in Table 3) likely to be involved in the detoxification of oxidative species as compared with cells incubated in  the  red  cell  fraction  or  plasma.  These  data  suggest  that generation of reactive oxygen species is a central mechanism  of  neutrophils  against C.  albicans  and  that C. albicans expresses a large set of genes that have the potential to counteract oxidative stress. Table 3. Upregulation of genes likely to be involved in the detoxification of oxidative species in C. albicans cells confronted with neutrophils as compared with cells incubated in other blood fractions or plasma (normalized data). By comparing the transcript profiles of C. albicans exposed to whole blood or the different cellular fractions or plasma, we concluded that neutrophils are the dominant blood cells that trigger a strong, adaptive gene response in C. albicans in human blood. To confirm the impact of neutrophils on the transcript profile of C. albicans, we partially depleted human blood of neutrophils with magnetic beads coated with antibody raised against CD15 antigen. This antigen is present on the surface of neutrophils, eosinophils and, to a varying degree, on monocytes. Treatment of whole blood with anti-CD15 beads removed 94.7% of all neutrophils (Table 1). C. albicans cells (5 × 106 ml−1) were incubated in blood depleted and blood not depleted of neutrophils for 30 min and the two transcript profiles were compared. In blood depleted of neutrophils, the induction of genes involved in protein synthesis, glycolysis and hypha-specific genes were upregulated compared with cells exposed to whole blood, confirming that these genes are repressed by neutrophils (Fig. 3; Table 2). Similarly, genes associated with the glyoxylate cycle, nitrogen and amino acid metabolism and the anti-oxidative response were expressed at higher levels by fungi in non-depleted blood (Tables 2 and 3). Therefore, these data confirm that neutrophils are responsible for the starvation and oxidative stress response in C. albicans when exposed to human blood. It should be noted that fungal cells incubated with MNC also expressed to higher level certain genes involved in nitrogen metabolism, the glyoxylate cycle and the anti-oxidative response. However, the expression of these genes was significantly lower than for C. albicans cells exposed to neutrophils (Tables 2 and 3). As described above, the majority of fungal cells were arrested in the yeast form after incubation with neutrophils. Consistent with this observation, most of the known hypha-specific genes, such as SAP4–6, HYR1, ECE1 and ALS3, were repressed in the PMN fractions. However, not all genes known to be hypha-associated in vitro were in fact downregulated in the PMN fraction. For example, the putative GPI-anchored superoxide dismutase gene SOD5 identical to IPF1222 in CandidaDB (http://genolist.pasteur.fr/CandidaDB/) and the stress gene DDR48 were shown to be hypha-associated (Lane et al., 2001; Nantel et al., 2002; Fradin et al., 2003). However, both SOD5 and DDR48 were not repressed, but rather induced in response to oxidative or general stress in the PMN fraction (Tables 2 and 3). This may also explain the surprising observation that SOD5 and DDR48 were more highly expressed by C. albicans in untreated whole blood as compared with blood depleted of neutrophils. To confirm that SOD5 is indeed regulated both during germ tube formation and in yeast cells in contact with neutrophils, we fused a green fluorescent protein (GFP) reporter gene to the promoter of the SOD5 gene. As expected, fluorescence of the GFP reporter was not detected in culture media that supported yeast growth, but was expressed in hypha-inducing medium (Fig. 4A). Furthermore, the expression of the GFP reporter was induced in the presence of PMN cells. Under these conditions, the GFP reporter was expressed in yeast cells phagocytosed by or attached to neutrophils (Fig. 4B and D). To analyse the dynamics of expression of SOD5, C. albicans cells were incubated with neutrophils for 10, 20 and 30 min and the number of cells inducing GFP expression was monitored. Almost no cells showed expression of GFP after 10 min, whereas 70.5% of C. albicans cells did express the GFP reporter after 30 min (Figs 2C and 4). As 23% of cells transformed with pACT1-GFP also showed no fluorescence after 30 min of incubation with neutrophils (Fig. 2C), we concluded that these cells were physiologically inactive or dead. SOD5 expression is induced in hyphal cells in culture medium or in yeast cells exposed to neutrophils. Expression was monitored using GFP as a reporter. C. albicans strain CAI-4 transformed with pACT1-GFP, pSOD5-GFP and pGFP plasmids was incubated under yeast- (SD, YPD) or hypha- (FCS) inducing conditions (A), with PMN cells for 30 min in plasma (B and D) or with PMN cells pretreated with cytochalasin D (C). For each sample, a micrograph of phase contrast and a GFP fluorescence micrograph are shown. Cells carrying pACT1-GFP show constitutive expression of GFP. Cells carrying promoterless GFP show no fluorescence. Cells containing pSOD5-GFP express GFP in hyphal cells (A, FCS-induced) or after interaction with neutrophils (B, middle, and D, middle). Staining with the vital dye trypan blue can discriminate between phagocytosed and attached C. albicans cells: non-phagocytosed C. albicans cells are stained red (D). Note that both stained and non-stained cells express SOD5. Cytochalasin D treatment represses SOD5 expression (C). Although our results demonstrate that neutrophils modify the transcriptional profile of C. albicans in human blood, it is not clear whether this occurs via intra- or extracellular activities of neutrophils. As mentioned above, 38% of all fungal cells were phagocytosed by and 57.5% attached to neutrophils in the PMN fraction. However, most of these cells showed SOD5 expression (as monitored by GFP fluorescence) and were arrested in the yeast form. These results suggest that the observed effects resulted from both intra- and extracellular activities of