Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Identification of immunogenic proteins of Candida parapsilosis by serological proteome analysis P.Y. Lee1, L.H. Gam2, V.C. Yong3, R. Rosli4, K.P. Ng5 and P.P. Chong1,6 1 2 3 4 5 6

Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia School of Biosciences, Taylor’s University (Lakeside Campus), Subang Jaya, Selangor, Malaysia Department of Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Department of Microbiology, Translational Infectious Diseases Program, Centre for Translational Medicine, National University of Singapore, Singapore

Keywords antigen, Candida parapsilosis, cell wall proteins, immunoproteomic. Correspondence Pei Pei Chong, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. E-mail: [email protected] 2013/2230: received 7 November 2013, revised 24 November 2013 and accepted 27 November 2013 doi:10.1111/jam.12408

Abstract Aims: Systemic candidiasis is the leading fungal bloodstream infection, and its incidence has been on the rise. Recently, Candida parapsilosis has emerged as an increasingly prevalent fungal pathogen, but little is known about its antigenic profile. Hence, the current work was performed to discover immunogenic proteins of C. parapsilosis using serological proteome analysis. Methods and Results: Cell wall proteins extracted from C. parapsilosis were resolved by two-dimensional electrophoresis followed by immunoblotting using antisera from experimentally infected mice. Mass spectrometry analysis of the 32 immunoreactive protein spots resulted in the identification of 12 distinct proteins. Among them, 11 proteins were known antigens of Candida albicans, whereas Idh2p was identified for the first time as an immunogenic protein of Candida species. Recombinant Idh2p was expressed in Escherichia coli, and its antigenicity was verified by immunoblot analysis. Conclusions: An immunoproteomic approach was successfully applied to identify immunogenic proteins of C. parapsilosis, with Idh2p as a novel candidate antigen. The identified antigens may serve as potential biomarkers for development of diagnostic assay and/or vaccine for C. parapsilosis. Significance and Impact of the Study: This work represents the first immunoproteomic analysis of C. parapsilosis, which provides new insights into host–pathogen interactions and pathogenesis of C. parapsilosis. The immunogenic proteins could be studied as biomarker candidates for C. parapsilosis.

Introduction Candida species is a member of normal human microflora that exists in healthy individuals without causing any disease. However, it is also an opportunistic fungal pathogen that can cause candidiasis, affecting mainly people with debilitated immune system. Candida spp. is the major human fungal pathogen and was ranked as the fourth organism causing nosocomial bloodstream infections in the United States (Wisplinghoff et al. 2004). Among all types of candidiasis, the systemic form is of foremost clinical importance as it is associated with high

mortality and morbidity rate (Ortega et al. 2011). The incidence of systemic candidiasis has increased markedly over the years with Candida albicans as the predominant species isolated. However, in recent years, there is a changing trend in the epidemiology of Candida infections worldwide (Pfaller and Diekema 2007). Of important note is the significant increase in the prevalence of Candida parapsilosis in several geographical locations across the globe (Forrest et al. 2008; Miceli et al. 2011). Likewise, the shift towards C. parapsilosis was also observed in Malaysia (Ng et al. 2001; Rahman et al. 2008; Siti Umairah et al. 2012).

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To date, diagnosis of candidiasis remains as a difficult task and current diagnosis still relies on microbiological blood culture method as the gold standard (Eggimann et al. 2011). This conventional diagnostic method is insensitive, time-consuming and often results in late diagnosis during advanced stages of infection. This subsequently causes delay in the antifungal treatment, which significantly reduces the survival rate of the patients. On top of that, current treatment strategies are also hampered by the adverse side effects of the antifungal drugs and emergence of resistant strains (Pappas et al. 2009). There is also no vaccine currently available for Candida spp. The outcome of infection is the result of complex interaction between the host and pathogen. Candida albicans and C. parapsilosis were shown to differ considerably in their pathogenicity and clinical outcomes (Arendrup et al. 2002; Bliss et al. 2012). This implies that each Candida spp. varies in its virulence factors and mechanism of pathogenesis. Identification of antigenic components that are able to elicit specific host immune response can facilitate the search of potential biomarkers for diagnostic and/or vaccine development. Cell wall proteins are potential source of useful disease biomarkers as they are important for initial contact with host cells and in mediating immune responses (Chaffin 2008). Serological proteome analysis or immunoproteomics is one of the key technologies that have the potential to identify disease-associated antigens as biomarkers. This approach can be used to discover novel candidate proteins that have not yet been identified. So far, most of the immunoproteomic studies focused primarily on C. albicans and have identified a number of antigens obtained from different protein preparations (Pardo et al. 2000; Pitarch et al. 2001, 2004; Hernando et al. 2007). Nevertheless, knowledge on antigenic proteins for other Candida spp. is limited, and their proteomes are not yet explored. In view of the emergence of C. parapsilosis and the lack of data regarding its antigenic proteins, there is a need to study its immunoproteome in order to characterize potential protein biomarkers. Advances in mass spectrometry and the availability of fully sequenced C. parapsilosis genome further encourage proteomic study of this species. In an attempt to discover antigenic proteins associated with C. parapsilosis, we embarked on an immunoproteomic approach using pooled sera from experimentally infected mice. Materials and methods Fungal strain Candida parapsilosis ATCC 22019 strain was used throughout the study. The strain was cultured and maintained on YPD agar plates (BD Difco, Sparks, MD). 1000

