Immunoproteomic Analysis of Antibody Responses ... - ACS Publications

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Immunoproteomic analysis of antibody responses to extracellular proteins of Candida albicans revealed the importance of glycosylation for antigen recognition Ting Luo, Thomas Krüger, Uwe Knüpfer, Lydia Kasper, Natalie Wielsch, Bernhard Hube, Andreas Kortgen, Michael Bauer, Evangelos J. GiamarellosBourboulis, George Dimopoulos, Axel A. Brakhage, and Olaf Kniemeyer J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b01065 • Publication Date (Web): 08 Jul 2016 Downloaded from http://pubs.acs.org on July 8, 2016

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Journal of Proteome Research

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Immunoproteomic analysis of antibody responses to extracellular proteins of Candida albicans revealed the

importance

of

glycosylation

for

antigen

recognition Ting Luo1,2, Thomas Krüger1,Uwe Knüpfer3, Lydia Kasper4, Natalie Wielsch5, Bernhard Hube2,4,6, Andreas Kortgen2,7, Michael Bauer2,7, Evangelos J. Giamarellos-Bourboulis2,8, George Dimopoulos9, Axel A. Brakhage1,6, Olaf Kniemeyer1,2,6* 1

Department of Molecular and Applied Microbiology, Leibniz-Institute for Natural Product

Research and Infection Biology – Hans Knöll Institute (HKI), 07745 Jena, Germany 2

Center for Sepsis Control and Care (CSCC), Jena University Hospital, 07747 Jena, Germany

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Bio Pilot Plant, Leibniz-Institute for Natural Product Research and Infection Biology (HKI),

07745 Jena, Germany 4

Department of Microbial Pathogenicity Mechanisms, Leibniz-Institute for Natural Product

Research and Infection Biology (HKI), 07745 Jena, Germany 5

Department of Mass spectrometry/Proteomics, Max-Planck-Institute for Chemical Ecology,

07745 Jena, Germany 6

Friedrich Schiller University Jena, Institute of Microbiology, 07743 Jena, Germany

ACS Paragon Plus Environment

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Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, 07747

Jena, Germany 8

The 4th Department of Internal Medicine, University of Athens, Medical School, Greece and

Hellenic Sepsis Study Group 9

The second Department of Critical Care Medicine, University of Athens, Medical School,

Greece and Hellenic Sepsis Study Group

ABSTRACT

During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues and modulates the host´s immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. Based on the screening of sera from candidemia and three groups of non-candidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1 and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from non-candidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the


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recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patients´ sera.

KEYWORDS: Candida albicans, secretome, yeast-to-hypha transition, extracellular proteins, antigens, immunoproteomics, glycosylation, SERPA

INTRODUCTION Candida albicans is a common opportunistic fungal pathogen, which normally resides on mucosal surfaces of healthy individuals as a harmless commensal. However, it can cause superficial infections and even disseminated candidemia in immunocompromised and critically ill patients. To adapt to the different host niches and bypass the host defense, C. albicans has developed different virulence traits. A main feature of C. albicans is its ability to switch from yeast to hyphal growth. It is generally believed that the yeast form is more associated with commensalism, whereas the hyphal form represents the invasive form which invades epithelial and endothelial cells thereby causing damage1-4. During the transition from yeast to hyphal growth not only the cell wall composition and stress tolerance of C. albicans changes, but also the type and number of secreted proteins5. Secreted hydrolytic enzymes of yeast and hyphal cells, like lipases and proteases, contribute to invasion, tissue degradation and nutrient acquisition6. Furthermore, they have been shown to induce apoptosis in epithelial cells and an inflammatory response in macrophages7, 8. Therefore, it is generally accepted that secreted components are involved in nutrient acquisition, immune evasion and the virulence of C. albicans. This view is consistent with the fact that C. albicans mutants with defective secretion pathways are less virulent in murine models of candidiasis9, 10.