neutrophils. Cytochalasin D, an inhibitor of actin polymerization, added at concentrations which inhibited phagocytosis but did not kill cells, not only blocked phagocytosis, but also repressed SOD5 expression (Figs 2D and 4C). Furthermore, most C. albicans cells incubated with neutrophils in the presence of cytochalasin D started to produce hyphae after 30 min of incubation indicating a role of actin in these processes. To further investigate the impact of extracellular compounds of neutrophils on C. albicans morphology, we incubated: (i) C. albicans and neutrophils together, (ii) C. albicans alone and (iii) neutrophils alone in plasma, removed cells by centrifugation and exposed C. albicans to the different supernatants. None of the supernatants inhibited the yeast-to-hypha transition, suggesting that the effect of neutrophils on extracellularly bound C. albicans depended on locally produced substances. In silico analysis of the deduced amino acid sequence of SOD5 (IPF1222) using PSORT (prediction of protein sorting signals and localization sites in amino acid sequences) (http://psort.nibb.ac.jp/), SIGFIND (signal peptide prediction server) (http://www.stepc.gr/~synaptic/sigfind.html) and DGPI (automatic detection of GPI-anchored protein) (http://129.194.185.165/dgpi/index_en.html) revealed a signal peptide corresponding to the first 15 amino acids and a putative ω cleavage site for GPI anchoring at amino acid 205. No basic residues were detected immediately upstream of amino acid 205, suggesting that Sod5 is probably incorporated into the cell wall (Caro et al., 1997; Vossen et al., 1997). To investigate whether Sod5 is in fact GPI-anchored and localized on the cellular surface, we used two different experimental approaches. First, a Sod5/GFP fusion protein cassette (pACT1-Sod5/GFP) was designed to obtain a fusion protein containing the first 16 amino acids of Sod5, the GFP protein and the last 38 amino acids of Sod5. The cassette was driven by the ACT1 promoter to avoid restricted expression condition of the protein fusion. Cells were transformed with pACT1-Sod5/GFP, pACT1-GFP or pGFP, and purified cell fractions of transformants were treated with GFP antiserum to immunolocalize the GFP protein. As expected, GFP expressed by pACT1-GFP transformants was localized in the cytoplasm of the cells. In contrast, GFP antigen expressed by C. albicans cells containing the pACT1-Sod5/GFP cassette was detected in the membrane and cell wall extracts (Fig. 5A). Localization of Sod5/GFP on the cell surface of transformants carrying pACT1-Sod5/GFP was further confirmed by fluorescent microscopy and immune electron microscopy (Fig. 5B and C). Second, we extracted cell walls from hyphal cells, treated the cell walls with HF-pyridine and analysed the released GPI-anchored proteins by mass spectrometry. HF-pyridine is known to specifically cleave phosphodiester bonds through which GPI proteins are linked to β-1,6-glucan chains (Groot et al., 2004). We detected two peptides whose sequences were identical with Sod5 peptides (Table 4). These data confirm the in silico prediction that Sod5 is a GPI-anchored cell wall protein of C. albicans. Sod5 is a GPI-modified cell wall protein. A. C. albicans CAI-4 cells transformed with pGFP (1), pACT1-GFP (2) and pACT1-Sod5/GFP (3) were lysed (total extract) and fractionated by centrifugation (13 000 g). The different samples were analysed by Western blotting using GFP antiserum. An isotype control showed no cross-reactivity. B. pACT1-GFP and pACT1-Sod5/GFP transformants were also visualized with a fluorescent microscope. C. Electron microscopy and post-embedding immunogold labelling of cells expressing pACT1-Sod5/GFP. Note that the fusion protein is predominantly located on the surface (arrows). Localization of the fusion protein was also found in vesicles (star) that are possibly part of the secretory pathway. No specific gold labelling was seen in control experiments without the addition of the GFP antibody and in the pGFP control. Table 4. Mass spectrometric identification of Sod5 in HF-pyridine-released cell wall extract. Candida albicans mutants lacking key proteins involved in virulence and oxidative stress response are hypersensitive to neutrophils The transcript profiling of C. albicans exposed to whole blood and blood fractions described above revealed that certain genes are upregulated in response to neutrophils. Furthermore, fungal morphology seems to play an important role during the C. albicans/host cell interactions in blood. To elucidate the relevance of selected upregulated genes and genes known to be key factors in the morphological transition or stress response, we studied the survival of C. albicans mutants lacking these genes when exposed to whole blood and blood fractions (Fig. 6). Survival assay of C. albicans wild-type (WT) and mutant strains lacking the superoxide dismutase Sod5, the proteinases Sap4–6, the isocitrate lyase Icl1, and the transcriptional regulators Efg1 or Cph1 exposed to blood cells and blood fractions. CAI-4 (WT, Ura+), Δsod5, Δsod5/SOD5, Δsap4–6, Δcap1, Δicl1, Δefg1 and Δcph1 strains were inoculated in blood, plasma and PMN and MNC fractions for 1 h at 37°C. Percentage survival for each strain in blood and PMN and MNC fractions was determined as follows: (cfu/cfuplasma) × 100. Values shown are the means and standard errors for three separate experiments (*, P   0.05, determined for each measurement compared with the wild type; ♯, P   0.05, determined for Δsod5 measurement compared with Δsod5/SOD5).CPH1 and EFG1 are known virulence regulators encoding key transcriptional factors of the dimorphic transition (Liu et al., 1994; Stoldt et al., 1997). When mutants lacking either EFG1 or CPH1 were exposed to blood, we monitored a markedly reduced survival of both mutants (Fig. 6). SAP4–6 are genes encoding hypha-associated secreted aspartic proteinases which are known to be important for systemic infections, tissue invasion and survival in macrophages (Sanglard et al., 1997; Borg-von Zepelin et al., 