Single colony was obtained by streaking onto YPD plates and incubated at 30°C. Infection conditions and generation of immune sera BALB/c male mice ranging in age from five to 7 weeks, with a weight of about 20–25 g, were used. All animal studies were carried out according to the guidelines for the care and use of animals approved by Animal Care and Use Committee (ACUC), Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM/FPSK/ PADS/UUH/F05). An experimental model of systemic candidiasis was generated in mice as described before (Clancy et al. 2009). Inocula were prepared by diluting C. parapsilosis cell suspension to the desired concentration in phosphate-buffered saline (PBS). Mice were randomly divided into 10 animals per group and challenged with inocula at a density of 1 9 107 colony-forming unit (CFU) via lateral tail vein injection. Another group of mice was injected with PBS alone as negative control. All mice were observed daily for any signs of distress. Blood was collected from the mice 20 days postinfection by cardiac puncture, and sera obtained were stored at 70°C prior to analysis. Besides, the kidneys were aseptically removed, weighed and homogenized in ice-cold PBS for determination of fungal burdens. The homogenate was serially diluted in PBS and plated onto Sabouraud dextrose agar (BD Difco) plates containing ampicillin (100 lg ml 1). The plates were incubated at 37°C for 2 days. The number of colonies was counted as CFU and calculated as log10 CFU g 1 kidney tissue. Protein sample preparation Cell wall-associated proteins were prepared as described before by Pitarch et al. (2008) with slight modifications. Briefly, C. parapsilosis were grown in YPD broth at 30°C to OD600 of 0
8–1
0. The yeast cells were harvested by centrifugation and resuspended in ice-cold lysis buffer (10 mmol l 1 Tris buffer, pH 7
5) containing protease inhibitor cocktail (Sigma, St Louis, MO). Equal volume of chilled glass beads (0
45–0
55 lm, Sigma) were added to the cell suspension, and the cells were lysed by repeated cycles of vortexing with intermittent cooling on ice. The lysate was then centrifuged, and the pellets were boiled in extraction buffer (50 mmol l 1 Tris-HCl, pH 8
0, 0
1 mol l 1 EDTA, 10 mmol l 1 DTT, 2% SDS) at 100°C for 10 min to extract cell wall-associated proteins. Supernatant containing cell wall proteins was collected and precipitated with trichloroacetic acid (TCA) and acetone. Briefly, protein samples were mixed with ice-cold 100% TCA diluted to a final concentration of 10% in prechilled acetone and incubated at 20°C for 30 min.

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The samples were centrifuged at 10 000 g for 10 min at 4°C, and the pellet was washed twice with prechilled acetone. The pellet was then air-dried and dissolved in the solution containing 8 mol l 1 urea, 2 mol l 1 thiourea, 4% CHAPS, 2 mmol l 1 tributylphosphine (TBP, Bio-Rad, Hercules, CA) and 0
5% IPG buffer (GE Healthcare, Uppsala, Sweden). Protein concentrations were determined using RC DC Protein Assay (Bio-Rad) using BSA as standard. Protein samples were stored at 70°C before analysis. Two-dimensional electrophoresis Two-dimensional electrophoresis (2-DE) was performed as described previously by G€ org et al. (2000) with minor modifications. Protein samples were dissolved in rehydration buffer (8 mol l 1 urea, 2 mol l 1 thiourea, 4% CHAPS, 2 mmol l 1 TBP, 0
5% IPG buffer and 0
002% bromophenol blue) and loaded onto IPG strips by passive rehydration for 16 h. Analytical run was first carried out using 7 cm Immobiline DryStrip pH 3–10 and pH 4–7 (GE Healthcare) for optimization purposes. For preparative run, 13 cm Immobiline DryStrip pH 4–7 (GE Healthcare) was used, and isoelectric focusing was performed using Ettan IPGphor 3 Unit (GE Healthcare) in the following steps: 500 V for 1 h (step mode), 1000 V for 1 h (gradient mode), 8000 V for 2
5 h (gradient mode) and 8000 V for total 16 000 Vh. The strips were either directly processed or stored at 70°C prior to subsequent analysis. The focused strips were treated with equilibration buffer (0
375 mol l 1 Tris buffer, pH 8
8, 6 mol l 1 urea, 2% SDS, 20% glycerol) containing 2% DTT for reduction followed by alkylation in the same buffer with 2
5% iodoacetamide. Both steps were carried out at room temperature for 15 min. Equilibrated strips were then placed on top of 10% SDS-PAGE gel and sealed with ReadyPrepTM Overlay Agarose (Bio-Rad). Electrophoresis was performed using SE 600 Ruby (GE Healthcare) at 100 V for 30 min followed by running at 160 V until the dye reached the bottom of the gels. Gels were stained with SimplyBlueTM SafeStain (Invitrogen, Carlsbad, CA) and destained with deionized water. Triplicate runs were carried out to ensure the reproducibility of the experiment. Gel images were digitized using ImageScanner III (GE Healthcare) and visualized using LABSCAN 6.0 software (GE Healthcare). Western blotting Another set of similar protein gels was used for Western blot analysis to identify immunogenic proteins. Proteins from the 2-DE gels were transferred onto ImmobilonP polyvinylidene difluoride (PVDF) membranes (Milipore, Billerica, MA) using HEP-1 PantherTM Semidry

