Immune evasion, stress resistance, and efficient nutrient acquisition are crucial for intracellular survival of Candida glabrata within macrophages.

Immune Evasion, Stress Resistance, and Efficient Nutrient Acquisition Are Crucial for Intracellular Survival of Candida glabrata within Macrophages

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Katja Seider, Franziska Gerwien, Lydia Kasper, Stefanie Allert, Sascha Brunke, Nadja Jablonowski, Tobias Schwarzmüller, Dagmar Barz, Steffen Rupp, Karl Kuchler and Bernhard Hube Eukaryotic Cell 2014, 13(1):170. DOI: 10.1128/EC.00262-13. Published Ahead of Print 20 December 2013.

Immune Evasion, Stress Resistance, and Efficient Nutrient Acquisition Are Crucial for Intracellular Survival of Candida glabrata within Macrophages

Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology—Hans Knoell Institute Jena (HKI), Jena, Germanya; Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University Vienna, Vienna, Austriab; Institute for Transfusion Medicine, University Hospital, Jena, Germanyc; Fraunhofer Institute for Interfacial Engineering, Stuttgart, Germanyd; Friedrich Schiller University, Jena, Germanye; Integrated Research and Treatment Center, Sepsis und Sepsisfolgen, Center for Sepsis Control and Care (CSCC), Universitätsklinikum Jena, Jena, Germanyf

Candida glabrata is both a human fungal commensal and an opportunistic pathogen which can withstand activities of the immune system. For example, C. glabrata can survive phagocytosis and replicates within macrophages. However, the mechanisms underlying intracellular survival remain unclear. In this work, we used a functional genomic approach to identify C. glabrata determinants necessary for survival within human monocyte-derived macrophages by screening a set of 433 deletion mutants. We identified 23 genes which are required to resist killing by macrophages. Based on homologies to Saccharomyces cerevisiae orthologs, these genes are putatively involved in cell wall biosynthesis, calcium homeostasis, nutritional and stress response, protein glycosylation, or iron homeostasis. Mutants were further characterized using a series of in vitro assays to elucidate the genes’ functions in survival. We investigated different parameters of C. glabrata-phagocyte interactions: uptake by macrophages, replication within macrophages, phagosomal pH, and recognition of mutant cells by macrophages as indicated by production of reactive oxygen species and tumor necrosis factor alpha (TNF-␣). We further studied the cell surface integrity of mutant cells, their ability to grow under nutrient-limited conditions, and their susceptibility to stress conditions mirroring the harsh environment inside a phagosome. Additionally, resistance to killing by neutrophils was analyzed. Our data support the view that immune evasion is a key aspect of C. glabrata virulence and that increased immune recognition causes increased antifungal activities by macrophages. Furthermore, stress resistance and efficient nutrient acquisition, in particular, iron uptake, are crucial for intraphagosomal survival of C. glabrata.

M

acrophages are an important part of the innate immune system and constitute, together with neutrophils, the front-line phagocytes which act to eliminate pathogens from the bloodstream and tissues. In addition, they are also important immune modulators, presenting antigens and secreting pro- and anti-inflammatory cytokines and chemokines, thereby activating the adaptive immune system. The first contact between a phagocytic cell and a microbe is mediated by host receptors detecting conserved, basic molecular components of microorganisms, the pathogen-associated molecular patterns (PAMPs) (1, 2). Once recognized, a pathogen is quickly engulfed and trapped within a phagosome, which then enters the phagocytic pathway and acquires a broad range of antimicrobial effectors (3, 4). The resultant phagolysosome is an extremely hostile compartment, deficient in nutrients and trace elements. The phagolysosome also undergoes acidification, accompanied by increased acidic hydrolase activity. Furthermore, a battery of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and antimicrobial peptides is transported into the organelle (3, 4). The combined action of these factors is normally sufficient to kill and degrade most phagocytosed microbes; however, some microorganisms employ strategies to avoid killing. Such survival activities include avoiding detection and phagocytosis, inhibition of phagosome maturation, neutralization of ROS or other antimicrobial mediators, and adaptation to the environment of the phagolysosome (for reviews, see references 4, 5, and 6). To subvert detection by immune cells, pathogenic fungal species such as Candida albicans and Histoplasma

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capsulatum are capable of concealing immunostimulatory PAMPs, such as ␤-1,3-glucan or chitin, under a mannoprotein coat (in the case of C. albicans) or under an ␣-1,3-glucan outer layer (as shown for H. capsulatum) (7, 8). However, such a strategy does not completely prevent uptake as the stimulation of other receptors, such as the mannose receptor, facilitates phagocytosis (9, 10). Candida species are common opportunistic fungal pathogens of humans, and Candida glabrata is the second most common cause of infection, surpassed only by C. albicans (11–13). Due to intrinsic antifungal resistance, which is often further increased, in the case of azoles, by adaptive resistance (14), C. glabrata infections are extremely difficult to treat, and systemic infections are associated with a high mortality rate (15). Despite its medical relevance, the pathogenicity mechanisms of C. glabrata are poorly understood. Mechanisms described so far include the abilities of C. glabrata to undergo phenotypic switching (16–18) and to regulate adherence by transcriptional silencing (19–21). Despite the high mortality of C. glabrata infections in humans, this yeast ex-

p. 170 –183

Received 27 September 2013 Accepted 11 November 2013 Published ahead of print 20 December 2013 Address correspondence to Bernhard Hube, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/EC.00262-13

January 2014 Volume 13 Number 1


Katja Seider,a Franziska Gerwien,a Lydia Kasper,a Stefanie Allert,a Sascha Brunke,a,f Nadja Jablonowski,a Tobias Schwarzmüller,b Dagmar Barz,c Steffen Rupp,d Karl Kuchler,b Bernhard Hubea,e,f

C. glabrata Genes Required for Phagocyte Survival

MATERIALS AND METHODS Ethics statement. Blood was obtained from healthy human donors with written informed consent. The blood donation protocol and use of blood for this study were approved by the institutional ethics committee (EthikKommission des Universitätsklinikum Jena, Permission no. 2207-01/08). Strains and growth conditions. C. glabrata mutant strains are derivatives of the laboratory strain ATCC 2001, harboring auxotrophies for histidine, leucine, and tryptophan. The strains were obtained from a novel genome-scale collection of C. glabrata deletion mutants. In each strain of the collection, a single open reading frame (ORF) was replaced with a bar-coded NAT1 resistance cassette in the triply auxotrophic derivative of strain ATCC 2001. The mutant library was kindly provided by T. Schwarzmüller and K. Kuchler, Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University Vienna, Vienna, Austria (submitted for publication). The triply auxotrophic wild-type (WT) strain was used for comparison. All yeast strains used in this study were routinely grown overnight in YPD (1% yeast extract, 1% peptone, 2% glucose) at 37°C in a shaking incubator. Prior to coincubation with neutrophils, C. glabrata strains were opsonized with 50% human serum (in phosphate-buffered saline [PBS; Sigma-Aldrich]) for 30 min at 37°C and resuspended in PBS. Macrophage culture. Human peripheral blood mononuclear cells (PBMC) were isolated by Histopaque-1077 (Sigma-Aldrich) density centrifugation from buffy coats donated by healthy volunteers. To differentiate PBMC into monocyte-derived macrophages (MDMs), 2 ⫻ 106 PBMC/ml were plated in RPMI 1640 media (with L-glutamine and 25 mM HEPES) (PAA Laboratories, Inc.) containing 10% heat-treated fetal bovine serum (PAA Laboratories, Inc.) in cell culture dishes. Macrophage colony-stimulating factor (M-CSF; ImmunoTools) (10 ng/ml) was included in the cultures to favor the differentiation of macrophages. After 5 days at 5% CO2 and 37°C, nonadherent cells were removed, and adherent MDMs were detached with 50 mM EDTA and plated in flat-bottom 96and 24-well plates to give a final concentration of approximately 3 ⫻ 104