1998; Felk et al., 2002). However, a mutant lacking all three proteinases was still able to survive contact with neutrophils in blood and the PMN fraction to an extent similar to wild-type cells. One of the key genes of the glyoxylate cycle, ICL1, was shown to be induced in both macrophages (Lorenz and Fink, 2001) and neutrophils (this study) suggesting a general role of this gene for survival after phagocytosis. However, mutants lacking ICL1 did not have reduced survival rates as compared with wild-type cells in our survival assay. CAP1, a gene encoding a key transcriptional regulator of oxidative stress response (Alarco and Raymond, 1999), was expressed during contact with neutrophils in the PMN fraction. A mutant lacking CAP1 had significantly reduced viability when exposed to whole blood or the PMN fraction (Fig. 6). SOD5 is regulated by both the yeast-to-hypha transition and exposure to neutrophils (see above). Using a GFP reporter we showed that a direct interaction between C. albicans and neutrophils is essential for the induction of SOD5 in yeast cells. Furthermore, the fact that Sod5 is exposed to the cell surface may suggest that this superoxide dismutase may act to detoxify exogenous reactive oxygen species. To study the relevance of SOD5 for survival in blood and during C. albicans/neutrophil interactions, we produced a mutant lacking this gene. Δsod5 was moderately but significantly attenuated in terms of mouse survival after intravenous challenge (data not shown). The same mutant had a significantly reduced ability to survive both incubation in whole blood and the PMN fraction, but showed no differences in viability in all other blood fractions as compared with the wild type (Fig. 6). Restoration of the native SOD5 gene into the Δsod5 mutant restored the ability to resist the attack of neutrophils suggesting that SOD5 plays a crucial role for survival in blood (Fig. 6). Candida albicans is an extremely successful pathogen which normally exists as a harmless human commensal, but may cause systemic or disseminated infection in immunocompromised patients and patients with central venous catheters. Therefore, C. albicans must have attributes and mechanisms by which the cells may enter and survive in the blood and penetrate from blood vessels into visceral tissues as essential steps during development of these life-threatening infections. Blood contains different components, cellular and soluble, that have the potential to affect C. albicans at different levels. Plasma contains a large number of immunoactive and anti-microbial proteins and peptides with bactericidal activities, including the entire complement system. The main function of erythrocytes is the transport of oxygen and carbon dioxide, but PMN and MNC fractions include phagocytic cells with the potential to kill microbes. Surprisingly, neither plasma components alone, nor mononuclear cells, seem to play a significant role in the immediate defence against C. albicans in whole blood, as the viability and growth of the fungus is not reduced in these fractions, and the transcript profile suggests that these fungal cells are in fact physiologically active. Several lines of evidence suggest that neutrophils dominate the influence on C. albicans gene expression in blood (Fig. 7). Global cluster analysis revealed that the expression of C. albicans in whole blood was very similar to the pattern of cells exposed to the PMN fraction. As neutrophils represent more than 90% of the PMN fraction, we concluded that these cells dominate the transcript profiling of C. albicans in blood. Furthermore, in neutrophil-depleted blood, C. albicans cells had an expression pattern which resembled the profile of cells exposed to plasma, rather than cells exposed to the PMN fraction. Influence of blood and blood cell fractions on the growth and gene expression pattern of C. albicans. Cells exposed to blood fractions containing no neutrophils were physiologically active, produced hyphae and showed a transcript profile resembling that of fungi grown in culture media. In contrast, neutrophils arrested cell growth, enhanced a fungal response to overcome nitrogen and carbohydrate starvation and induced the expression of genes involved in oxidative stress response. Cells incubated in blood showed an intermediate growth with shorter hyphal cells and an expression pattern which reflects two populations of cells: those in close contact or phagocytosed by neutrophils and those not attached to neutrophils. Clearly, neutrophils dominate the viability, morphology and transcript profile of C. albicans in several ways: (i) arrest of growth and inhibition of hypha formation, (ii) oxidative stress, (iii) carbohydrate starvation, (iv) nitrogen starvation and (v) anti-microbial peptides. Inhibition of hypha formation and amino acid starvation in blood may predominantly occur in cells which are phagocytosed by neutrophils as recently observed by Rubin-Bejerano et al. (2003), who investigated the transcriptional profile of S. cerevisiae and C. albicans exposed to purified neutrophils and monocytes and concluded that the neutrophil phagosome, but not the monocyte phagosome, is amino acid deficient. However, as discussed below, it is not only phagocytosis by neutrophils which inhibits hypha formation in blood. The yeast-to-hypha transition is regulated by a number of environmental conditions such as pH, temperature or oxygen content or by specific inducers (Brown and Gow, 1999). For example, as soon as C. albicans is exposed to plasma, the transcriptional programme is activated which modulates the transition from yeast to hyphal cells. Several known genes encoding hypha-associated factors, including HWP1, HYR1, ALS3, SAP4–6, SOD5 and DDR48, were co-regulated with hypha formation when cells were incubated in plasma (Fradin et al., 2003). Hypha formation and hypha-associated factors seem to have several functions for C. albicans at the different stages of infection (including commensal growth), but some of them seem to be particularly