Iden of imm prot of Cp by SERPA

Electroblotter (Owl Separation Systems, Asheville, NC). Protein transfer was performed according to manufacturer’s recommendations under constant current of 3 mA cm 2 using Towbin buffer (25 mmol l 1 Tris, 192 mmol l 1 glycine and 20% methanol, pH 8
3). The membranes were blocked with 3% BSA in TBST (10 mmol l 1 Tris-HCl, 150 mmol l 1 NaCl, 0
05% Tween-20, pH 7
6) for 1 h at room temperature. After blocking, the membranes were washed with TBST for 5 min. The membranes were then probed with pooled sera from C. parapsilosis-infected mice diluted 1 : 1000 in TBST for overnight at 4°C. Pooled sera from uninfected mice at same dilution were used as negative control. After that, the membranes were washed three times with TBST for 5 min each and incubated with goat anti-mouse IgG peroxidase conjugate antibody (Calbiochem, San Diego, CA) diluted 1 : 40 000 in TBST for 1 h at room temperature. After incubation, the membranes were washed three times for 5 min each in TBST. The membranes were developed with SuperSignalâ West Femto Chemiluminescent substrate (Pierce, Rockford, IL), and signals generated were captured using FluorChem 5500 imaging system (Alpha Innotech, San Leandro, CA). To ensure reproducibility of the results, triplicates were performed. In-gel digestion Protein spots corresponding to the immunoreactive spots were manually excised from the Coomassie-stained 2-DE gels and prepared for in-gel digestion according to the method described by Gam et al. (2006). Briefly, the gel pieces were first dehydrated in 100% acetonitrile (ACN) and rehydrated in 100 mmol l 1 ammonium bicarbonate (NH4HCO3). This process was repeated twice. The gel pieces were then dried completely in a speed-centrifuge under vacuum (Eppendorf). The dried gel pieces were rehydrated in 12
5 ng ll 1 trypsin (Sigma) in digestion buffer containing 5 mmol l 1 CaCl2 and 50 mmol l 1 NH4HCO3 and incubated at 4°C for 45 min. Excess trypsin solution was discarded, and digestion buffer without trypsin was added to the gel pieces. After overnight digestion at 37°C, the supernatant was recovered and transferred to a new microcentrifuge tube. Peptides were extracted by incubating the gel pieces with 20 mmol l 1 NH4HCO3, followed by three times extraction with 5% (v/v) formic acid in 30 : 70 of ddH2O:ACN for 20 min each. Collected supernatants were pooled and dried under nitrogen flow. The peptide samples were stored at 20°C before analysis. Mass spectrometry analysis Peptide samples were reconstituted in 0
1% (v/v) formic acid in 85:15 ddH2O:ACN and analysed using