MDMs/well and 2 ⫻ 105 MDMs/well in RPMI 1640 without serum. For microscopic analysis, MDMs were allowed to adhere to coverslips within a 24-well plate. To test purity, MDMs were stained with an anti-CD14 antibody and analyzed by flow cytometry. The proportion of CD14-positive cells was ⱖ80%. All experiments were performed with cells isolated from at least three different donors. Screening a mutant library (survival assay 1). Mutant strains were grown in 96-well plates and diluted in RPMI 1640 (1:100), and 10 ␮l of each strain was added to one well with a confluent layer of MDMs in 100 ␮l RPMI 1640 in another 96-well plate (giving a multiplicity of infection [MOI] of approximately 1). Another 10 ␮l of each strain was inoculated in 100 ␮l YPD, and growth curves were measured by recording the optical density at 600 nm (OD600) as a function of time (control curves). After 3 h of coincubation, non-cell-associated yeasts were removed by washing with RPMI 1640, MDMs were lysed with 20 ␮l Triton X-100 per well, and, after the addition of 100 ␮l of YPD, yeast growth (OD600) was again measured as a function of time. The time to reach the mid-log phase was dependent on the number of viable yeast cells associated with MDMs (or, in the case of control curves, on the number of cells inoculated) and was taken to compare wild-type and mutant strains. Mutants with decreased growth rates after incubation with MDMs, but not within control plates, were further tested. Survival assay 2. Mutant strains identified in the survival assay described above were further tested in an additional survival assay. Mutant strains were washed in RPMI 1640, and total numbers of cells were assessed by the use of a hemocytometer. MDMs in 96-well plates were infected at an MOI of 1, and after 3 h of coincubation at 37°C and 5% CO2, non-cell-associated yeasts were removed by washing with RPMI 1640. To measure yeast survival in MDMs, lysates of infected MDMs were plated on YPD plates to determine CFU. Adhesion and uptake by MDMs. Next, mutant strains identified in the survival assay(s) described above were analyzed for altered properties with respect to adherence to macrophages and for altered phagocytosis rates. MDMs, seeded in 96-well plates, were infected with wild-type or mutant cells at an MOI of 2 and coincubated for 30 min and 90 min. Uptake by or adhesion to MDMs was assessed by plating supernatants and lysates on YPD plates to determine CFU. Replication within MDMs. To quantify yeast intracellular replication, yeast cells were labeled with 100 ␮g/ml fluorescein isothiocyanate (FITC) (Sigma-Aldrich) in carbonate buffer (0.1 M Na2CO3, 0.15 M NaCl, pH 9.0) for 30 min at 37°C, followed by washing with PBS. MDMs were infected at an MOI of 2 for 30 min and washed to remove unbound yeast cells, followed by an additional incubation step of 6 h at 37°C and 5% CO2. Cells were fixed with 4% paraformaldehyde, stained 30 min at 37°C with 25 ␮g/ml Alexa Fluor 647-conjugated concanavalin A (ConA) (Molecular Probes) to visualize nonphagocytosed yeast cells, and mounted cell side down in ProLong Gold antifade reagent (Molecular Probes). As FITC is not transferred to daughter cells, differentiation of mother and daughter cells was possible and intracellular replication was quantified by fluorescence microscopy (Leica DM5500B and Leica DFC360) and by counting at least 200 phagocytosed yeast cells, scored for FITC staining or no staining. Quantification of phagolysosomal fusion. Acidification of the phagosomes was assessed by use of the acidotropic dye LysoTracker Red DND-99 (Molecular Probes). LysoTracker (diluted 1:10,000 in RPMI 1640) was added 1 h prior to infection and during the incubation with yeast cells (MOI of 2). After 1 h of infection, the cells were fixed, stained with Alexa Fluor 647-conjugated ConA, and mounted. Yeast-containing phagosomes were analyzed for lysosomal fusion or no fusion by fluorescence microscopy (Leica DM5500B and Leica DFC360). Chitin and ␤-glucan exposure. To measure chitin content, yeasts from an overnight culture were washed in 100 mM Tris-HCl (pH 9.0) and incubated with 25 ␮g/ml calcofluor white diluted in 100 mM Tris-HCl (pH 9.0) for 1 h at 37°C. After washing with water, fluorescence was quantified by flow cytometry (BD FACSCanto). For ␤-glucan staining,

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hibits low virulence in animal infection models. Indeed, mice survive hematogenously disseminated candidiasis following infection with high C. glabrata doses and produce only low levels of proinflammatory cytokines (22–24). Nevertheless, C. glabrata can be reisolated from immunocompetent animals weeks after infection, suggesting that the fungus employs efficient immune evasion strategies. This view is further supported by the fact that C. glabrata can survive attacks by macrophages in vitro and can even replicate within these phagocytes (25–27). The members of a family of glycosylphosphatidylinositol-linked aspartic proteases (Yapsins) have been implicated in survival within macrophages and in virulence (26). Additionally, recycling of cellular components (autophagy) may contribute to the survival of C. glabrata when nutrient availability is limited, as is the case inside phagosomes (27). Our own studies have shown that survival within macrophages is associated with arrest of phagosomal maturation in a late endosomal stage, accompanied by inhibition of acidification, reduced ROS production, and low levels of proinflammatory cytokines (28). Furthermore, chromatin remodeling, which facilitates a reprogramming of cellular energy metabolism and provides protection against DNA damage, seems to be central for survival of macrophage-internalized C. glabrata cells (29). To further elucidate factors and activities which contribute to the capacity of C. glabrata to avoid, resist, or neutralize host defense mechanisms of macrophages, we screened a library of 433 C. glabrata gene deletion mutants for survival within human primary macrophages. A total of 23 mutants with altered abilities to survive the challenge with macrophages were further analyzed in a set of in vitro assays to functionally characterize these genes.

Seider et al.

TABLE 1 Composition of media used for determination of growth under conditions of nutrient starvation Componentsa

SD Glucose deficit Nitrogen deficit Iron deficit

1⫻ YNB, 2% glucose, 0.5% (NH4)2SO4, 1.5% HLT 1⫻ YNB, 0.05% glucose, 0.5% (NH4)2SO4, 1.5% HLT 1⫻ YNB, 2% glucose, 0.05% (NH4)2SO4, 1.5% HLT 1⫻ YNB, 2% glucose, 0.5% (NH4)2SO4, 1.5% HLT, 5 ␮M BPS 1% yeast extract, 1% peptone, 2% glucose 0.1% yeast extract, 0.1% peptone, 0.2% glucose

YPD 10% YPD

a 100% HLT, mixture of 30 mM histidine, 80 mM leucine, and 50 mM tryptophan; BPS, bathophenanthrolinedisulfonate.

yeast cells were washed with PBS and incubated with 5% bovine serum albumin (BSA) for 30 min at 37°C, followed by a first step of 1 h of incubation with a monoclonal anti-␤-glucan antibody (Biosupplies) (diluted 1:400 in 2% BSA) and a second step of 1 h of incubation with an Alexa Fluor 488 conjugate secondary antibody (Molecular Probes) (diluted 1:400 in 2% BSA). Fluorescence was again quantified by flow cytometry (BD FACSCanto). TNF-␣ production. MDMs were infected in 24-well plates at an MOI of 5. Lipopolysaccharide (LPS) (Sigma-Aldrich) was used as a control and applied at a concentration of 1 ␮g/ml. After 24 h, samples of surrounding medium were collected and centrifuged (10 min, 1,000 ⫻ g). The amount of TNF-␣ secreted by MDMs was determined by enzyme-linked immunosorbent assay (ELISA) according to the protocol of the manufacturer (eBioscience). The cytokine amount was measured spectrophotometrically at 450 nm and calculated from a standard curve obtained from dilutions of recombinant proteins (supplied by the manufacturer). All experiments were performed in triplicate and normalized to wild-type samples. Detection of ROS in MDMs. ROS production was measured by luminol-enhanced chemiluminescence quantification, and all cells and reagents were prepared in RPMI 1640 without phenol red. MDMs were grown in white 96-well plates in 100 ␮l medium per well. Overnight yeast cultures were washed in RPMI 1640 and counted, and 50 ␮l was added to MDMs (giving an MOI of 10). For control experiments, MDMs were left untreated in 150 ␮l RPMI 1640 or 100 nM phorbol myristate actetate (PMA) (Sigma-Aldrich) was added to 50 ␮l RPMI 1640. All samples were prepared in triplicate. Fifty microliters of a mixture containing 200 ␮M luminol and 16 U horseradish peroxidase in RPMI 1640 was immediately added prior to quantification. Luminescence was measured every 3 min over a 3-h period of incubation at 37°C using a microplate reader (Tecan Infinite 200). Susceptibility to oxidative stress. Sensitivity of C. glabrata mutants to oxidative stress was tested by spotting serial dilutions (1 ⫻ 105 to 1 ⫻ 101) of yeast cells on SD or YPD plates containing H2O2 (Roth) (7.5 mM) or menadione (Sigma-Aldrich) (60 ␮M) followed by incubation for 1 to 2 days at 30°C. Growth under conditions of nutrient starvation. Mutants from an overnight culture were washed in 1⫻ YNB (BD Bioscience) and counted. Next, 5 ⫻ 105 yeast cells/ml were inoculated in 100 ␮l minimal SD, YPD, or nutrient-deficient medium (Table 1). Growth curves were measured by recording OD600 as a function of time (using a Tecan Infinite 200 reader), and curves were compared by analyzing the time required to reach the mid-log phase [t (ODmax/2)] and the ODmax. Isolation of neutrophils. The isolation of neutrophils was performed as previously described by Miramón et al. (30). Neutrophil survival assay. Neutrophils (106 cells/ml) were infected with opsonized C. glabrata cells at an MOI of 1 (200 ␮l final volume) and coincubated for 3 h at 37°C and 5% CO2. After neutrophil lysis with 0.25% SDS (Roth) and treatment with 20 U DNase I (Invitrogen) to dissolve any neutrophil extracellular trap (NET) structures that might have formed, yeast survival was measured by plating on YPD plates to determine CFU.