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In eukaryotes, like C. albicans, most secreted proteins enter the lumen of the rough endoplasmic reticulum (ER) as the first organelle of the secretory pathway via an N-terminal hydrophobic signal peptide. There, the secretory proteins are properly folded and post-translationally modified. Subsequently, they are translocated to the Golgi complex, where they are further modified and packed in vesicles for the transport to the cell surface (or the vacuole). Transmembrane proteins and glycosylphosphatidylinositol (GPI)-anchored proteins are retained in the cell membrane or further moved to the cell wall and covalently attached to glucans via a truncated GPI anchor (GPI-proteins)

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. The soluble secreted proteins are either continuously

released (constitutive secretory pathway) or stored in granules for rapid discharge upon certain stimulatory signals (regulated secretory pathway)12. According to in silico prediction, the putative secretome of C. albicans comprises 225 different proteins (3.1% of the proteome)5. It is therefore slightly larger than those of other yeasts, for instance C. glabrata (121), Pichia pastoris [Komagataella pastoris] (105), and Saccharomyces cerevisiae (156)5, but considerably smaller in comparison to saprophytic filamentous fungi like Aspergillus niger with 832 predicted secreted proteins (5.9% of the genome)5, 13. In a previous C. albicans LC-tandem mass spectrometry-based secretome analysis, Sorgo et al. reported about 44 secretory proteins, but also 28 cytosolic proteins without predicted ER signal peptides detected in the growth medium of C. albicans14. Recently, Gil-Bona et al. presented evidence, that extracellular vesicles (EV) of C. albicans carry cytosolic proteins to the outside of cells, which represents an alternative transport mechanism for the secretory pathway15. It was shown, that many so-called moonlighting proteins like the glycolytic enzymes Eno1, Pgk1 and Tdh3 as well as the translation elongation factor Tef2 are exclusively detected in extracellular vesicles of C. albicans (EV), but not in EV-free supernatants15. Furthermore, C. albicans EVs are


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immunologically active and have the potential to modulate the host immune response16. Alternatively, these cytosolic proteins could be released by damaged or dying cells within a natural population of cells. Extracellularly secreted proteins of C. albicans were shown to interact with host immune cells7, 8. Although C. albicans protease-specific IgG antibodies have long been detected in sera of candidemia patients17, until now there are no global proteomic studies focusing on the host serological response to the C. albicans secretome. Most studies have mainly investigated the serological responses of candidemia patients to C. albicans cell wall-associated surface and intracellular proteins18-20. The results of these investigations suggested that monitoring C. albicans directed antibodies could help to diagnose Candida infections and predict their outcome. Pitarch et al.

21

demonstrated that anti-Hsp90 and anti-Eno1 antibodies could serve as

potential biomarkers for invasive candidiasis (IC) diagnosis in non-neutropenic patients. Likewise, a protein microarray study identified 13 cell surface antigens, which could discriminate acute candidemia from healthy individuals and non-infected patients22. Most of these proteins were associated with the oxidative stress response or drug resistance. The same study revealed stage-specific antibody responses during the acute, early and mid-convalescence phase of candidemia22. In short, immunoproteomic methods can contribute to diagnosing candidemia and predicting the patient outcome, but also provide valuable information about the type of C. albicans proteins expressed during the infection. However, all of these identified antigen-specific antibody patterns still have not led to a reliable tool to improve diagnosis of candidemia. The diagnostic “gold-standard” for Candida infections still remains the blood culture test. In addition, ELISA assays for the detection of circulating fungal antigens, such as ßglucan or mannan are frequently applied23.


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Here we present the first C. albicans yeast and hyphal secretome analysis by a combined 2D gel and LC-MS/MS approach. Further, we focused on the profiling of serological responses to C. albicans secreted proteins, which revealed a core set of 19 C. albicans-reactive IgGs with diagnostic value. Intriguingly, protein glycan moieties of some secreted proteins, such as Sap6, are involved in antibody recognition.