advantageous for the fungus when entering the bloodstream. First, factors secreted from hyphal cells have the potential to inhibit killing by neutrophils (Smail et al., 1992). Second, hypha formation may help the fungus escape from monocytes after phagocytosis (Borg-von Zepelin et al., 1998; Lorenz and Fink, 2001). Hypha-associated factors such as the secreted proteinases Sap4–6 are crucial for this process (Borg-von Zepelin et al., 1998). Third, hyphal cells have stronger adherence properties that may help the fungus to adhere to endothelial cells (Rotrosen et al., 1985) and hyphal cells, but not yeast cells, have been shown to induce phagocytosis by endothelial cells, a mechanism which is discussed as a potential strategy of C. albicans to escape from the bloodstream (Zink et al., 1996; Phan et al., 2000). Finally, hyphal cells are known to have greater invasive properties in tissue which would assist the fungus in penetrating into the surrounding tissue of blood vessels (Calderone and Fonzi, 2001). These functions are likely to be supported by both physical forces and hypha-associated factors. However, hypha formation is inhibited in blood as opposed to plasma, suggesting that certain blood cells repress hypha formation to some extent. Erythrocytes and mononuclear cells are unlikely causes of this repression as, as in plasma, almost 80% of C. albicans cells formed germ tubes in the presence of these blood cells. In contrast, polymorphonuclear cells seem to inhibit this transition completely and consequently reduce the expression of most hypha-associated genes. For example, expression of SAP4–6, which has been associated with survival in macrophages (see above), was reduced. This is in agreement with the observation that the survival rate of the Δsap4–6 mutant was similar to the wild type in blood or the PMN fraction. As the same mutant was strongly attenuated in systemic infection of mice (Sanglard et al., 1997), it can be concluded that these genes are crucial for survival in macrophages, but not neutrophils, and at later stages of systemic infection when cells invade tissues (Borg-von Zepelin et al., 1998; Felk et al., 2002). The fact that the Δefg1 mutant, lacking a key regulator of the yeast-to-hypha transition, whose ability to produce hyphal cells is completely blocked in plasma, has reduced abilities to survive in blood, further supports the view that hypha formation and/or associated factors regulated by Efg1 are important for survival in blood. Similarly, the reduced survival of the Δcph1 mutant can be explained by a Cph1-dependent regulation of factors which are crucial for the interaction with neutrophils. This inhibition of hypha formation can be correlated with the dramatic decrease of mRNA levels of genes involved in protein production of C. albicans cells incubated in  the PMN fraction. Clearly, neutrophils also reduced the expression of these genes and inhibited hypha transition of C. albicans in blood. However, a notably higher number (42.3%) of C. albicans cells produced hyphal forms in blood as compared with the PMN fraction even though these filaments were considerably shorter compared with hyphal cells incubated in plasma, red cells or MNC fractions. Similarly, C. albicans genes involved in protein synthesis were less repressed in blood compared with the PMN fraction. Therefore, it can be concluded that certain mechanisms or factors in blood counteract the complete repression of hyphae by neutrophils. The oxidative burst of neutrophils is essential for killing many microorganisms (Hampton et al., 1998) and is likely to add to the efficient killing of C. albicans by neutrophils. However, C. albicans is not defenceless to the attack of reactive oxidative species. C. albicans genes encoding a cytoplasmic superoxide dismutase, a catalase, the glutathione peroxidase/glutathione reductase complex and the thioredoxin peroxidase/thioredoxin reductase complex were up to sevenfold upregulated in the presence of neutrophils. Three out of the 18 antioxidant genes identified in our study were also found to be induced by C. albicans in response to neutrophils by Rubin-Bejerano et al. (2003). In addition, several of these genes (such as TTR1, TRX1, CTA1, CAP1) were identified by Enjalbert et al. (2003) who investigated the transcriptional profile of C. albicans exposed to oxidative stress. This suggests that reactive oxygen species produced by neutrophils act on C. albicans. The expression of the cell surface-associated superoxide dismutase gene SOD5 was not repressed in the PMN fraction (where C. albicans cells were arrested in the yeast form), although SOD5 was shown to be upregulated during the yeast-to-hypha transition (Nantel et al., 2002; Fradin et al., 2003). Furthermore, this gene was expressed more in C. albicans exposed to blood than in blood depleted of neutrophils. SOD5 belongs to a family of six genes encoding superoxide dismutases (SOD1–6) in C. albicans. While SOD1–3 encode internal superoxide dismutases (Hwang et al., 1999; Lamarre et al., 2001), the  gene  products  of SOD4–6  (Sod4,  Sod5  and Sod6) are putative cell surface-located GPI-anchored proteins (http://genolist.pasteur.fr/CandidaDB/, Nantel et al., 2002; De Groot et al., 2003). In this study, we used a Sod5/GFP fusion protein to show that Sod5 is localized on the cell surface of C. albicans. Furthermore, mass spectrometry analysis of HF-pyridine-released cell wall proteins demonstrated that (i) Sod5 is associated with the cell wall and (ii) this association to the cell wall is mediated by a GPI anchor. The existence of cell surface-associated superoxide dismutases in C. albicans may suggest that these enzymes are involved in external oxidative stress rather than the detoxification of internal oxidative species. Mutants lacking SOD5 were more sensitive to neutrophils confirming the importance of this gene for the survival of C. albicans to external oxidative stress. In contrast, SOD5 was not important for the survival of the fungus in macrophages (Martchenko et al., 