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RESULTS

Screening of a C. glabrata targeted gene deletion library for survival within macrophages and identification of mutants with reduced survival. In order to identify C. glabrata genes specifically involved in surviving phagocytosis by macrophages, a threestage approach was developed and a library of 433 mutants tested (mutant generation by T. Schwarzmüller, B. Ma, and K. Kuchler; submitted for publication). The members of this set of strains lack genes which are putatively involved in antifungal drug resistance, environmental stress sensing, and signaling as determined on the basis of sequence similarity with Saccharomyces cerevisiae and which may play a role in host-pathogen interactions. First, survival of all mutants was assessed by measuring growth rates following coincubation with human monocyte-derived macrophages (MDMs). Second, those mutants with reduced growth in standard liquid medium were discarded. Finally, those mutants with specific growth defects following coincubation with MDMs were independently tested for survival by plating CFU. A total of 23 mutants, representing mutations of genes involved in cell wall biosynthesis, calcium homeostasis, nutritional response, response to stress, protein glycosylation, and iron homeostasis, were defective for surviving interactions with MDMs (Table 2). Beyond that, the importance of five of these genes (CCH1, SLM1, CAGL0M12496g, FRE8, and PMT4), deleted in the same but protophic background strain, was assessed in independent survival tests. For four of these mutants, survival was still reduced compared to that of the corresponding wild type and, thus, was independent of auxotrophies (for auxotrophic versus protrophic cch1⌬ strain, 64.9% versus 44.5%; for slm1⌬ strain, 42.0% versus 41.3%; for CAGL0M12496g⌬ strain, 22.3% versus 32.3%; and for fre8⌬ strain, 75.2% versus 83.8%). The prototrophic pmt4⌬ mutant, however, was no longer attenuated in

Eukaryotic Cell


Medium

Uptake and replication within neutrophils. To quantify phagocytosis and replication, a 24-well plate was precoated with 0.1% gelatin overnight and washed with PBS. Neutrophils (1.25 ⫻ 106 cells/ml [400 ␮l]) were infected with FITC-stained C. glabrata strains at an MOI of 1, followed by coincubation for 30 min and 3 h at 37°C and 5% CO2. Fixation, ConA staining, and mounting were conducted as described for the MDMs. To determine the phagocytosis rate, all neutrophils with internalized yeast cells were counted microscopically and compared to those with cells which did not phagocytose. Extracellular (ConA-positive) and intracellular (ConA-negative) replication rates were calculated after 3 h via counting daughter (FITC-negative) cells and mother (FITC-positive) cells. Detection of reactive oxygen species in neutrophils. ROS production was measured in a manner analogous to the method used for the MDMs by luminol-enhanced chemiluminescence. Neutrophils (5 ⫻ 105 cells/ml) were seeded in white 96-well plates in 100 ␮l RPMI 1640 1640 –2% autologous serum per well and incubated for 45 min at 37°C and 5% CO2. A 50-␮l volume of C. glabrata overnight cultures (5 ⫻ 106 cells/ml) was added to neutrophils (MOI of 5). For further treatment, see above (“Detection of ROS in MDMs”). Statistical analysis. All experiments were performed with MDMs isolated from at least three different donors. Unless otherwise stated, all data are reported as means ⫾ standard deviations (SD). The data were analyzed using a two-tailed, unpaired Student’s t test for intergroup comparisons. Statistical significant results were marked with a single asterisk (or, in the tables, a single x) representing P ⬍ 0.05, two asterisks (“xx” in the tables) representing P ⬍ 0.01, or three asterisks (“xxx” in the tables) representing P ⬍ 0.005. Contingency analyses were performed by using Fisher’s exact test, and correlation analyses were performed with the help of the GraphPad Prism 5 software.


TABLE 2 Screening a C. glabrata-targeted gene deletion library for survival within MDMs revealed 23 mutants with reduced survivala Screening result (survival in MDMs [% WT])

Gene designation/ORF

Key word(s)

Function (based on homology to S. cerevisiae)

Genes involved in response to stress 1

CCH1/CAGL0B02211g

Calcium channel

2

SLM1/CAGL0G02827g

TOR pathway

3

SHO1/CAGL0G03597g

Osmosensor

Voltage-gated high-affinity calcium channel involved in calcium influx in response to some environmental stresses Phosphoinositide binding protein, regulating actin cytoskeleton organization in response to stress, phosphorylated by TORC2 Transmembrane osmosensor; participates in activation of both the Cdc42- and MAP kinase-dependent pathways

ARG81/CAGL0H06875g

Transcription factor

5

GPR1/CAGL0K01507g

GPCR

6

GPA2/CAGL0I08195g

Alpha subunit of GPCR

7 8

FRE8/CAGL0M07942g CAGL0M12496g

Iron/copper reductase Response to low biotin

LRG1/CAGL0C05599g

␤-Glucan synthesis

10

GNT1/CAGL0I09922g

Modification of N-linked glycans

11

ERG5/CAGL0M07656g

Ergosterol biosynthesis

12

SLG1/CAGL0F01507g

Stress-activated PKC1MPK1 kinase pathway

OST6/CAGL0G07040g

Glycosylation

14

MNN4/CAGL0H09130g

15 16

MNS1/CAGL0M00528g PMT2/CAGL0J08734g

Mannosyl phosphoryation of N-linked oligosaccharides Alpha-1.2-mannosidase Protein O-mannosyltransferase

17

PMT4/CAGL0M00220g

Protein O-mannosyltransferase

YGR106C/CAGL0G07887g

Genes involved nutritional sensing or signaling 4

Genes involved in cell membrane and cell wall structure 9

Genes involved in protein glycosylation 13

Genes with other functions 18 19

BAR1/CAGL0J02288g

Assembly of vacuolar ATPase Aspartyl protease

20

CDC12/CAGL0I01188g

Cytokinesis

21

CKA2/CAGL0G02035g

Casein kinase 2

Zinc-finger transcription factor involved in the regulation of arginine-responsive genes Plasma membrane GPCR; integrates nutritional signals with the modulation of cell fate via PKA and cAMP Alpha subunit of the heterotrimeric G protein that interacts with the receptor Gpr1p; signaling role in response to nutrients Iron/copper reductase involved in iron homeostasis Transcriptional activator; required for the VHREmediated induction of VHT1 and BIO5 in response to low biotin concns

Putative GAP involved in the Pkc1-mediated signaling pathway that controls cell wall integrity; appears to specifically regulate 1.3-␤-glucan synthesis N-Acetylglucosaminyltransferase capable of modification of N-linked glycans in the Golgi apparatus C-22 sterol desaturase; a cytochrome P450 enzyme involved in ergosterol biosynthesis Sensor-transducer of the stress-activated PKC1-MPK1 kinase pathway involved in maintenance of cell wall integrity