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nanoACQUITY UPLC system coupled to Xevo quadrupole time-of-flight (Q-TOF) mass spectrometer (Waters, Mildford, MA, USA). The nanoACQUITY UPLC system was equipped with trapping column nanoACQUITY UPLC Symmetry C18, 5 lm, 180 lm I.D. 9 20 mm, and the nanobore column was a nanoACQUITY UPLC BEH C18, 1
7 lm particle size, pore size 130  A, 75 lm I.D. 9 100 mm. Mobile phase A consisted of 0
1% formic acid in LC-MS grade H2O, while mobile phase B was 0
1% formic acid in 100% ACN. Sample (4 ll) was injected and loaded onto trapping column at an isocratic flow of 7 ll min 1 with a mobile phase of 99 : 1 ratio of A:B for 3 min. Eluted peptides were then separated in the nanobore column by a gradient mode of 3
0% B–95
0% B in 45 min at a flow rate of 0
45 ll min 1. The column effluent was interfaced to a Q-TOF mass spectrometer with electrospray spray ionization device. Mass spectrometer was operated in a positive ion mode at capillary voltage of 3
0 kV, sampling cone was at 40 V, and extraction cone at 4 V and source temperature was 80°C. TOF was operated in continuous extraction mode. Glu-Fibrinopeptide B (m/z 785
84206) was used as mass calibrant for lock mass correction in MS/MS mode. Fragmentation of ion was achieved when minimum intensity was detected for the three most abundant ions. Protein identification and database searches Instrument control, data acquisition and processing were performed using MASSLYNX 4.1 software (Micromass, UK). Fragmentation ion spectra raw data files were processed with the software that converts MS/MS raw data to peak lists. The resulting data were searched against Swiss-Prot database release 2013_07 (containing 540 546 sequence entries) using Protein Lynx Global Server (PLGS) v2.4 search engine (Waters, Mildford, MA). Databank search parameters include trypsin specificity, maximum of one missed cleavage, peptide tolerance of 100 ppm, MS/MS tolerance of 0
1 Da, one minimum peptide to match, 200 maximum hits to return, modification by carbamidomethylation of cysteine as fixed modification and monoisotopic mass values. The generated pkl files were also used to search against National Center for Biotechnology Information nonredundant (NCBI-nr) database ver. 20130725 (containing 32 504 738 protein entries) using MASCOT software ver. 2.4.01 (http://www. matrixscience.com). The search parameters were specified as follows: trypsin enzyme, maximum of one missed cleavage, carbamidomethyl as fixed modification, peptide tolerance of 2 Da, MS/MS tolerance of 0
8 Da and monoisotopic mass value. The significance threshold for protein identification was set at P < 0
05.

1002

Bioinformatics analysis Sequences of the identified proteins were analysed with several bioinformatics tools. BLASTP (http://www.ncbi. nlm.nih.gov/BLAST/) was used to search for homology sequences. Functional information of the proteins was obtained based on the prediction of the protein functions from UniProt database (http://www.uniprot.org/). SignalP webserver (http://www.cbs.dtu.dk/services/SignalP/) was used to predict the presence of signal peptide, whereas CELLO (http://cello.life.nctu.edu.tw/) was employed to predict protein subcellular localization. Production of recombinant protein and 1-D immunoblot analysis IDH2 gene was amplified from the genomic DNA of C. parapsilosis by PCR using gene-specific primers (Forward: 5′-ATGTTGAGACAAATTGCTAAATC-3′, Reverse: 5′-GTACAAGTTTTTGATAACTTCTTCAG-3′). The gene was cloned into pBAD-TOPOâ vector and transformed into E. coli cells according to manufacturer’s protocol (pBAD TOPOâ TA Expression Kit, Invitrogen). The clones were verified by PCR and DNA sequencing. Expression of the target protein was performed by induction with 20% arabinose for 4 h at 37°C. The histidine-tagged fusion protein was purified using ProBondTM Purification System (Invitrogen) following manufacturer’s instructions. The purified protein was analysed by SDS-PAGE and Western blot. Protein was transferred onto PVDF membranes and blocked with 3% BSA in TBST for 1 h at room temperature. The membranes were subsequently incubated with sera from mice infected with C. parapsilosis at a dilution of 1 : 2500 in TBST for 2 h and washed three times with TBST for 5 min each. The secondary antibody used was goat anti-mouse IgG peroxidase conjugate (Calbiochem) diluted 1: 100 000 in TBST, and incubation was carried out for 1 h. After washing three times with TBST, the membranes were treated with SuperSignalâ West Pico Chemiluminescent Substrate (Pierce) and imaged. Results Mouse model of systemic C. parapsilosis infection In mouse model of systemic candidiasis, fungi were accumulated in the kidneys as the major target organ due to inherent susceptibility to Candida infection (Lionakis et al. 2011). In this work, fungal burdens were determined through CFU counts by culturing the kidney homogenates. The results showed that fungal cells were consistently recovered from the kidneys of C. parapsilosis-infected

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mice, with mean log10 CFU g 1 of 3
42  0
26. On the contrary, no tissue colonization was detected for the control uninfected mice. This provides evidence on Candida dissemination within the mice and thus successful establishment of systemic infection. 2-DE proteome map Samples enriched with cell wall proteins from C. parapsilosis were separated by 2-DE to provide a comprehensive overview of the protein composition. Preliminary analysis of the protein samples was conducted using 7-cm wide range pH gradient strips of pH 3–10 (result not shown). As most of the protein populations were clustered in the centre region, 13 cm strips of pH 4–7 were used for subsequent analysis to obtain better protein separation and resolution. Separated proteins were visualized by Coomassie blue staining (Fig. 1). Majority of the spots were well-resolved into discrete protein spots. The proteomics map allowed detection of c. 262 protein spots of varying abundance with molecular weight ranging from 20 to 120 kDa. Triplicates were performed, and the 2-DE protein pattern was highly reproducible.