2004). This observation is not surprising as neutrophils and macrophages are known to have different defence mechanisms against C. albicans (Lehrer et al., 1975; Diamond et al., 1980; Diamond and Haudenschild, 1981). It may be possible that superoxide anions are important for killing of C. albicans by neutrophils, but not by macrophages. Interestingly, we observed that SOD4 was upregulated in the Δsod5 mutant in blood, suggesting that C. albicans may compensate to some extent for the deletion of an important SOD gene by the upregulation of alternative SOD genes. However, SOD4 was expressed at significantly lower levels compared with SOD5. Compensation by other SOD genes may explain why the Δsod5 mutant was only moderately attenuated in the murine model of systemic candidiasis. Following contact with neutrophils, a significant portion of C. albicans cells are phagocytosed. Phagocytosis and killing of an ingested microorganism by neutrophils is a very complex mechanism involving oxidative and non-oxidative agents that act in concert or independently of each other. The phagosome of neutrophils constitutes an extremely hostile environment with an acidic or alkaline pH, anti-microbial peptides, reactive oxidative species and relatively nutrient-poor conditions (Hampton et al., 1998; Faurschou and Borregaard, 2003). Rubin-Bejerano et al. (2003) have demonstrated that phagocytosis of C. albicans by neutrophils induces a fungal amino acid starvation response. Our present study confirms that genes involved in the regulation, biosynthesis and transport of amino acids were induced in response to neutrophils, but also show that C. albicans in fact faces a more general nitrogen deprivation. For example, genes encoding vacuolar proteases, possibly involved in the recruitment of internal nitrogen sources, were expressed more in the presence of neutrophils as compared with fractions lacking neutrophils. Furthermore, genes encoding ammonium transporters were upregulated in blood and the PMN fraction. It is not clear whether neutrophils actively produce a nitrogen-poor environment or block uptake of nitrogen. However, as nitrogen starvation is known to enhance hypha formation (Tripathi et al., 2002), it is less likely that this starvation is responsible for the observed growth arrest. Furthermore, mononuclear cells which did not repress hypha formation by C. albicans also induced a nitrogen starvation response to some extent. Similarly, C. albicans cells exposed to neutrophils seem to face a carbohydrate-poor environment which is reflected by a strong upregulation of genes encoding key enzymes of the glyoxylate cycle. Activation of the glyoxylate cycle may compensate for the lack of carbohydrate in the phagosome as was shown for C. albicans cells after phagocytosis by macrophages (Lorenz and Fink, 2001). However, the observation that the survival rate of the Δicl1 mutant was similar in blood and the PMN fraction compared with the wild type suggests that carbohydrate starvation was unlikely to be the main reason for the cell growth inhibition caused by neutrophils. Although the switch to a starvation growth mode is caused by exposure to neutrophils, it is not clear whether phagocytosis is essential for the observed transcriptional response, because only 38% of the cells in the PMN fraction were in fact phagocytosed by 30 min. We conclude that either the starvation response resulted from a strong gene expression of phagocytosed cells or non-phagocytosed cells in contact with neutrophils may also face nutrient deprivation. It is also theoretically possible that the phagosome or the neutrophil local environment may in fact contain sufficient glucose and nitrogen sources and that the neutrophils have developed mechanisms to block the sensing of glucose or nitrogen sources which would signal to the cells that no nutrients are available and thus induce the glyoxylate cycle or a nitrogen starvation response. Clearly, our results suggest that neutrophils also act extracellularly on C. albicans in human blood. For example, the SOD5 expression pattern of individual cells suggest that oxygen species were produced intracellular (in the phagosome) and released to, or produced on, the cell surface. This is in agreement with several other studies that demonstrated extracellular release of oxygen metabolites upon contact with microbes (Hampton et al., 1998). Furthermore, the vast majority of C. albicans cells attached to neutrophils were blocked in the yeast form, suggesting that locally produced extracellular compounds of neutrophils caused growth arrest. However, when C. albicans cells were incubated in supernatants from neutrophil co-cultures, the yeast-to-hypha transition was not inhibited. This suggests that close contact between C. albicans and neutrophils is crucial for inhibition of the transition. One possible explanation for this observation may be that high concentrations of extracellular compounds are essential for their effect. Such a view is in agreement with the recent discovery of ‘Neutrophil Extracellular Traps’ (NETs) (Brinkmann et al., 2004), consisting of extracellular fibres produced by neutrophils which bind microbes and may keep anti-microbial compounds from diffusing away. Interestingly, cytochalasin D not only blocked phagocytosis, but also repressed a possibly actin cytoskeleton-dependent release of Sod5 and repressed the yeast-to-hypha transition. Similarly, it has been shown that the extracellular oxidative burst of professional phagocytes induced by Helicobacter pylori was inhibited by cytochalasin D (Ramarao et al., 2000). In addition to the secretion of oxidative species, neutrophils have other potent anti-microbial properties (Henson et al., 1988) which probably also act on C. albicans. For example, granules released to the phagosome and the surrounding area contain a number of anti-microbial agents such as proteinases and lactoferrin. Lactoferrin is known to inhibit growth and germ tube formation in C. albicans (Okutomi et al., 1997; Wakabayashi et al., 