Subunit of the oligosaccharyltransferase complex of the ER lumen; glycosylation of newly synthesized proteins Putative positive regulator of Mnn6; involved in mannosylphosphorylation of N-linked oligosaccharides Alpha-1.2-mannosidase involved in ER quality control Protein O-mannosyltransferase; transfers mannose residues from dolichyl phosphate-D-mannose to protein serine/threonine residues Protein O-mannosyltransferase; transfers mannose residues from dolichyl phosphate-D-mannose to protein serine/threonine residues

Endoplasmic reticulum protein that functions in assembly of the V-ATPase Aspartyl protease secreted into the periplasmic space of mating type a cells; cleaves and inactivates alpha factor, allowing cells to recover from alpha-factorinduced cell cycle arrest Component of the septin ring of the mother-bud neck that is required for cytokinesis Alpha catalytic subunit of casein kinase 2, with roles in cell growth and proliferation

Assay 2 [CFU]

62.5 ⫾ 19.4

64.9 ⴞ 18.6

44.0 ⫾ 4.3

42.0 ⴞ 13.9

84.5 ⫾ 7.1

75.9 ⴞ 27.4

42.5 ⫾ 33.3

79.7 ⴞ 6.3

41.1 ⫾ 29.7

76.1 ⴞ 10.0

65.4 ⫾ 9.2

68.4 ⴞ 10.8

78.7 ⫾ 31.8 23.9 ⫾ 9.2

75.2 ⴞ 21.3 22.3 ⴞ 17.7

48.0 ⫾ 19.3

50.5 ⴞ 20.8

49.4 ⫾ 32.8

51.7 ⴞ 2.1

61.8 ⫾ 17.1

60.5 ⴞ 11.6

54.8 ⫾ 29.1

66.0 ⴞ 26.2

46.6 ⫾ 25.8

39.3 ⴞ 9.5

51.7 ⫾ 43.9

30.0 ⴞ 15.2

50.5 ⫾ 34.7 66.3 ⫾ 76.3

43.2 ⴞ 17.4 57.2 ⴞ 30.8

31.2 ⫾ 17.2

66.4 ⴞ 24.4

70.5 ⫾ 19.1

52.2 ⴞ 3.5

48.9 ⫾ 15.7

41.0 ⴞ 11.5

54.9 ⫾ 51.1

74.7 ⴞ 26.4

54.8 ⫾ 56.2

56.5 ⴞ 21.3

(Continued on following page)


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Gene no.

Assay 1 [growth curve]

Seider et al.

TABLE 2 (Continued) Screening result (survival in MDMs [% WT])

Gene no.

Key word(s)

Function (based on homology to S. cerevisiae)

22

HEK2/CAGL0L06226g

RNA binding protein

23

MPS3/CAGL0G06864g

SPB duplication and nuclear fusion

RNA binding protein involved in the asymmetric localization of ASH1 mRNA; regulates telomere position effect and length Nuclear envelope protein required for SPB duplication and nuclear fusion

Assay 2 [CFU]

52.0 ⫾ 45.1

36.0 ⴞ 19.1

54.9 ⫾ 16.4

68.8 ⴞ 20.9

a cAMP, cyclic AMP; ER, endoplasmic reticulum; GAP, GTPase-activating protein; GPCR, G protein-coupled receptor; MAP, mitogen-activated protein; Mnn6, mannosylphosphate transferase; MPK1, mitogen-activated protein kinase 1; PKA, protein kinase A; PKC1, protein kinase C 1; SPB, spindle pole body; TOR, target of rapamycin; TORC2, TOR complex 2; V-ATPase, vacuolar ATPase; VHRE, vitamin H-responsive element.

survival (auxotrophic versus protrophic pmt4⌬ strain, 66.4% versus 93.3%). Therefore, the possibility of a cumulative effect of gene deletion in addition to the three auxotrophies for survival in macrophages cannot be excluded, even though in vivo studies suggested that histidine, leucine, or tryptophan auxotrophy, as well as a combination of these auxotrophies, does not influence fitness in a murine model of infection (24). We next sought to determine why the set of 23 defective mutants exhibited reduced survival by dissecting the phagocytic process and analyzing the behavior of mutants under in vitro conditions, which mimic certain aspects of the interaction with macrophages. Table 3 provides an overview of all assays and results obtained for the mutants.

Uptake and intracellular replication. Survival of C. glabrata cells challenged with macrophages may depend not only on intracellular survival per se but also on the efficiency of phagocytosis. Since our screening protocol did not discriminate between a lower survival rate inside the macrophages and a modified initial uptake rate (or contact with macrophages), which might possibly influence survival, we determined the MDM adhesion and uptake rates of all 23 mutants after 30 min and 90 min of coincubation with MDMs. In general, MDMs internalized wild-type yeast cells avidly (with ⬎85% cells found intracellularly after 60 min and more than 92% after 120 min), in agreement with a previous study (10). No mutant exhibited reduced adhesion or uptake rates after 30 min, confirming that the observed reduced survival was not due to

TABLE 3 Summary of mutant phenotypes obtained by all assays of this studya

Gene Gene no. designation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

CCH1 SLM1 SHO1 ARG81 GPR1 GPA2 FRE8 CAGL0M12496g LRG1 GNT1 ERG5 SLG1 OST6 MNN4 MNS1 PMT2 PMT4 CAGL0G07887g BAR1 CDC12 CKA2 HEK2 MPS3

Susceptibility to caspofungin, Oxidative osmotic, cell Surface TNF-␣ ROS stress C and N Iron wall, or heat Neutrophil Uptake Replication integrity production production susceptibility starvation deprivation stressb killing x

x x

xx x

x

x x

x x xx x xx x xx x xx

xx x x x x

x x x

x xx

x x xx xx xx x

x x

x x x x xx

x x x

x x x x

x x

x x x

xxx

x x

(x) x

x x x

x

xx

x

x x x xx

x x (x)

x

(x) x x xx

xx x xx

x

x

x x

x x

x

x x

x x

(x)

a Mutants with significant differences (unpaired Student’s t test) compared to wild-type C. glabrata cells are marked with x (P ⬍ 0.05), xx (P ⬍ 0.01), or xxx (P ⬍ 0.005). (x), the result was not statistically significant but was used for further analyses. b The experiment was performed by T. Schwarzmüller, B. Ma, and K. Kuchler (submitted for publication).

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Gene designation/ORF

Assay 1 [growth curve]


90 min postinfection (MOI of 2). (Results shown represent means ⫾ SD, n ⱖ 3.) For statistics (unpaired Student’s t test), single asterisks ⫽ P ⬍ 0.05 (in comparison to wild-type C. glabrata cells).

higher numbers of yeast cells being removed during the wash steps of the survival assays. However, four mutants showed a significantly higher rate of early adhesion or uptake (Fig. 1). By 90 min, adhesion or uptake of three of these four mutants reached levels comparable to wild-type levels, while adhesion or internalization of one mutant (pmt4⌬) remained significantly higher at this later time point. As the survival rates of these four mutants were lower than that of the wild type (Table 2), it would appear that higher phagocytic activity correlates with more-efficient killing. Different laboratories have shown that C. glabrata not only survives phagocytosis but can even replicate inside macrophages (25, 26). We therefore determined intracellular replication for each mutant at 6 h postinfection by prestaining C. glabrata with FITC a dye which is not transferred to daughter cells (Fig. 2) (28). Intracellular replication (the production of daughter cells) was observed for all strains; however, for 16 mutants, replication was significantly lower than wild-type replication. The reduced replication rates of these mutants, however, cannot fully account for their reduced survival, as by 3 h postinfection (the time when survival was assayed), daughter cells accounted for only ⬃20% of the phagocytosed yeast population (28). Phagosome maturation. Our own previous data suggest that normal phagosome maturation is actively disrupted by phagocytosed C. glabrata cells (28). Specifically, C. glabrata is able to prevent phagosome acidification (keeping the pH value at 6.1)


and block phagosome maturation at a late endosomal stage. To determine whether reduced survival of the 23 mutants was associated with phagosomal acidification, MDMs were stained with LysoTracker Red (DND-99) after coincubation. This membrane-diffusible dye is trapped within intracellular acidic compartments and can therefore be used to visualize phagosomes with a pH below 5.5 to 6.0 (31, 32). Interestingly, all 23 mutants resided in nonacidified phagosomes in a manner comparable to that seen with the wild type (not shown). Therefore, reduced survival was not due to increased phagosomal acidification. Fungal recognition by macrophages. We next sought to determine whether higher killing of the mutants was due to altered recognition of C. glabrata by macrophages. Cell surface alterations. Cell wall changes may result in different host-receptor interactions and may lead to an altered route of uptake, potentially resulting in more-potent killing (33, 34). Alternatively, lack of a particular gene may result in higher sensitivity to the phagosomal defense mechanisms (see below). To determine whether the disruption of any of the 23 genes of interest caused cell wall alterations, we examined ␤-glucan exposure and chitin content on the surface of the mutants (Table 4). Nine mutants exhibited significantly increased ␤-glucan accessibility, and nine mutants had a higher chitin content in the cell wall. Six mu-