provide sufficient sera for immunoblotting. The result revealed that immunoblotting analysis using sera from control uninfected mice showed no significant reactivity (Fig. 2a). On the other hand, about 32 immunoreactive proteins were discovered following incubation of the blot with pooled sera from C. parapsilosis-infected mice (Fig. 2b). Similar pattern of reactive proteins was obtained in three independent Western blotting runs. Identification of immunogenic proteins and in silico analysis The immunogenic protein spots were selected after matching the immunoblot reactivity pattern with their Mr (kDa) 135 (a)

IEF SDS

95 72 52 42

Western blotting analysis For screening of immunogenic proteins, the 2-DE separated proteins were transferred to membranes and incubated with immune sera obtained from mice. Sera samples from mice of the same group were pooled to Mr (kDa) 200 150 120 100 85 70 60

IEF SDS

34 5

50

1 2

Mr (kDa) 135

IEF SDS

(b)

141516

72 20

34 5

52

22 23 6 7 8 27

1 2

24 25

29 12 13

42

21

22 23 6 7 8 26 28

9 10 11

30

20

17 18 19

21

26 28

40

7

95

141516 17 18 19

pI 4

24 25 27 29 12 13

9 10 11 30 31 32

30 31 32

25 20 pI 4 pI 4

7

7

Figure 1 Representative 2-DE gel profile of Candida parapsilosis cell wall proteins. Protein sample was separated in the first dimension by isoelectric focusing using 13 cm IPG strip pH 4–7 followed by separation by 10% SDS-PAGE in second dimension. The gel was visualized with Coomassie blue staining. Numbered spots were immunogenic proteins identified by mass spectrometry.

Figure 2 Representative immunoblot pattern of the 2-DE separated proteins from Candida parapsilosis. Identical gels were transferred onto PVDF membrane and used for immunoblotting. The blot was probed with pooled sera from uninfected mice (a) and C. parapsilosis-infected mice (b), respectively. No significant reactivity was detected for sera from negative control. Labelled spots were immunogenic proteins identified by mass spectrometry.

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respective Coomassie-stained 2-DE gels that were run in parallel to the blotted gels. Protein spots of interest were excised from the gels, subjected to tryptic digestion and analysed by mass spectrometry. The mass spectra were then searched against databases for protein identification. Proteins were identified based on the best hit that matched proteins from C. parapsilosis or other fungal species. This led to the identification of 32 protein spots representing 12 distinct proteins (Table 1). It is worth mentioning that although complete genome sequences of C. parapsilosis are available, its proteins are poorly characterized and annotated. As a result, seven of the proteins were identified as hypothetical proteins of C. parapsilosis. BLASTP search was therefore conducted for the hypothetical proteins, and the results revealed that the proteins shared sequence similarities with known proteins from other Candida spp. Another four proteins were identified as proteins of C. albicans and one as protein of yeast Saccharomyces cerevisiae. The identified immunogenic proteins were eukaryotic initiation factor 4A or translation initiation factor eIF4A subunit (Tif1p), ATP synthase subunit beta (Atp2p), enolase (Eno1p), glyceraldehyde-3-phosphate dehydrogenase (Gap1), heat shock protein 70 (Ssb1p), pyruvate decarboxylase (Pdc11p), ATP synthase subunit alpha (Atp1p), phosphoglycerate kinase (Pgk1p), alcohol dehydrogenase (Adh1p), fructose-bisphosphate aldolase (Fba1p), isocitrate dehydrogenase (Idh2p) and guanine nucleotidebinding protein subunit beta-like protein (Bel1p). Biological functions of the proteins were obtained based on the literature search and Gene Ontology analysis. Generally, the proteins are involved in carbohydrate and energy metabolism, protein synthesis and cellular transport. Most of the proteins were classified as metabolic enzymes. The results of signal peptide prediction revealed that none of the identified proteins possess a signal peptide. On the other hand, subcellular localization prediction analysis demonstrated that Tif1p was localized in the plasma membrane, whereas Atp1p, Atp2p, Idh2p and Bel1p were localized in the mitochondria. Besides, the results also predicted that Eno1p, Gap1p, Ssb1p, Pdc11p, Pgk1p, Adh1p and Fba1p were localized in the cytoplasm. Antigenicity of recombinant Idh2p Our findings revealed that Idh2p is a new antigenic protein, but its role in pathogenesis of Candida spp. is not well understood. Thus, the IDH2 gene from C. parapsilosis was cloned and expressed as recombinant protein in E. coli to confirm the MS result. Expression of the target protein with estimated molecular weight of 44 kDa was observed in the induced sample, and the recombinant 1004