1998) and thus probably contributes to yeast cell arrest of C. albicans after phagocytosis and to the reduced germ tube formation in blood. Although the C. albicans : neutrophil ratio was identical in the PMN fraction and in blood, we observed that fungal cells were physiologically more active in blood and that the survival rate of C. albicans in blood was markedly higher (56%) compared with cells exposed to the PMN fraction (37%) after 1 h of incubation. This may be explained by the fact that C. albicans cells in blood also have contact with erythrocytes or monocytes and lymphocytes and are to some extent able to produce hyphal forms which may inhibit phagocytosis and/or reduce killing by neutrophils. This observation was valuable in terms of understanding the pathogenesis of infections by C. albicans (and other pathogenic microbes) as it shows the importance of studying a model of infection in the presence of all of the host protagonists. Even when phagocytosed by monocytes the survival rate of C. albicans was almost identical to C. albicans exposed to plasma. Therefore, monocytes ingest C. albicans, but the fungus can survive, which also may explain the higher survival rate in blood. Furthermore, reduced survival of mutants lacking Sod5, Cap1 or Efg1 in the PMN fraction shows that C. albicans is not defenceless in the presence of neutrophils. In summary, our data suggest that neutrophils play a key role in bloodstream infections with C. albicans. This observation is in line with the high susceptibility of neutropenic patients to disseminated candidosis. However, we also show that C. albicans is equipped with a large set of genes and transcriptional programmes which may help the fungus to avoid contact with neutrophils, to counteract the attack of neutrophils and to escape from the bloodstream. These factors may be of key importance during intravascular catheter-related candidemia in patients without neutropenia or major immunodeficiency. Candida albicans SC5314 (Gillum et al., 1984) or CAI-4 carrying CIp10 (see below) were used as wild-type strains in all experiments. Mutants lacking SAP4–6, CAP1, ICL1, EFG1 and CPH1 genes and strains CAI-4 or CAF4-2 (derived from SC5314) (Fonzi and Irwin, 1993) were obtained from Dominique Sanglard, Alistair Brown, Mike Lorenz, Joachim Ernst and Gerry Fink. A possible URA3 positional effect (Brand et al., 2004) has not been systematically tested for these mutants. Human blood was collected from a single healthy donor as described previously (Fradin et al., 2003). Red blood cells, polymorphonuclear cells and mononuclear cells were isolated from whole human blood by gradient density centrifugation in Histopaque 1077 and Histopaque 1119 (Sigma) according to the manufacturer\'s instructions. The PMN cells were incubated for 7 min at 37°C in 0.83% (w/v) NH4Cl, 10 mM Hepes-NaOH, pH 7.0 to lyse residual red cells. The different blood cells were resuspended in plasma prepared as described previously (Fradin et al., 2003) by reconstituting their original concentration in blood. The quality of the purification of each fraction was verified by staining slides containing cells of each fraction with May-Grünwald-Giemsa and microscopy examination. To compare the transcriptional profile from C. albicans exposed to blood and plasma from the single donor with blood and plasma from a pool of donors, we collected blood from six further healthy blood donors (three male, three female). CD15+ cells were depleted from fresh human blood with Dynabeads M-450 CD15 (Dynal) according to the manufacturer\'s instructions, except that blood was not diluted. To remove CD15+ cells, 2 × 107 beads in 50 µl of PBS, 0.1% bovine serum albumin, were used per millilitre of blood. In a control experiment, the same volume of blood was incubated with buffer only. Candida albicans strain SC5314 was grown overnight in YPD medium (1% yeast extract, 2% Bacto-peptone and 2% glucose) at 37°C. The cells were washed once and suspended in PBS. Candida cells were inoculated in whole blood, plasma or blood depleted of CD15+ cells and in the three different blood fractions and incubated for 30 min at 37°C. The final C. albicans:leukocyte ratios were: whole blood 1:1, neutrophil-depleted blood 2.4:1, PMN fraction 1.5:1, MNC fraction 3:1. Cells were collected by centrifugation at 4°C for 2 min, and the pellets shock frozen in liquid nitrogen. Cells of the different co-cultures were visualized microscopically and the relative length of the germ tubes formed in plasma and blood measured using the inverted microscope Axiowert 200 and the AxioVision 3.1 software (Zeiss). Frozen cells were lysed and homogenized by vortexing in PeqGOLD RNApure reagent (Peqlab) with acid-washed glass beads (0.4–0.6 mm; B. Braun Biotech International) for 20 min. Total RNA was extracted and mRNA isolated as described previously (Fradin et al., 2003). Cy3- and Cy5-labelled cRNA were prepared from 5 µg of mRNA by use of the Agilent fluorescent linear amplification kit and protocol (Agilent technologies). For transcript profiling we used C. albicans microarrays (Eurogentec) containing 6039 open reading frames and  27  control  genes  spotted  in  duplicate  on  glass  slides.  