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FIG 1 Internalization of C. glabrata mutants by MDMs. The uptake of the C. glabrata wild type (WT) and mutants was measured by CFU plating 30 min and

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tants showed significantly increased accessibility of both ␤-glucan and chitin compared to wild-type cells. Induction of TNF-␣. To assess whether these cell wall alterations affected immune recognition of C. glabrata, we measured

the release of the TNF-␣ proinflammatory cytokine from cell culture supernatants after 24 h of infection of MDMs with C. glabrata mutants (Table 4). We observed increased TNF-␣ production for 8 of 23 mutants. Interestingly, seven (87.5%) of the eight mutants also had altered cell wall compositions (␤-glucan or chitin or both). Therefore, cell wall alterations correlate with higher TNF-␣ production. The correlation between chitin exposure and TNF-␣ release was statistically significant (P ⫽ 0.0203) (Fig. 3). ROS production. Following phagocytosis, the production of reactive oxygen species (ROS) by macrophages is thought to contribute to killing of microorganisms (3, 4). However, C. glabrata is

TABLE 4 Mutants exhibiting elevated chitin/␤-glucan exposure and TNF-␣ and ROS production by MDMs % WT ⫾ SD Gene no.

Gene designation

Chitin exposurea

␤-Glucan exposurea

TNF-␣ productionb

ROS productionc

1 2 4 5 6 8 9 10 11 12 14 15 16 17 19 20 21 22 23

CCH1 SLM1 ARG81 GPR1 GPA2 CAGL0M12496g LRG1 GNT1 ERG5 SLG1 MNN4 MNS1 PMT2 PMT4 BAR1 CDC12 CKA2/ HEK2 MPS3

209.5 ⫾ 31.8* 102.8 ⫾ 5.0 103.8 ⫾ 12.8 114.3 ⫾ 12.1 128.0 ⫾ 14.9 189.7 ⫾ 25.2* 122.9 ⫾ 8.4* 108.1 ⫾ 8.3 100.9 ⫾ 9.6 134.8 ⫾ 11.3* 284.6 ⫾ 55.3* 144.1 ⫾ 12.6* 97.1 ⫾ 10.8 266.4 ⫾ 26.7** 143.8 ⫾ 9.3* 103.4 ⫾ 13.1 92.5 ⫾ 2.3 116.4 ⫾ 24.4 208.1 ⫾ 10.5**

254.2 ⫾ 43.5* 95.3 ⫾ 2.9 172.3 ⫾ 9.1** 170.7 ⫾ 25.6* 206.1 ⫾ 32.9* 421.7 ⫾ 174.3* 137.4 ⫾ 17.1 136.1 ⫾ 33.1 150.2 ⫾ 21.2 262.6 ⫾ 58.3* 124.9 ⫾ 45.9 101.8 ⫾ 22.8 72.4 ⫾ 34.4 277.1 ⫾ 88.5* 246.9 ⫾ 54.4* 158.8 ⫾ 42.3 118.9 ⫾ 39.0 288.9 ⫾ 95.0 163.0 ⫾ 17.9*

248.0 ⫾ 84.3** 173.2 ⫾ 34.8** 130.4 ⫾ 34.8 254.8 ⫾ 69.9** 322.1 ⫾ 95.6** 160.7 ⫾ 34.2** 104.6 ⫾ 23.8 151.3 ⫾ 43.0 102.2 ⫾ 42.0 141.1 ⫾ 27.7 149.5 ⫾ 49.2 193.1 ⫾ 49.6** 125.6 ⫾ 74.2 253.4 ⫾ 76.5** 161.8 ⫾ 92.1 74.3 ⫾ 30.6 111.9 ⫾ 27.8 119.3 ⫾ 95.3 298.7 ⫾ 81.8**

194.9 ⫾ 5.2** 176.0 ⫾ 40.9* 113.4 ⫾ 20.8 134.3 ⫾ 19.7* 141.0 ⫾ 23.9* 134.5 ⫾ 24.0 171.8 ⫾ 49.1* 132.0 ⫾ 6.0* 141.6 ⫾ 23.7* 154.2 ⫾ 18.8** 135.6 ⫾ 24.5* 154.7 ⫾ 36.8* 156.5 ⫾ 31.4* 166.2 ⫾ 20.4** 138.7 ⫾ 30.4 126.6 ⫾ 16.6* 171.9 ⫾ 51.7* 184.9 ⫾ 39.7** 173.8 ⫾ 91.4

a

Chitin/␤-glucan exposure was as analyzed by fluorescence-activated cell sorter (FACS) analysis upon staining with calcofluor white or an anti-␤-glucan antibody. Results shown represent means (n ⱖ 3). For statistics (unpaired Student’s t test), single asterisks ⫽ P ⬍ 0.05 and double asterisks ⫽ P ⬍ 0.01 (in comparison to wild-type C. glabrata cells). b The release of TNF-␣ was measured by ELISA. c ROS production by MDMs was measured by luminol-enhanced chemiluminescence.

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FIG 2 Mutant replication within MDMs. (A) Representative microscopy pictures of C. glabrata phagocytosed by MDMs. The bright-field picture shows all phagocytosed yeasts. Yeast cells were stained with FITC (shown in green) prior to internalization. The dye is not transferred to daughter cells, allowing differentiation of mother cells (red arrow) and daughter cells (white arrow) within MDMs in the merged picture. DIC, differential interference contrast. (B) Quantification of intracellular replication for wild-type C. glabrata and mutant cells 6 h after infection (MOI of 2). (Results shown represent the means ⫾ SD; n ⱖ 3.) For statistics (unpaired Student’s t test), single asterisks ⫽ P ⬍ 0.05 and double asterisks ⫽ P ⬍ 0.01 (in comparison to wild-type C. glabrata cells).

FIG 3 Correlation between disturbed surface integrity and enhanced TNF-␣ production by MDMs. A scatter plot of chitin/␤-glucan exposure measurements from a TNF-␣ release assay for each mutant is shown. By correlation analysis (GraphPad Prism 5), the relationship between chitin exposure and TNF-␣ results were statistically significant. ns, not significant; ⴱ, P ⬍ 0.05.


able to inhibit ROS production or to detoxify ROS (28). We therefore tested macrophage ROS levels in response to each mutant. Interestingly, the majority (15 of 23) of the mutants stimulated ROS production to a significantly higher degree than the wild type (Table 4). This included eight mutants with increased exposure of either ␤-glucan or chitin. Therefore, the reduced survival of several identified C. glabrata mutants is associated with cell surface alterations, atypical cytokine induction, and elevated levels of reactive oxygen species.

TABLE 5 Mutants with a reduced growth ability under nutrient-limited conditions % WT ⫾ SDa Gene no.