EXPERIMENTAL PROCEDURES Organism and growth conditions Candida albicans strain SC5314 was used throughout this study24. Yeast cells were pre-cultured overnight in liquid YPD medium (10 g/l yeast extract, 10 g/l peptone and 20 g/l glucose) on a rotary shaker at 200 rpm and 30 °C, later transferred to flasks containing YNBS (5 g/L ammonium sulfate, 1.7 g/L nitrogen base, 20 g/L sucrose, 100 mM citric acid – sodium citrate buffer, pH 4). Precultures in YNBS were used to inoculate a fermenter (Sartorius Stedim biotech Biostat® D-DCU 10L, Göttingen, Germany) for a batch cultivation of C. albicans yeast or hyphal cells. Fermenter cultivation of C. albicans For batch cultivation of yeast cells, a fermenter containing YNBS (5 g/L ammonium sulfate, 1.7 g/L nitrogen base, 20 g/L sucrose and 100 mM citric acid – sodium citrate buffer, pH 4) was inoculated with C. albicans yeast cells at an initial OD600 of 0.05. Cultivation was performed in a volume of 6 L at 30 °C, at a constant pressure of 400 mbar and at a maximum stirring rate of 400 rpm for 15 hours. For hyphal induction, 6L YNBS medium (5 g/L ammonium sulfate, 1.7 g/L


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nitrogen base, 20 g/L sucrose, 75 mM MOPS [3-(N-morpholino) propanesulfonic acid], pH 7.4), supplemented with 5 mM N-acetylglucosamine (GlcNAc) was inoculated with a preculture of C. albicans yeast cells as described above at a temperature of 37 °C, a constant stirring rate of 100 rpm and a continuous pressure of 400 mbar for 18 hours. Isolation of secreted proteins from growth media Harvested C. albicans yeast and hyphal cells were visually checked under the microscope. Cell viability tests were performed with propidium iodide staining. For the isolation of extracellular proteins, C. albicans cells were spun down at 12,000 × g and the supernatant was sterile-filtered with a 0.2 µm filter (PES, Millipore). Secreted proteins were precipitated overnight at 4 °C by the addition of 10 % (w/v) trichloroacetic acid (TCA) and 3 mg/ml DTT. After centrifugation for 1 hour at 30,000 × g at 4 °C the supernatant was discarded and protein pellets were washed two times with 90% (v/v) ice-cold acetone and centrifuged at 13,000 × g for 1 hour at 4°C. The pellets were air-dried for 10 min and resuspended in lysis buffer [7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.8% (v/v) pharmalyte (GE healthcare Life Sciences)] for 2D gel electrophoresis as described in Wartenberg et al.25. After solubilization of the protein pellet the sample was centrifuged at 20,000 × g for 15 min at room temperature and the supernatant was transferred to a new microcentrifuge tube. The protein concentration was measured using the BioRad Protein Assay Dye Reagent Concentrate (Bio-Rad, Munich). Gel-based Proteomics Two-dimensional gel electrophoresis (2-DE) For yeast and hyphal secretome analysis, 2D gel electrophoresis was carried out as described in Wartenberg et al. (2011)25 with slight modifications. The amount of 350 µg proteins per sample