The C. albicans microarray has been developed in collaboration with the European Galar Fungail Consortium (http://www.pasteur.fr/recherche/unites/Galar_Fungail/). The Stanford Genome Technology Center generated the nucleotide sequence data for C. albicans with funding from the NIDCR, NIH and the Burroughs Wellcome Fund. Information about coding sequences and proteins was obtained from the CandidaDB database (http://genolist.pasteur.fr/CandidaDB/). Arrays were designed as described under http://www.pasteur.fr/recherche/unites/Galar_fungail/arrays.html. Two micrograms of the Cy3 and Cy5 probes were hybridized to the C. albicans arrays overnight at 42°C in DIG Easy Hyb solution (Roche). The blood, plasma, PMN and MNC samples were all co-hybridized with the RC sample. The slides were washed at room temperature for 5 min in 2× SSC, 0.06% SDS, for 5 min in 0.4× SSC and for 5 min in 0.1× SSC and dried by centrifugation for 7 min. Hybridized slides were scanned with an Axon 4000B scanner at a 10 µm resolution. Data were extracted by GenePix 4.1 software (Axon). An intensity-dependent data normalization (LOWESS) was performed in GeneSpring 6.0. The different sets of data were compared with each other by one-way analysis of variance (anova) test with a P-value cut-off of 0.05 for genes that had an intensity in both channels higher than 100. Each gene that passed this test and showed 1.5-fold changes in two experiments was defined as differentially expressed. The data presented correspond to the average of three biological replicates including a dye swap. The complete transcriptional profiling data are available at http://www.pasteur.fr/recherche/unites/Galar_Fungail/array_data.html. The differentially expressed genes were organized in GeneSpring by a hierarchical clustering algorithm (Eisen et al., 1998) based on the standard correlation. These regulated genes were also divided into five groups after k-means clustering. Northern analysis (Fradin et al., 2003) was used to confirm the expression pattern of selected genes in blood, plasma and each blood fraction. Equal loading and quality of each blot was verified by ethidium bromide staining of the rRNA bands. To construct a pSOD5-GFP gene fusion, the promoter region of SOD5 (IPF1222) was polymerase chain reaction (PCR) amplified with the primer pair IPF1222P1 (5′-TCCGCTC GAGAGAAATGATGAAAACCTGGT-3′) and IPF1222P2 (5′-CATACCAAGCTTGATGAATGGTAAGTTAGA-3′) containing XhoI and HindIII sites respectively (underlined). This PCR fragment was digested by XhoI and HindIII and used to replace the ACT1 promoter in the pACT1-GFP plasmid kindly provided by C. Barelle and A. Brown (Barelle et al., 2004). A plasmid containing GFP without a promoter was obtained by digesting pACT1-GFP with XhoI and HindIII to remove the ACT1 promoter. Cohesive ends were filled with Klenow fragment and blunt end ligated. C. albicans strain CAI-4 was transformed (Gietz and Woods, 2001) with either StuI-linearized pSOD5-GFP, pACT1-GFP or pGFP. A single integration of these plasmids at the RPS10 locus in the same allele was confirmed by Southern blot analysis. Transformants with integrated plasmids were incubated in different media (YPD, SD and FCS) and with the PMN fraction for 10, 20 and 30 min at 37°C. Cells were centrifuged and mounted in mounting fluid to be visualized under a Axiowert 200 fluorescent microscope (Zeiss). To inhibit phagocytosis, the PMN fraction was incubated for 15 min at 37°C with 10 µg ml−1 cytochalasin D (stock solution in DMSO) before the addition of the different C. albicans transformants. As a control, PMN cells were also pre-incubated with DMSO alone. Cells from the different co-cultures were fixed on glass coverslips overnight at 4°C in 4% formalin, stained with May-Grünwald-Giemsa and used for microscopic examinations. The percentage of C. albicans unbound, bound to or phagocytosed by neutrophils was determined for the three different time-course. Furthermore, the phagocytosis rate of C. albicans by neutrophils was monitored by using a modified method of Busetto et al. (2004). For this study, the pACT1-GFP transformants incubated with the PMN fraction were mounted with a mounting solution containing trypan blue and the cells were visualized under a fluorescent microscope. The percentage of green/red (non-phagocytosed cells) and only green (phagocytosed cells) fluorescent yeast cells were then determined. The same experiment was performed using SC5314 and the percentage of red fluorescent cells (non-phagocytosed cells) and non-fluorescent cells (phagocytosed cells) were monitored. To construct pACT1-Sod5/GFP, GFP was fused to a 5′ and a 3′ fragment of SOD5. First, SOD5 (bp 1 to +439) was cloned into pCR2.1-TOPO vector and a reverse PCR product of this vector [using primers IPF1222Fus1 (5′-GCATGC AACACTTCTTCTAGTATGGCTTCT-3′) and IPF1222Fus2 (5′-CTGCAGACCAGCCAAAGCAAAAGTAGCAAG-3′)] was ligated with the GFP gene (amplified with primers GFP1  (5′-GATCCTGCAGATGTCTAAAGGTGAA-3′) and GFP2 (5′-GATCGCATGCTTTGTACAATTCATC-3′) after digestion with SphI and PstI (sites underlined). pCR2.1-TOPO-Sod5/GFP  was  amplified  using  the  primers  IPF1222Fus3 (5′-ACCCCAAGCTTATGAAGTATTTGTCCATT-3′) and IPF1222Fus4 (5′-AGCTAGCTAGCTGCGAGAATCGGCATTCA-3′) containing a HindIII and a NheI site respectively (underlined). This fragment was used to replace GFP in the pACT-GFP construct to obtain pACT1-Sod5/GFP. The plasmid was integrated into the RPS10 locus of CAI-4 cells as described above. Transformants carrying pGFP, pACT1-GFP or pACT1-Sod5/GFP were pre-cultured overnight in YPD, transferred to a fresh 50 ml YPD culture and grown for 2 h. Cells were washed twice in PBS, resuspended in 1 ml of cold PBS containing protease inhibitor cocktail set II (Calbiochem) and vortexed for 30 min at 4°C in the presence of acid-washed glass beads (0.4–0.6 mm; B. Braun Biotech International). Samples from this step were taken and correspond to the total-cell extract. The remaining extracts were centrifuged for 15 min at 13 000 g at 4°C. The supernatant corresponds to the cytoplasm. The pellets, containing membranes and cell wall, were washed twice in cold PBS and resuspended in 500 µl of cold PBS. Samples (30 µg) were separated on 12% SDS-polyacrylamide gels and transferred to PVDF membrane (Roche). Membranes were incubated for 1 h at room temperature in PBS-T [PBS, 0.1% Tween 20 (Merck)] containing 5% of non-fat dry milk. GFP was detected with NBT/BCIP (Roche) using a rabbit polyclonal GFP antiserum and alkaline phosphatase-conjugated anti-rabbit antibodies diluted 1/5000 and 1/10 000, respectively, in PBS-T and incubated for 2 h at room temperature for each antibody. A rabbit polyclonal antiserum not directed against GFP was used as isotype control. In addition, aliquots of each culture were centrifuged, mounted in mounting fluid to be visualized by fluorescent microscopy. Immunoelectron