protein was purified by affinity chromatography. The antigenicity of the purified protein was then investigated by Western blotting analysis. The results showed that the target protein was specifically recognized by pooled sera from mice infected with C. parapsilosis but not by the control sera (Fig. 3). Discussion In this study, an immunoproteomic analysis was carried out in search for immunogenic proteins of C. parapsilosis that were recognized by sera from experimentally infected mice. Mice model of systemic candidiasis was established by lateral tail vein challenge to produce immune sera. In this model of infection, the process of colonization and invasion of the fungal pathogen are comparable to the clinical course of infection in human (Pappas 2006). In this approach, the 2-DE separated proteins were screened by circulating IgG antibodies in the mice sera to identify immunogenic proteins. The results revealed that about 32 protein spots were specifically recognized by sera from C. parapsilosis-infected mice with different levels of immunoreactivities. It is noteworthy to mention that strong immune signal was also observed for low-abundance proteins (protein spots 1 and 2). This implies that the interaction between the antigens and antibodies is specific. The recognition of this subset of proteins by antibodies suggests that these proteins induce specific immune response in the host during infection. The immunoreactive protein spots were further analysed by mass spectrometry for protein identification. Intriguingly, protein spots that appeared as horizontal cluster in a line at different positions on the gel were identified as identical protein. Gap1p was found in five different spots, while Atp2p, Eno1p, Ssb1p and Pdc11p in three spots each and the remaining proteins appeared in two spots. Similarly, the detection of these proteins from several spots has also been reported previously in C. albicans (Pitarch et al. 2004; Martınez-L opez et al. 2008). This could be attributable to the well-known ability of 2-DE with high resolution power to separate proteins according to the two distinct biochemical properties. The existence of the same protein in multiple spots suggests the presence of putative post-translational modifications (PTMs) or protein processing which cannot be predicted from the gene sequences. Yet, it could also due to protein modifications that occurred during sample preparation and separation. It is also interesting to point out that some of the immunogenic proteins have a predicted cytosolic localization as they lack conventional signal peptides. Nevertheless, consistent with our findings, homologs of all of

Journal of Applied Microbiology 116, 999--1009 © 2013 The Society for Applied Microbiology

Journal of Applied Microbiology 116, 999--1009 © 2013 The Society for Applied Microbiology

P07251

P46273

gi|354547299

gi|354547586

gi|354546785

P83774

20,21

22,23

24,25

26,27

28,29

30–32

Hypothetical protein CPAR2_207210 [Candida parapsilosis] Hypothetical protein CPAR2_808670 [Candida parapsilosis] Heat shock protein SSB1 OS Candida albicans strain WO 1 GN SSB1 PE 1 SV 2 Hypothetical protein CPAR2_501020 [Candida parapsilosis] ATP synthase subunit alpha mitochondrial OS Saccharomyces cerevisiae strain ATCC 204508 S288c G Phosphoglycerate kinase OS Candida albicans strain SC5314 ATCC MYA 2876 GN PGK1 PE 1 SV 1 Hypothetical protein CPAR2_502580 [Candida parapsilosis] Hypothetical protein CPAR2_401230 [Candida parapsilosis] Hypothetical protein CPAR2_211610 [Candida parapsilosis] Guanine nucleotide-binding protein subunit beta-like protein OS Candida albicans strain SC5314 AT

ATP dependent RNA helicase eIF4A OS Candida albicans strain SC5314 ATCC MYA 2876 GN TIF1 PE 3 SV Hypothetical protein CPAR2_402220 [Candida parapsilosis]

Protein name

Alcohol dehydrogenase (Adh1p) Fructose-bisphosphate aldolase (Fba1p) Isocitrate dehydrogenase (Idh2p) Guanine nucleotide-binding protein subunit beta-like protein (Bel1p)

Phosphoglycerate kinase (Pgk1p)

Glyceraldehyde-3-phosphate dehydrogenase (Gap1p) Heat shock protein 70 (Ssb1p) Pyruvate decarboxylase (Pdc11p) ATP synthase subunit alpha (Atp1p)

Enolase (Eno1p)

ATP synthase beta subunit (Atp2p)

Eukaryotic initiation factor 4A (Tif1p)

BLAST result†

†

*Proteins were identified based on the best hit from Swiss-Prot or NBCI database. The top rank protein from BLAST search result was shown.

gi|354547143

gi|354545590

9–13

17–19

gi|354546348

6–8

P87222

gi|354547686

3–5

14–16

P87206

Swiss-Prot/ NBCI accession no*

1,2

Spot no.