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was loaded via sample cups onto the 24 cm IPG strips (Immobiline DryStrip, GE Healthcare, Germany) that covered a pH range of either 4-7 or 7-11NL (non-linear). Isoelectric focusing was performed using an IPGphor II device (GE Healthcare, Germany) with the following program: 4 h for gradient 300V, 4 h for gradient 600V, 4 h for gradient 1000V, 4 h for gradient 8000V and 48,000Vh at 8000V. Then IPG strips were equilibrated with SDS equilibration buffer (30% (v/v) glycerol, 6M urea, 75mM Tris-HCl, pH 8.8, 2% (w/v) SDS and 0.002% (w/v) bromophenol blue) containing 1% (w/v) DTT for 15 min with gentle agitation, followed by equilibration with SDS equilibration buffer containing 2.5% (w/v) iodoacetamide. The second dimension was performed using self-casted 12.5% (w/v) polyacrylamide Tris/HCl gels and an Ettan Dalt Six electrophoresis unit (GE Healthcare, Germany). Gel electrophoresis was performed at 15W/gel at 20 °C for 4 hours. Gels were stained by colloidal Coomassie Blue G25026. Protein identification using MALDI-TOF/TOF analysis Excised protein spots were in-gel digested with trypsin as described by Shevchenko et al. 199627. The tryptic peptides were extracted from gel pieces with extraction buffer (50% ACN in 0.05% TFA) and then dried down in a vacuum centrifuge. For MALDI-TOF/TOF analysis samples were reconstructed in 10 mg/ml HCCA resolved in solution [30% / 70% (v/v) ACN / 0.1% TFA]. The samples were measured on an ultrafleXtreme MALDI-TOF/TOF device (Bruker Daltonics, Germany). MS spectra (m/z 700-3500 Da) were acquired and the 5 most intense precursor ions were selected for MS/MS fragmentation. FlexControl 3.3 software was used for data collection and flexAnalysis 3.3 for spectra analysis and peak list generation. The generated MS and MS/MS spectra were analyzed by searching the Candida Genome Database (http://www.candidagenome.org/) using MASCOT 2.4.1 server


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(Matrix Science, UK). Search parameters were the following: oxidized methionine as variable modification, carbamidomethyl cysteine as fixed modification, one miscleavage and a peptide mass tolerance of 100 ppm and a fragment mass tolerance of 0.6 Da. Results were regarded as significant with an allowed likelihood for a random hit of p ≤ 0.05, according to the Mascot score. Database searches were performed and archived on a Proteinscape 3.0 database server (Bruker Daltonics, Germany). Three to four biological replicates of secretome samples from each morphological form were analyzed. Protein identification using LC-MS/MS analysis Samples not identified by MALDI TOF-TOF, were further analysed by LC-MS/MS. They were reconstructed in 10 µL aqueous 1% formic acid. Depending on the protein amount, 5-10 µL of the sample were injected into LC-MS/MS system consisting of a nanoAcquity UPLC system (Waters, Manchester, UK) on-line coupled to a Synapt HDMS tandem mass spectrometer (Waters, Manchester, UK). LC-MS/MS data were collected using data-dependent acquisition (DDA). Each acquisition cycle consisted of a survey scan covering the range of m/z 400-1500 amu followed by MS/MS fragmentation of the four most intense precursor ions collected over a 1 sec interval in the range of 50-1700 m/z. Dynamic exclusion was applied to minimize multiple fragmentations for the same precursor ions. DDA raw files were collected using MassLynx v4.1 software and processed using ProteinLynx Global Server Browser (PLGS) v.2.5 software (Waters, Manchester, UK) under baseline subtraction, smoothing, deisotoping, and lockmass-correction. Pkl files of MS/MS spectra were generated and searched against NCBInr database (updated December 5, 2012, installed on a local server) using MASCOT version 2.4. Mass tolerances for precursor and fragment ions were 15


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ppm and 0.03 Da, respectively. Other search parameters were: instrument profile, ESI-Trap; fixed modification, carbamidomethyl (cysteine); variable modification, oxidation (methionine); up to one missed cleavage was allowed. Gel-free LC-MS/MS analysis Protein sample preparation The secretome of C. albicans was also characterized by LC-MS/MS analysis. Protein pellets obtained after TCA precipitation were resolubilized in 50 µl 50 mM NH4HCO3 in 50%/50% (v/v) trifluoroethanol /water and ultrasonicated for 15 min and subsequently denaturated at 90 °C for 15 min. Cysteine residues were reduced by addition of 5 µL 200 mM dithiothreitol (DTT) and incubation for 20 min at 50 °C. Subsequently, reduced cysteine residues were carbamidomethylated for 30 min at room temperature in the dark by applying 5 µL 1 M iodoacetamide. Afterwards, samples were diluted approximately 1:10 with 445 µl 50 mM NH4HCO3 (pH 8) to gain a final TFE concentration of

Immunoproteomic Analysis of Antibody Responses ... - ACS Publications

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