microscopy of C. albicans strains containing pACT1-Sod5/GFP, pACT-GFP (control) and pGFP (control) was carried out for ultrastructural localization of Sod5. Cells were fixed, centrifuged, and the sediment was embedded in 3% agarose at 37°C then cooled on ice. Small parts of agarose blocks were embedded in Lowicryl (Polysciences). Ultrathin sections (50 nm) were mounted on Formvar-coated nickel grids and incubated with rabbit anti-GFP (Abcam) followed by 10 nm gold-conjugated goat anti-rabbit IgG (Auroprobe EM). In control samples the primary antibody was omitted. Grids were counterstained with uranyl acetate and lead citrate and examined using a Zeiss 109 transmission electron microscope (Zeiss). Candida albicans strain SC5314 was inoculated in salt-based medium [0.45%  (w/v)  NaCl,  0.25%  (w/v)  NH4SO2,  0.085% (w/v) yeast nitrogen base without amino acids and without (NH4)2SO2 (Becton Dickinson and Company) and 2.5 mM N-acetyl glucosamine] to a final concentration of OD600 = 1 and incubated for 5 h at 37°C. Extraction of GPI cell wall proteins (GPI-CWPs) and their preparation for mass spectrometric analysis were performed as described previously (Groot et al., 2004). Briefly, GPI-CWPs were released from isolated cell walls by HF-pyridine treatment. Extracted GPI-CWPs were reduced with DTT, S-alkylated with iodoacetamide (Shevchenko et al., 1996) and digested with trypsin. Tryptic digests were analysed by LC/MS/MS using an Ultimate nano-LC system (Dionex, Sunnyvale, CA) and a MicroMass Q-TOF mass spectrometer (Waters, Milford, MA) as previously described (Groot et al., 2004). Resulting MS/MS spectra were processed and analysed with Biolynx and Masslynx Pepseq software. For protein identification, the resulting spectra and amino acid sequences were analysed using MASCOT software and compared with in silico digests of the CandidaDB protein database. The Ura-blaster protocol (Fonzi and Irwin, 1993) was used to disrupt IPF1222/SOD5. The 3 kb hisG-URA3-hisG sequence of pMB7 was flanked at the 3′ end with a PCR product corresponding  to  the  IPF1222  region  from  positions −472 to  96  and  at  the  5′  end  with  a  PCR  product  corresponding  to the IPF1222 region from positions 500 to +170. PCR fragments were amplified using primers IPF1222P3   (5′-GACGTGGTACCCCAGATTATATGTTGACCA-3′) and IPF1222P4 (5′-ATCGGAAGATCTTAGCAATTAATGATGGAC-3′) containing a KpnI and BglII site respectively (underlined) and primers IPF1222P5 (5′-TGCACTGCAGCTTGACGAGG GACACGGC-3′) and IPF1222P6 (5′-ACCCAAGCTTTAAT GTGCTACTGTTTGG-3′) containing a PstI and HindIII site respectively (underlined). This plasmid was linearized and transformed into strain CAI-4. Two rounds of Ura-blasting were performed to disrupt both IPF1222/SOD5 alleles to give Δsod5, which was confirmed by PCR and Southern blot. A reconstituted strain was constructed by cloning a PCR fragment containing the native IPF1222/SOD5 gene into CIp10 (Murad et al., 2000). The PCR fragment containing the IPF1222 region from positions −976 to +439 was ligated into CIp10 and the resulting pCIp-SOD5 was transformed into Ura–Δsod5 after linearization. To give the corresponding controls, linearized CIp10 alone was transformed into CAI-4 and Ura–Δsod5. A single integration of these plasmids at the RPS10 locus in the same allele was confirmed by Southern blot analysis. To investigate the survival of mutants in blood and the blood cell fractions, CAI-4 + CIp10, Δsod5 + CIp10, Δsod5 + CIp10-SOD5, Δsap4–6, Δcap1, Δicl1, Δefg1 and Δcph1 were incubated in blood, plasma and the MNC and PMN fractions for 1 h at 37°C at a 1:1 ratio of Candida : leukocytes in each sample. Cells were centrifuged, resuspended in water, vigorously vortexed, plated on YPD plates and incubated overnight at 37°C. The colony-forming units (cfu) were counted and the percentages of survival determined as follows: (cfu/cfuplasma) × 100. To control for proper plating of yeast and hyphal cells, suspensions of C. albicans cells cultured in YPD and plasma were counted with a haemocytometer and plated on YPD plates. A good correlation between the haemocytometer counts and the cfu number was found. For inoculum preparation, C. albicans strains were grown in NGY medium [0.1% Neopeptone (Difco), 0.1% yeast extract (Difco), 0.4% glucose] at 30°C for 18–24 h, with shaking at 200 r.p.m. Cells were harvested, washed twice with sterile, physiological saline, and resuspended in sterile, physiological saline to produce inocula of 2.0 × 104 cfu per gram mouse body weight in 100 µl. For each C. albicans strain tested, six female BALB/c mice were injected intravenously with 100 µl of cell suspension into the lateral tail vein. Mice were monitored daily and humanely killed when they showed signs of distress or were unable to freely reach food and water. All animal work was carried out under the terms and conditions stipulated by the Home Office, UK. This work was supported by the European Commission (QLK2-2000-00795; ‘Galar Fungail consortium’) and the Deutsche Forschungsgemeinschaft (Hu 528/10). We thank Christophe d’Enfert for creating and maintaining the web page for public data of the Galar Fungail Consortium (http://www.pasteur.fr/recherche/unites/Galar_Fungail). We thank Alistair Brown and Caroline Barelle, Joachim Ernst, Gerry Fink, Mike Lorenz, Dominique Sanglard for providing mutant strains, Alistair Brown for pACT1-GFP, Neil Gow and Julian Naglik for critical reading of the manuscript, Oliver Liesenfeld and Hans Möllenkopf for their valuable contributions. We also wish to thank all blood donors of this study and Antje Erler, Ines Korsukéwitz and Wiebke Thoma for taking blood. Sequence data from Candida  albicans  were  obtained  from  the  Stanford  DNA Sequencing and Technology Center website at http://www-sequence.stanford.edu/group/candida. Sequencing of C. albicans at the Stanford DNA Sequencing and Technology Center was accomplished with the support of the NIDR and the Burroughs Wellcome Fund. Alarco, A.M., and Raymond, M. 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