Table 1 Identification of immunogenic proteins of Candida parapsilosis by mass spectrometry

293
589

62

74

71

75
064

390
044

218

6052
509

116

321

553

975
319

Score

20

9

7

15

3

18

12

43

11

16

16

14

No. of peptides matched

9
78

27

22

34

14
56

12
48

29

41
11

30

36

50

24
94

Sequence coverage (%)

6
07/34555

6
62/39916

5
37/39819

8
57/43520

6
07/45179

9
06/58608

5
28/62603

5
25/66479

6
19/36266

5
59/46995

5
23/53446

5
22/44599

Theoretical pI/Mw (Da)

Carbohydrate metabolism (Fermentative enzymes) Carbohydrate metabolism (Glycolytic enzyme) Carbohydrate metabolism (Tricarboxylic acid cycle) Respiration and fermentation, translation apparatus, ribosomal protein

Respiration and fermentation, cellular transport Carbohydrate metabolism (Glycolytic enzyme) Carbohydrate metabolism (Glycolytic enzyme) Protein folding and stabilization Carbohydrate metabolism (Fermentative enzymes) Respiration and fermentation, cellular transport Carbohydrate metabolism (Glycolytic enzyme)

Translation apparatus

Functions

P.Y. Lee et al. Iden of imm prot of Cp by SERPA

1005

Iden of imm prot of Cp by SERPA

(a) kDa

M

P.Y. Lee et al.

1

2

3

200 150 120 100 85

(b) kDa 170

M

1

M

2

130 95 72

70 60

55

50

43

40 34 30

26

25

Figure 3 Recombinant expression of Idh2p in E. coli and immunoblotting analysis. (a) SDS-PAGE analysis of purification of recombinant Idh2p by affinity chromatography. Lane M: molecular size standard (PageRulerTM Unstained Protein Ladder from Fermentas); Lane 1: lysate of induced E. coli cells; Lane 2: flow through fraction; Lane 3: wash fraction; Lane 4: purified protein from the elution fraction. (b) Western blot analysis of purified protein using mice sera. Lane M: molecular size standard (PageRulerTM Prestained Protein Ladder from Fermentas); Lane 1: incubation with pooled sera from control uninfected mice; Lane 2: incubation with pooled sera from Candida parapsilosis-infected mice.

these proteins have been detected in the cell wall fractions of C. albicans in previous studies (Pitarch et al. 2002; Ebanks et al. 2006), signifying their true localization in the cell wall. Besides, some of the proteins (Adh1p, Atp1p, Eno1p, Fba1p, Gap1p, Pdc11p, Pgk1p, Ssb1p and Tpi1p) were reported to be associated with cell surface of C. albicans (Nombela et al. 2006), whereas some of them (Adh1p, Eno1p, Fba1p, Gap1p, Pdc11p, Pgk1p and Ssb1p) have also been detected in the plasma membrane of C. albicans (Cabez on et al. 2009). As such, these proteins have been referred to as atypical cell wall proteins and might have multiple cellular localizations. It is postulated that this type of proteins was exported by alternative secretory pathways, but the exact mechanism is still not well understood. In the current work, immunoproteomic analysis of C. parapsilosis resulted in the identification of a total of 12 immunogenic proteins. Among them, 11 of the identified proteins have been previously reported as antigens of C. albicans (Pitarch et al. 2001, 2004, 2006; FernandezArenas et al. 2004; Martınez-L opez et al. 2008). These include Adh1p, Atp1p, Atp2p, Eno1p, Tif1p, Fba1p, Gap1, Bel1p, Ssb1p, Pgk1p and Pdc11p. Apart from that, some of these proteins have also been implicated as antigenic proteins in other pathogens such as Aspergillus fumigatus (Singh et al. 2010), Cryptococcus neoformans (Young et al. 2009) and Mycobacterium (Deenadayalan et al. 2010). Remarkably, some of the immunogenic proteins have also been detected in patients with systemic candidiasis and were targeted by human antibodies, supporting the 1006

findings from the animal model. Eno1p and Ssb1p were among the protein antigens that were found in the blood circulation of patients with systemic candidiasis (Walsh et al. 1991; Giraldo et al. 1999). On the other hand, Pgk1p and Fba1p were major protein antigens that reacted with human salivary IgA (Calcedo et al. 2012). Proteins that are seroreactive to sera from patients are potentially valuable for development of serological test and immunotherapeutics. Besides, some of the antigens were reported to bind to host proteins and involve in pathogenesis of C. albicans. Eno1p and Pgk1p have been reported to bind plasminogen and plasmin, which was associated with invasion of host tissues (Crowe et al. 2003). Gap1p was reported to bind to fibronectin and laminin (Gozalbo et al. 1998), suggesting its role in adhesion and dissemination during infection. Adh1p was recognized by antibodies to human integrins (Klotz et al. 2001), implying that it might be receptor for fibronectin or vibronectin and play role in adhesion to host cells. On the other hand, Bel1p was shown to confer virulence in mice as null mutant was defective in adhesion and invasion (Kim et al. 2010). Among the identified proteins, Eno1p and Gap1p have been studied for their potential as biomarkers for candidiasis. Eno1p was shown to be the most immunogenic antigen in mice infected with C. albicans (Pitarch et al. 2001; Fernandez-Arenas et al. 2004; Martınez-L opez et al. 2008). Besides, Eno1p showed great potential for serodiagnosis of invasive candidiasis (Laın et al. 2007) and was suggested as a potential target for vaccine development