Gene designation

1 2 4 7 8 14 20 21 22 23

CCH1 SLM1 ARG81 FRE8 CAGL0M12496g MNN4 CDC12 CKA2 HEK2 MPS3

a

t (ODmax/2)

ODmax

10% YPD

N deficit

C deficit

Fe deficit

10% YPD

N deficit

C deficit

Fe deficit

107.14 ⫾ 4.17 102.65 ⫾ 3.45 106.49 ⴞ 2.08* 101.20 ⫾ 3.54 101.87 ⫾ 5.35 98.91 ⫾ 3.92 103.09 ⫾ 2.99 101.50 ⫾ 2.26 101.88 ⫾ 4.84 91.50 ⫾ 9.03

98.77 ⫾ 13.74 93.23 ⫾ 10.55 92.68 ⫾ 3.68 99.04 ⫾ 14.15 93.62 ⫾ 9.94 101.34 ⫾ 1.23 101.75 ⫾ 3.74 106.99 ⴞ 0.73* 100.55 ⫾ 2.56 104.34 ⫾ 4.50

94.18 ⫾ 11.21 90.79 ⫾ 13.45 89.74 ⫾ 17.16 90.67 ⫾ 19.83 88.28 ⫾ 16.60 96.72 ⫾ 6.57 94.40 ⫾ 12.39 88.13 ⫾ 4.28 85.28 ⫾ 5.43 86.82 ⫾ 17.39

131.95 ⴞ 23.87* 105.13 ⫾ 36.43 70.71 ⫾ 5.19 127.36 ⴞ 7.65* 146.64 ⴞ 12.70* 111.24 ⫾ 19.44 98.48 ⫾ 8.13 100.11 ⫾ 14.27 115.45 ⴞ 7.36* 134.26 ⴞ 24.37*

83.09 ⫾ 13.17 108.63 ⫾ 29.22 103.78 ⫾ 15.65 103.24 ⫾ 20.04 97.45 ⫾ 15.79 107.55 ⫾ 5.12 96.62 ⫾ 14.89 92.96 ⫾ 16.43 89.52 ⫾ 14.04 83.87 ⫾ 7.30

96.10 ⫾ 28.18 74.76 ⫾ 15.71 89.13 ⴞ 3.97* 101.97 ⫾ 3.41 106.84 ⫾ 7.97 89.29 ⫾ 3.13 81.79 ⫾ 8.99 84.55 ⫾ 6.94 99.37 ⫾ 9.08 85.10 ⴞ 4.20*

91.45 ⫾ 20.72 86.13 ⫾ 43.60 104.18 ⫾ 20.86 114.29 ⫾ 16.83 122.71 ⫾ 8.11 90.65 ⫾ 12.30 99.22 ⫾ 12.72 126.00 ⫾ 25.23 128.45 ⫾ 36.99 115.25 ⫾ 14.50

72.75 ⴞ 12.55* 67.67 ⴞ 8.55* 92.31 ⫾ 7.61 83.88 ⴞ 11.06* 45.86 ⴞ 16.57* 84.44 ⴞ 7.02* 83.80 ⴞ 6.23* 101.85 ⫾ 12.79 74.30 ⴞ 7.05* 83.40 ⫾ 18.32

Results shown represent means (n ⱖ 3). For statistics (unpaired Student’s t test), single asterisks ⫽ P ⬍ 0.05 (in comparison to wild-type C. glabrata cells).


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FIG 4 Mutants with enhanced oxidative stress susceptibility. In drop tests, serial dilutions (1 ⫻ 105 to 1 ⫻ 101) of wild-type and mutants cells were spotted on YPD or SD agar plates containing H2O2 (7.5 mM) or menadione (60 ␮M). n ⱖ 3; representative pictures are shown.

Resistance to phagosome-simulative conditions. The macrophage phagosome is a hostile environment for microorganisms, and responding to or counteracting this environment has been shown to be crucial for survival and replication of several intracellular pathogens (4–6). Therefore, we tested all mutants under defined in vitro conditions which resembled different aspects of the environment within a phagosome. Oxidative stress resistance. The oxidative-stress sensitivity of mutants was tested by plating serial dilutions of C. glabrata mutants on SD or YPD agar plates, each containing either H2O2 (7.5 mM) or menadione (60 ␮M). Nine of 23 mutants showed reduced growth when exposed to one or both stressors in comparison to the wild type (Fig. 4). Five of these mutants also induced higher ROS production in MDMs, suggesting a role for oxidative stress in the poorer survival of these mutants. To further examine this correlation, we repeated the survival assay based on CFU readout in the presence of apocynin, a specific NADPH oxidase inhibitor. Apocynin had no inhibitory effect on growth of C. glabrata alone (not shown). The effectiveness of this substance in blocking ROS production by MDMs was confirmed by a lack of luminol-enhanced chemiluminescence in MDMs stimulated with PMA. However, even in the presence of the NADPH oxidase inhibitor, the survival rate of the mutants which induced higher ROS levels was still decreased (not shown). Therefore, elevated ROS production by MDMs alone, or in combination with a higher susceptibility to ROS, cannot be the sole explanation for the decreased survival of the mutants. Nutrient starvation. It is generally believed that the phagosome is a nutrient-poor environment (4). To analyze replication of the identified 23 mutants under conditions of nutrient starvation, we measured growth rates by recording growth curves (OD at 600 nm) in diluted complex medium (10% YPD) and in minimal medium with reduced concentrations of ammonium sulfate (nitrogen source) or glucose (carbon source) (Table 1). In these media, the wild type showed reduced but measurable growth. As measures of growth, the maximum OD (ODmax) and the time to reach half this value [t (ODmax/2)] were calculated for each condition. Unexpectedly, only one mutant (arg81⌬) grew slower than the wild type in diluted YPD, and only three mutants had a significantly longer generation time than the wild type under conditions of nitrogen or glucose limitation (Table 5). In addition to carbon and nitrogen, the trace element iron is essential for intracellular growth, and pathogens employ a num-

Seider et al.

ber of strategies to acquire iron in the host. We therefore investigated the effect of iron chelation in minimal medium on growth of the mutants. Growth of eight of the mutants was strongly inhibited under conditions of iron limitation (Table 5). Since iron limitation inside the phagosome may contribute to the intracellular antimicrobial conditions, a reduced ability to acquire iron likely contributes to the reduced survival of these mutants after coincubation with MDMs. Interaction with human neutrophils. To analyze whether the reduced ability to survive within macrophages was specific to macrophage-generated stresses or to a general disability to deal with phagocytic cells, the 23 mutants were also exposed to human neutrophils (Fig. 5). Only two mutants were significantly reduced in survival after 3 h of coincubation, and a further four mutants had moderate survival rates (Fig. 5A). These six mutants were subjected to further analyses. The erg5⌬ and pmt4⌬ mutants were less efficiently phagocytosed by human neutrophils (after 3 h and after 30 min and 3 h, respectively) (Fig. 5B). Quantification of fungal replication showed that growth of wild-type cells occurred outside neutrophils (about 40% daughter cells 3 h after infection); however, in contrast to macrophages, intracellular growth was strongly inhibited (only about 8% wild-type daughter cells) (Fig. 5C). There was no significant difference measurable between the wild type and the six selected mutants. Production of reactive oxygen species by neutrophils was induced by viable wild-type C. glabrata cells, but the level of produc-

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tion was surpassed even by heat-killed wild-type cells and all mutants tested (Fig. 6). Five mutants showed significant elevated levels of ROS compared to the wild type, with the pmt4⌬ mutant being the most potent stimulant.

FIG 6 Production of reactive oxygen species (ROS) by neutrophils infected with C. glabrata wild-type and mutant cells. ROS production was measured by luminol-enhanced chemiluminescence upon infection of neutrophils with C. glabrata wild-type and mutant cells (MOI of 5). Unstimulated neutrophils and neutrophils treated with phorbol myristate actetate (PMA) served as controls. (Results shown represent means ⫾ SD; n ⱖ 3). For statistics (unpaired Student’s t test), triple asterisks ⫽ P ⬍ 0.005 (in comparison to wild-type C. glabrata cells).

Eukaryotic Cell


FIG 5 Survival, uptake, and replication of mutants during interaction with human neutrophils. (A) Neutrophils were infected with opsonized C. glabrata cells at an MOI of 1. Killing was measured by CFU plating 3 h postinfection, and values are presented as percent wild type. (B) To quantify uptake, neutrophils were infected with opsonized C. glabrata cells (MOI of 1) and neutrophils with internalized yeast cells were counted microscopically upon differential staining. (C) Quantification of extra- and intracellular replication of FITC-labeled wild-type and mutant cells 3 h after infection (MOI of 1). (Results shown represent means ⫾ SD; n ⱖ 3.) For statistics (unpaired Student’s t test), single asterisks ⫽ P ⬍ 0.05, double asterisks ⫽ P ⬍ 0.01, and triple asterisks ⫽ P ⬍ 0.005 (in comparison to wild-type C. glabrata cells).


DISCUSSION


phage, the majority (16 of 23) had undergone fewer cell divisions than the wild type at that time point. The role of cell wall integrity for recognition and intracellular survival. The fungal cell wall constitutes the interface between fungal and host cells and mediates the initial steps of host-fungus interactions: mutual recognition and activation of signal transduction via host receptors. Since such interactions likely lead to the induction of antimicrobial mechanisms, we investigated whether cell wall disturbances might contribute to enhanced macrophage activation in response to the presence of C. glabrata mutants. The cell walls of fungi are normally composed of an inner skeletal layer consisting of chitin and ␤-glucan linked to the outer layer, which is made up of heavily mannosylated proteins and phospholipomannan (36–39). These cell wall layers not only provide cells with structural support and protection but can also prevent or evoke strong immunostimulatory responses. Alterations in the accessibility of immunostimulatory cell wall components, as we observed for 12 of the mutants, may be responsible for stronger macrophage activation and more-potent antimicrobial activity. In addition, macrophages can be activated by a proinflammatory response. In particular, ␤-glucan plays a key role in immune recognition. Recognition of this cell wall component by macrophages and neutrophils can trigger phagocytosis, the induction of proinflammatory cytokines, and the generation of ROS (40–44). Recent studies have also demonstrated an immune-modulatory role for chitin (45). Intact yeast cells of C. albicans or S. cerevisiae have low reactivity with dectin-1, the receptor which mediates ␤-glucan recognition. However, when the integrity of the cell wall is perturbed and ␤-glucan is exposed, binding of dectin-1 facilitates recognition and elicits macrophage activation and production of cytokines such as TNF-␣ (43). To investigate the impact of cell wall alterations of C. glabrata mutants on fungal recognition by MDMs, we monitored TNF-␣ production upon cocultivation. We found that unmasking C. glabrata ␤-glucan and/or chitin caused an altered inflammatory response by MDMs, indicated by TNF-␣ induction. Indeed, a statistically significant correlation between increased chitin content and enhanced TNF-␣ production was observed. In addition, half of the mutants with increased ␤-glucan exposure and two-thirds of the mutants with increased chitin content also induced higher ROS production. Cell surface compositions and altered recognition of cell wall components, therefore, have a major impact on subsequent steps of the macrophage response. Together, our data support the hypothesis that ␤-glucan and/or chitin accessibility in the cell wall of C. glabrata is important for recognition and activation of the host immune system. In contrast, Keppler-Ross et al. (2010) have reported cell wall mannan, rather than ␤-glucan or chitin, as a key ligand for recognition of S. cerevisiae, C. glabrata, and C. albicans (10). This observation may explain why C. glabrata and S. cerevisiae are more efficiently phagocytosed by macrophages than C. albicans, as both species contain a much higher mannose-to-glucose ratio than C. albicans. In fact, modified levels of mannoproteins in the cell wall can alter the immune response and reduce fungal survival (46). In our study, we found a link between dysfunctions in protein glycosylation and decreased phagosomal survival. A Gene Ontology (GO)-term analysis of all disrupted genes revealed a significant enrichment of genes associated with protein glycosylation (GO: 0006493) in our subset of mutants with decreased intracellular

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Previous studies have shown that the success of C. glabrata as a pathogen is due not only to its high antifungal resistance but also to the fact that this yeast has successfully developed adaptation and immune evasion strategies, enabling the fungus to survive, disseminate, and persist within mammalian hosts (22–24, 26). Like many other facultative intracellular pathogens, C. glabrata is able to survive phagocytosis by macrophages (25–27). Intracellular survival mechanisms have been described for a number of pathogenic microorganisms (4, 6–8); however, the survival mechanisms of C. glabrata are less well described. Rai et al. demonstrated a role of chromatin remodeling and associated reprogramming of cellular energy metabolism for C. glabrata phagocytosed by a human macrophage cell line (29). These mechanisms seem to facilitate long-term adaptation of internalized C. glabrata. Using a C. glabrata knockout library, we aimed at identifying factors that (i) are crucial for short-term survival of the fungus and which may allow early adaptation to primary human macrophages or (ii) may counteract killing mechanisms of these phagocytes or (iii) are involved in recognition by primary human macrophages. Of 433 gene deletion mutants, 23 exhibited decreased survival. Eight of the corresponding genes were upregulated (at least 1.5fold differences at one or more time points [10 min, 30 min, and/or 180 min]) during interaction with MDMs in a previously performed transcriptional analysis (28). The induction of these genes (CCH1, SLM1, ARG81, GNT1, MNN4, BAR1, CDC12, and MPS3) suggested a putative function of their gene products in response to phagocytosis by macrophages. Enhanced uptake and reduced intracellular replication may contribute to reduced survival. All 23 mutants were efficiently internalized by macrophages. Indeed, four mutants were internalized in significantly greater numbers than the wild type. For these four mutants, reduced survival at 3 h postinfection may be explained by an extended time for the macrophages to apply their antimicrobial armamentarium or by increased recognition and stimulation. For example, an acapsular Cryptococcus neoformans strain which is more efficiently phagocytosed by macrophages results in higher proinflammatory cytokine production (35). Therefore, avoidance of uptake may be a key strategy of C. glabrata wild-type cells which prevents killing by macrophages. However, as wild-type C. glabrata cells are efficiently internalized by macrophages in vitro (even more so than S. cerevisiae and C. albicans cells [10]), avoidance of phagocytosis is less likely to represent an immune evasion mechanism of C. glabrata. In contrast, the ability of C. glabrata cells to subsequently resist killing and multiply within macrophages argues in favor of an intracellular survival strategy. Why then are the mutants identified in this study less able to survive inside the phagosome? Survival rates of C. glabrata mutants within MDMs 3 h after infection are the net result of killing and intracellular replication. Our own previous experiments showed that the wild type produces only a few (⬃20% [28]) daughter cells within this early time period. Therefore, a lower level of intracellular replication can have only a minor impact on net survival at this time point. However, replication deficiencies may reflect a general impairment with respect to withstanding antimicrobial effectors within the phagosome. We therefore examined replication at 6 h of infection, a time point at which around 30% of the wild-type intracellular population consisted of daughter cells. Although all mutants replicated within the macro-

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hypersensitive to at least two of the following three conditions: osmotic stress, cell wall stress, and heat stress. Interestingly, all of these mutants were more susceptible to caspofungin. As T. Schwarzmüller, B. Ma, and K. Kuchler (Schwarzmüller et al., submitted) tested the whole 433-mutant library under these stress conditions, we were able to perform contingency analyses using Fisher’s exact test. We found a significant enrichment of caspofungin-sensitive mutants in the macrophage-attenuated subset compared to the entire C. glabrata mutant library (P ⫽ 0.0037). It can be concluded that these mutants have defects in the assembly, remodeling, or stress response of the cell wall. However, we did not observe a strong correlation between sensitivity to cell wall stresses and ␤-glucan or chitin content. Overall, 75% of the survival-deficient mutants exhibited altered cell wall composition and/or enhanced sensitivity to cell wall stress, suggesting that maintenance of a robust cell wall is crucial for surviving the hostile environment of the phagosome. Intracellular survival and nutrient acquisition. The microbicidal activities of the phagosome as well as its maturation process have been extensively studied (3, 4). The nutritional status of the phagosome, however, is still rather unclear. It is generally believed that the phagosome is a nutrient-poor environment. For instance, Mycobacterium tuberculosis secretes a siderophore to acquire iron within the phagosome (55), and phagocytosed strains of both M. tuberculosis and C. albicans exhibit a starvation response, with induction of the glyoxylate cycle instead of glycolysis (56, 57). In addition, Roetzer et al. (2010) have demonstrated the importance of autophagy for C. glabrata intracellular survival within macrophages (27). Thus, we reasoned that adaptation to nutrient limitation is essential for C. glabrata intraphagosomal replication. However, upon exposure to carbon or nitrogen limitation, very few (three) mutants exhibited moderately reduced growth parameters. It seems unlikely that these moderate growth defects under conditions of carbon or nitrogen limitation account for the reduced survival of these mutants. Iron limitation, on the other hand, had a much stronger effect on growth of the mutants with reduced survival in macrophages. Altogether, 11 of these mutants were not able to cope efficiently with iron restriction. Within the phagosome, iron is sequestered from microbes via the action of intraphagosomal scavengers and transporters in the phagosomal membrane, such as lactoferrin or natural resistance-associated macrophage protein 1 (NRAMP1) (58–60). Facultative intracellular pathogens have developed different mechanisms to counteract such “nutritional immunity”: H. capsulatum, for instance, secretes a siderophore which is indispensable for growth within macrophages and for virulence in a mouse infection model (61). Replicating C. glabrata cells, accordingly, should possess one or more mechanisms to obtain iron within the phagosome. Of the 23 tested C. glabrata mutants, only 1 lacks a gene (FRE8) annotated as involved in iron acquisition; in fact, this mutant exhibited reduced growth upon iron limitation. The requirement of the other genes for growth under conditions of limited iron availability is less obvious, as they are annotated as involved in diverse cellular processes; however, the observed phenotypic correlation strongly suggests that iron acquisition is an important event for intracellular survival and replication of C. glabrata within the phagosome of macrophages. Survival of C. glabrata mutants in neutrophils. Even though