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(Li et al. 2011). Antibodies to another major immunogenic protein, Gap1p, were reported to possess discriminating ability and correlated with poor prognosis of invasive candidiasis (Pitarch et al. 2011). However, a study on its potential immunotherapeutic use showed that antibodies against Gap1p did not confer protection in murine model of systemic candidiasis (Gil et al. 2006). Besides, some of the immunogenic proteins were also implicated in stress and drug responses. The abundance of Atp2p and Tif1p was increased after treatment with peroxide stress, while increased abundance of Atp1p, Atp2p, Eno1p, Fba1p, Gap1p, Pgk1p and Ssb1p was observed after exposure to cadmium stress (Yin et al. 2009). On the other hand, ATP1 gene expression was upregulated after exposure to antifungal drugs ketoconazole and flucytosine (Liu et al. 2005). Besides, FBA1 gene expression was reported to be upregulated in biofilm and in response to antifungal drug fluconazole (Garcıa-Sanchez et al. 2004). This suggests that these proteins might serve as potential antifungal drug targets for candidiasis. Remarkably, the antigenicity of Idh2p for Candida spp. was first reported in our present study. Idh2p is an enzyme that is involved in the tricarboxylic acid cycle. It catalyses the conversion of isocitrate to alpha-ketoglutarate (a-ketoglutarate) and CO2 in a two-step process. As mentioned before, many of the known intracellular proteins with important metabolic functions have been recovered from the cell wall compartment and were reported to be immunogenic. Similarly, Idh2p was identified as an antigen in the current work and has been found in the C. albicans cell wall-associated fraction (Ebanks et al. 2006). Thus, it is possible that these antigens are multifunctional proteins that confer metabolic roles in the cytosol and are trafficked to the surface to participate in virulence functions. Higher IDH2 gene expression has been observed in high iron condition (Lan et al. 2004). Its homologous protein was identified as antigen of pathogenic fungus A. fumigatus (Singh et al. 2010) and Gram-negative bacterium Helicobacter pylori (Mcatee et al. 1998). Idh2p was also shown to elicit strong B cell response and was a potential immune marker for serodiagnosis of tuberculosis (Florio et al. 2002). Importantly, this study showed that the recombinant Idh2p reacted specifically with pooled sera from mice infected with C. parapsilosis on immunoblot, therefore validating its antigenicity. This further signifies that Idh2p is a novel antigen and could be a potential biomarker candidate for C. parapsilosis. Besides, the recombinant protein represents interesting target molecule that can be exploited for further characterization and analysis. To our knowledge, this is the first study that reported immunogenic proteins of C. parapsilosis. The results from

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this study provided evidence of in vivo expression of the immunogenic proteins during infection and their ability to elicit humoral immune response. The current work identified several candidate antigens, with Idh2p as a novel immunogenic protein that may serve as specific biomarker for C. parapsilosis. However, the potential of the identified immunogenic proteins as targets for the development of diagnostic assay and/or vaccine needs to be assessed and confirmed in further work. In conclusion, an integrated approach using proteomics, Western blotting and mass spectrometry was successfully employed to identify protein antigens from C. parapsilosis. A total of 32 immunogenic protein spots were detected using antisera from mice with systemic C. parapsilosis infection. This distinct antibody response was absent in sera from control mice. Mass spectrometry analysis of the immunoreactive spots led to the identification of 12 proteins, with Idh2p as a new immunogenic protein. Our findings provide an insight into the immune pathogenesis of C. parapsilosis, and the immunogenic proteins can be considered as potential diagnostic and/or vaccine candidates for C. parapsilosis. Acknowledgements This work was supported by the E-Science Fund, Malaysia (Project No. 02-01-04-SF0761) sponsored by Ministry of Science, Technology and Innovation (MOSTI), Malaysia. Conflict of interest No conflict of interest declared. References Arendrup, M., Horn, T. and Frimodt-Møller, N. (2002) In vivo pathogenicity of eight medically relevant Candida species in an animal model. Infection 30, 286–291. Bliss, J.M., Wong, A.Y., Bhak, G., Laforce-Nesbitt, S.S., Taylor, S., Tan, S., Stoll, B.J., Higgins, R.D. et al. (2012) Candida virulence properties and adverse clinical outcomes in neonatal candidiasis. J Pediatr 161, 441–447. Cabez on, V., Llama-Palacios, A., Nombela, C., Monteoliva, L. and Gil, C. (2009) Analysis of Candida albicans plasma membrane proteome. Proteomics 9, 4770–4786. Calcedo, R., Ramirez-Garcia, A., Abad, A., Rementeria, A., Pont on, J. and Hernando, F.L. (2012) Phosphoglycerate kinase and fructose bisphosphate aldolase of Candida albicans as new antigens recognized by human salivary IgA. Rev Iberoam Micol 29, 172–174. Chaffin, W.L. (2008) Candida albicans cell wall proteins. Microbiol Mol Biol Rev 72, 495–544.

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