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survival and/or replication compared to the whole set of 433 deletion mutants (P ⫽ 0.011). In general, it would appear that no single gene or cell wall component is responsible for resistance of C. glabrata to killing by macrophages. Instead, we propose a model whereby an intact outer network (probably consisting of mannan) shields the inner layer from the immune system and that this surface triggers a specific recognition and uptake mechanism, which results in efficient phagocytosis but not killing of C. glabrata. Concurrently, if this shield is disturbed, the ability of C. glabrata to survive and replicate in macrophages is severely impaired. Reduced stress resistance correlates with reduced intracellular survival. In order to assess the resistance of our mutants to stress, we analyzed the growth of each strain under conditions of oxidative stress. Furthermore, Schwarzmüller et al. (T. Schwarzmüller, B. Ma, and K. Kuchler, submitted for publication) kindly provided data on the ability of mutants to withstand osmotic stress (NaCl), cell wall stress (caspofungin, Congo red), and heat stress (42°C) (results are summarized in Table 3). The majority (15/23) of mutants exhibited growth defects under at least one of these conditions, suggesting that, in general, lower tolerance to environmental stress attenuates the ability to survive the harmful conditions within the phagosome. The production of reactive oxygen metabolites is a key event following phagocytosis (47), and, as described above, the majority of the mutants induced ROS. Both C. glabrata and C. albicans produce enzymes and molecules to evade oxidative killing, which may represent an important immune evasion mechanism (48). For C. albicans, fungal detoxifying enzymes, such as superoxide dismutases, were shown to be crucial for counteracting the oxidative burst (49–51). Moreover, mutants lacking these enzymes were more susceptible to killing by macrophages and neutrophils, suggestive of ROS-dependent killing activity against this fungal pathogen. However, C. glabrata is highly resistant to oxidative killing compared to C. albicans and S. cerevisiae (52). This is substantiated by our finding that, while many mutants induced an enhanced ROS release by MDMs, none of these mutants showed higher survival rates when ROS production was inhibited. Hence, killing or inhibition of replication must occur via additional mechanisms. In vitro, resistance to oxidative stress is mediated mainly by activation of the transcription factors Skn7p, Yap1p, Msn2p, and Msn4p and the production of detoxifying enzymes such as the catalase Cta1p (52). Interestingly, of all the genes required for wild-type ROS resistance or detoxification, only the ortholog of SHO1 has previously been implicated in oxidative stress resistance in C. albicans (53). In addition, none of the mutants lacking a gene with a known function in ROS detoxification (e.g., genes encoding catalase, superoxide dismutases, peroxidases, or thioredoxin proteins) which were included in our mutant collection were found to have reduced survival following coincubation with MDMs. This suggests that C. glabrata possesses robust and redundant antioxidant systems and that ROS play a relatively minor role in killing of this fungus. However, since some of the 23 mutants with reduced survival in MDMs were more susceptible to H2O2 or menadione in vitro, it is possible that ROS act in combination with other stresses or microbicidal mechanisms inside the phagosome and thus may at least partially contribute to killing of C. glabrata (54). Several of the mutants with reduced intracellular survival were



ACKNOWLEDGMENTS This work was supported by the German Federal Ministry of Education and Health (BMBF) via the Center for Sepsis Control and Care (CSCC) (FKZ: 01EO1002) and the ERA Pathogenomics program (0313931B) and by the Deutsche Forschungsgemeinschaft (DFG) via the SPP1580 Intracellular Compartments as Places of Pathogen-Host Interaction (Hu528/ 15-1 and 16-1) and via the DACH program (Hu528/17-1). S.B. was supported by a grant from the Studienstiftung des deutschen Volkes; K.K. was supported by the Fonds zur Förderung der wissenschaftlichen Forschung (FWF) via the ERA Pathogenomics program (FWF-API0125-B08) and the DACH program (FWF-I746-B11) and in part by a grant of the Christian Doppler Society. We thank Duncan Wilson for critical reading of the manuscript, Marie Kristin Vorberg for technical support, and Betty Hebecker, Anja Lüttich, and Antje Heyken and the members of the FunPath consortium (www .pathogenomics-era.net) for helpful discussions.

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neutrophils do not appear to be responsible for the reduction of the C. glabrata burden during experimental infections of mice (24, 62), neutropenia is a major risk factor for life-threatening systemic fungal infections of human patients (63). As key players during C. albicans infections (49, 64), these phagocytes have also been shown to kill 62% of wild-type C. glabrata cells within 1 h in vitro (65). This is in contrast to the observed survival and replication of C. glabrata within macrophages in vitro and suggests different recognition and/or killing mechanisms by neutrophils. We were therefore interested in determining whether factors important for macrophage survival are also crucial for the interaction with neutrophils. Only 2 of the 23 mutants shown to be attenuated in surviving phagocytosis by macrophages in this study (the CAGL0M12496g⌬ and erg5⌬ mutants) were significantly impaired in resistance to killing by neutrophils, and a further 4 mutants showed moderate attenuated survival rates (below 80% of wild-type levels). We were not able to detect differences in extraor intracellular replication of these six mutants, and replication within neutrophils was overall low (⬍10%) for wild-type and mutant cells, suggesting that the conditions caused by the presence of neutrophils were too harsh for C. glabrata to multiply. We also tested the activation state of infected neutrophils by measuring the production of the cytokine interleulin-8 (IL-8), myeloperoxidase concentrations, and the expression of surface markers (CD66b and CD11b) (not shown). However, no differences after infection with wild-type or mutant cells were detected. Oxidative-killing mechanisms, however, seem to play a role in killing by neutrophils more prominent than that of macrophages: all mutants analyzed induced significantly more ROS in neutrophils compared to the wild type, and killing of wild-type yeasts was invertibly blocked by addition of the NADPH oxidase inhibitor apocynin (not shown). The highest induction was observed for the mutant pmt4⌬ lacking a gene potentially involved in protein mannosylation. This mutant is characterized by an elevated exposure of ␤-glucan and chitin content and enhanced ROS and TNF-␣ production by macrophages (Table 4), supporting the view that an intact cell wall of C. glabrata is required for survival of the hostile environment of phagocytes. Conclusions. In summary, the screening of 433 C. glabrata mutants for survival within macrophages revealed a set of 23 genes (no. 1 to 18) with putative functions in response to stress, cell wall and membrane architecture, glycosylation, and nutritional sensing and acquisition—all functions predicted to be involved in resistance to the harsh conditions of the phagosome. Other genes (no. 19 to 23) have diverse functions, and their importance for intracellular survival was unexpected. Extensive phenotypic screening of mutants with defective survival in macrophages revealed a relationship between survival and growth under conditions of iron but not carbon or nitrogen limitation. Genes involved in cell surface integrity and stress resistance appeared to be particularly important for macrophage survival. Alterations of cell wall compositions induced both ROS and TNF-␣ production, highlighting the importance of the cell wall as a barrier shielding yeasts from immune recognition. Experiments with isolated neutrophils also substantiate this hypothesis. Together, our data significantly extend the number of known genes required for successful intracellular survival of C. glabrata and highlight some of the cellular processes involved.

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Establishment of an in vitro system to study intracellular behavior of Candida glabrata in human THP-1 macrophages.

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Acquired Flucytosine Resistance during Combination Therapy with Caspofungin and Flucytosine for Candida glabrata Cystitis.

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Proteomic Analysis of Intracellular and Membrane Proteins From Voriconazole-Resistant Candida glabrata.